Erythropoietic Potential of CD34+ Hematopoietic Stem Cells ...
10
Research Article Erythropoietic Potential of CD34+ Hematopoietic Stem Cells from Human Cord Blood and G-CSF-Mobilized Peripheral Blood Honglian Jin, 1 Han-Soo Kim, 2 Sinyoung Kim, 1 and Hyun Ok Kim 1 1 Division of Transfusion Medicine and Cell erapy, Department of Laboratory Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea 2 Innovative Cell and Gene erapy Center, International St. Mary’s Hospital, 25 Simgok-ro, 100 beon-gil, Seo-gu, Incheon 404-834, Republic of Korea Correspondence should be addressed to Hyun Ok Kim; [email protected] Received 23 February 2014; Accepted 30 March 2014; Published 5 May 2014 Academic Editor: Mina Hur Copyright © 2014 Honglian Jin et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Red blood cell (RBC) supply for transfusion has been severely constrained by the limited availability of donor blood and the emergence of infection and contamination issues. Alternatively, hematopoietic stem cells (HSCs) from human organs have been increasingly considered as safe and effective blood source. Several methods have been studied to obtain mature RBCs from CD34+ hematopoietic stem cells via in vitro culture. Among them, human cord blood (CB) and granulocyte colony-stimulating factor- mobilized adult peripheral blood (mPB) are common adult stem cells used for allogeneic transplantation. Our present study focuses on comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs. By using CD34+ cells from cord blood and G-CSF mobilized peripheral blood, we showed in vitro RBC generation of artificial red blood cells. Our results demonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under the same conditions, but differ considerably with respect to expression levels of various genes and hemoglobin development. is study is the first to compare the characteristics of CB- and mPB-derived erythrocytes. e results support the idea that CB and mPB, despite some similarities, possess different erythropoietic potentials in in vitro culture systems. 1. Introduction Red blood cell transfusion is a well-established and essen- tial therapy for patients with severe anemia. However, the worldwide supply of allogeneic blood faces a serious shortage, and there are many patients around the world whose survival depends on blood transfusion. Around 92 million blood donations are collected annually from all types of blood donors (voluntary unpaid, family/replacement, and paid), but in the report of 39 counties of 159 countries on their collections, donated blood is still not routinely tested for transfusion-transmissible infections (TTIs) including HIV, hepatitis B, hepatitis C, and syphilis [1]. Nevertheless, blood transfusion saves lives, but the transfusion of unsafe blood puts lives at risk because HIV or hepatitis infections can be transmitted to patients through transfusion. However, the financial consequence of discarding unsafe blood creates yet another burden in developing countries. Research performed on stem cells, specifically hematopoietic stem cells (HSCs), holds promise for the production of mature red blood cells in large quantities through differentiation induction. e classic source of HSCs has been the bone marrow, but bone marrow procurement of cells is an invasive process with risks. e artificial RBCs from stem cells in vitro culture can be generated from sources such as embryonic stem cells (ESCs) [2], induced pluripotent stem cells (iPSs) [3], cord blood (CB) [4–6], and peripheral blood (PB) [7]. Of these, ESCs and iPSCs are the least promising due to the low generation efficiency and long-term in vitro culture cost hindrances. Currently, granulocyte colony-stimulating factor- (G-CSF-) mobilized peripheral blood (mPB) and CB are therefore Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 435215, 9 pages http://dx.doi.org/10.1155/2014/435215
Transcript of Erythropoietic Potential of CD34+ Hematopoietic Stem Cells ...
Research ArticleErythropoietic Potential of CD34+ HematopoieticStem Cells from Human Cord Blood and G-CSF-MobilizedPeripheral Blood
Honglian Jin1 Han-Soo Kim2 Sinyoung Kim1 and Hyun Ok Kim1
1 Division of Transfusion Medicine and Cell Therapy Department of Laboratory Medicine Yonsei University College of Medicine50 Yonsei-ro Seodaemun-gu Seoul 120-752 Republic of Korea
2 Innovative Cell and Gene Therapy Center International St Maryrsquos Hospital 25 Simgok-ro 100 beon-gil Seo-guIncheon 404-834 Republic of Korea
Correspondence should be addressed to Hyun Ok Kim hyunok1019yuhsac
Received 23 February 2014 Accepted 30 March 2014 Published 5 May 2014
Academic Editor Mina Hur
Copyright copy 2014 Honglian Jin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Red blood cell (RBC) supply for transfusion has been severely constrained by the limited availability of donor blood and theemergence of infection and contamination issues Alternatively hematopoietic stem cells (HSCs) from human organs have beenincreasingly considered as safe and effective blood source Several methods have been studied to obtain mature RBCs from CD34+hematopoietic stem cells via in vitro culture Among them human cord blood (CB) and granulocyte colony-stimulating factor-mobilized adult peripheral blood (mPB) are common adult stem cells used for allogeneic transplantation Our present study focuseson comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs By using CD34+ cells fromcord blood and G-CSF mobilized peripheral blood we showed in vitro RBC generation of artificial red blood cells Our resultsdemonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under thesame conditions but differ considerably with respect to expression levels of various genes and hemoglobin development Thisstudy is the first to compare the characteristics of CB- and mPB-derived erythrocytes The results support the idea that CB andmPB despite some similarities possess different erythropoietic potentials in in vitro culture systems
1 Introduction
Red blood cell transfusion is a well-established and essen-tial therapy for patients with severe anemia However theworldwide supply of allogeneic blood faces a serious shortageand there are many patients around the world whose survivaldepends on blood transfusion Around 92 million blooddonations are collected annually from all types of blooddonors (voluntary unpaid familyreplacement and paid)but in the report of 39 counties of 159 countries on theircollections donated blood is still not routinely tested fortransfusion-transmissible infections (TTIs) including HIVhepatitis B hepatitis C and syphilis [1] Nevertheless bloodtransfusion saves lives but the transfusion of unsafe bloodputs lives at risk because HIV or hepatitis infections can betransmitted to patients through transfusion However the
financial consequence of discarding unsafe blood creates yetanother burden in developing countries
Research performed on stem cells specificallyhematopoietic stem cells (HSCs) holds promise for theproduction of mature red blood cells in large quantitiesthrough differentiation induction The classic sourceof HSCs has been the bone marrow but bone marrowprocurement of cells is an invasive process with risksThe artificial RBCs from stem cells in vitro culture can begenerated from sources such as embryonic stem cells (ESCs)[2] induced pluripotent stem cells (iPSs) [3] cord blood(CB) [4ndash6] and peripheral blood (PB) [7] Of these ESCsand iPSCs are the least promising due to the low generationefficiency and long-term in vitro culture cost hindrancesCurrently granulocyte colony-stimulating factor- (G-CSF-)mobilized peripheral blood (mPB) and CB are therefore
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 435215 9 pageshttpdxdoiorg1011552014435215
2 BioMed Research International
widely researched as a potential alternate source for stemcell procurement However this has not been a widespreadstandard of therapy and the characteristics of mature redblood cells derived from HSCs after mass production are notyet well known Our study focuses on comparing CB- andmPB-derived stem cells with respect to their characteristicsand function after differentiation
2 Materials and Methods
21 CD34+ HSC Isolation Culture and Erythropoietic Dif-ferentiation CB samples from normal full-term deliveries(119899 = 7) were collected in a bag (Green Cross CorpYong-in Korea) containing 245mL of citrate phosphatedextrose A (CPDA-1) Five milliliters of G-CSF-mPB wasobtained (119899 = 7) with the written informed consent ofnormal voluntary allogeneic HSC donors This study wasapproved by Severance Hospital IRB (IRB number 4-2011-0081)TheCD34+ cells from both sources were isolated usinga MACS isolation kit (density 1077 Pharmacia BiotechUppsala Sweden) using an antibody against CD34 accordingto the manufacturerrsquos instructions And the sorted CD34+cells were cultured at a density of 1 times 105 cellsmL in astroma-free condition for 17ndash21 days as described previously[8 9] Briefly from day 0 to 7 sorted CD34+ cells werecontinually cultured in serum-free conditioned erythrocyteculture medium with 100 ngmL SCF (Peprotech RehovotIsrael) 10 ngmL IL-3 (Peprotech) and 6 IUmL recombinantEPO (Recormon Epoetin beta Roche) with a half-volumemedium change twice a week Serum-free culture mediumconsisted of StemPro-34 SFM Complete Medium (GibcoGrad Island NY) supplemented with 1 bovine serum albu-min (Sigma) 150 120583gmL iron-saturated human transferrin(Sigma) 50120583gmL insulin (Sigma) 90 ngmL ferrous nitrate(Sigma) 2mMolL L-glutamine (Sigma) 16 times 10minus4molLmonothioglycerol (Sigma) 308 120583ML vitamin C (Sigma)2 120583gmL cholesterol (Sigma) and 1 penicillin-streptomycinsolution (Gibco) In the second 7-day period of culture themedium was replaced with serum-free conditioned mediumwith 3 IUmL of recombinant EPO 50 ngmL of SCF and10 ngmL of IL-3 for expansion and differentiation Duringdays 15ndash18 of culture only one cytokine (EPO at 2 IUmL)was used for erythrocyte differentiation and poloxamer 188(Pluronic F68 (F68) Sigma MW 8400) was added at aconcentration of 005No cytokineswere added during days19ndash21 of culture and only poloxamer 188 was added duringthis period At the end of each phase cultured cells werecounted using a hemocytometer The trypan blue stain wasused in all cell counts and only viable cells are included in thefold expansion results All cultures were maintained at 37∘Cin a humidified atmosphere of 5 CO
2
22 Assessment of Cell Morphology Cell morphology wasassessed using slides prepared by Cytospin using a cyto-centrifuge (Cytospin 3 Shandon Scientific Tokyo Japan)at 800 rpm for 4min followed by Wright-Giemsa stainingPictures of the stained cells were taken with a digital camera(DP70 Olympus Tokyo Japan) at 400x magnification
23 Differential Counting of Cultured Erythroblasts Fivedifferential countings were enumerated as proerythroblastsearly and late basophilic erythroblasts polychromatic ery-throblasts and orthochromatic erythroblasts at 1000x mag-nification
24 Flow Cytometric Analyses of Erythroid Markers For flowcytometric analyses of cell surface antigens a total of 1 times 105cells were stained with phycoerythrin- (PE-) or fluoresceinisothiocyanate- (FITC-) conjugated mouse anti-human anti-bodies against CD45 CD34 CD71 and glycophorin A (GpA)for 15min washed resuspended in FACS buffer and analyzedusing aCell LabQuanta SC (BeckmanCoulter Fullerton CAUSA) using a 488 nm wavelength laser Cells were analyzedusing two-color flow cytometry through WinMDI 29 Theantibody combinations usedwere CD45-FITCCD34-PE andCD71-FITCGpA-PE using G1-FITCG1-PE as a control Allfluorescent conjugated monoclonal antibodies used werepurchased from BD Biosciences (San Jose CA)
25 Quantitative Real-Time Polymerase Chain Reaction Toevaluate gene expression levels during erythrocyte differen-tiation from different sources we harvested over 1 times 106cells from cultured erythrocytes at 7 10 14 and 17 days andisolated total RNA for quantitative polymerase chain reaction(PCR) Gene expression levels were quantified using the LightCycler 480 Real-time PCR System (Roche Applied Science)Quantitative real-time polymerase chain reaction (qPCR)was performed using Light Cycler 480 SYBR Green I Mastermix (Roche Applied Science) according to themanufacturerrsquosinstructions Primers were designed [10 11] and generatedby Bioneer (Korea) (Table 1) Total RNA (800 ng) was usedto generate first-strand cDNA using the Maxime RT PremixKit (Intron Biotech) Differences between the Cp (crossingpoint) values of actin and target mRNAs for each samplewere used to calculate ΔCp values The ΔCp values derivedfrom the isolated undifferentiated CD34+ cells were usedas control ΔCp values Relative expression levels betweensamples and controls were determined using the formularelative expression level = 2minus(119878ΔCpminus119862ΔCp) Comparative real-time PCR with primers specific for GATA1 GATA2 EKLFeALAS and SCLTall (Table 1) was performed in triplicateReactions were performed at 95∘C for 10min followed by 45cycles of 95∘C for 30 s 60∘C for 30 s and 72∘C for 30 s
26 Functional Analysis of Hemoglobin We used a Hemox-Analyzer (TCS Medical Products Division SouthamptonPA) to measure the oxygen binding and dissociation abilitiesof the hemoglobin produced in mature erythrocytes derivedfrom the mPB and cord blood Hemox-Analyzer is an auto-matic system for recording blood oxygen equilibrium curvesand related phenomena [12] The operating principle of theHemox-Analyzer is based on dual-wavelength spectropho-tometry for the measurement of the optical properties ofhemoglobin and a Clark electrode for measuring the oxygenpartial pressure in millimeters of mercury The resultingsignals from both measuring systems are fed to the 119883-119884recorder Both the P
50value and observation of the fine
BioMed Research International 3
1
10
100
1000
10000
100000
1000000
Cel
l num
bers
mPBCB
1010
5022
411481
11087 98386 498114 83256218277 177284 259210 636038
Comparison of cultivative cell growth
0d 4d 7d 10d 14d 17d 21d
(a)
mPB 100 502 820 2695 887 506 167CB 10 35 137 380 97 146 25
000
1000
2000
3000
4000
5000
6000
Fold
indu
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Comparison of cell fold induction
0d 4d 7d 10d 14d 17d 21d
(b)
Figure 1 Comparison of cell growth in PB- and CB-derived CD34+ cell cultures Stem cells were cultured for 21 days and counted at the endof each phase (a) Erythroid cell amplification (mean plusmn standard deviation) of mPB- and CB-derived CD34+ cells (b) Numbers of total cellsexpanded from cultures of mPB- and CB-derived CD34+ cells CB-CD34+ cells exhibit higher amplification efficiency than mPB cells lowast119875 lt0001
Table 1 Real-time polymerase chain reaction primers
Gene Primer sequence120573-actin
Forward primerReverse primer
51015840-ATTGGCAATGAGCGGTTC-3101584051015840-GGATGCCACAGGACTCCAT-31015840
GATA-1Forward primerReverse primer
51015840-CACTGAGCTTGCCACATCC-3101584051015840-ATGGAGCCTCTGGGGATTA-31015840
GATA-2Forward primerReverse primer
51015840-GGCAGAACCGACCACTCATC-3101584051015840-TCTGACAATTTGCACAACAGGTG-31015840
eALASForward primerReverse primer
51015840-GATGTGAAGGCTTTCAAGACAGA-3101584051015840-GGAAAATGGCTTCCTTAGGC-31015840
EKLFForward primerReverse primer
51015840-ATCGAGTGAAGAGGAGACCTTCC-3101584051015840-TGAAGATACGCCGCACAACTT-31015840
SCLTal1Forward primerReverse primer
51015840-ACACACAGGATGACTTCCTC-3101584051015840-CCCATGTCCTGCGC-31015840
structure of the curve can furnish information about thedelivery of oxygen to tissues CD34+ cells derived from CBand mPB that were cultured for 17 days in three separatephases were analyzed using this system Normal red bloodcells were used as a control
27 Capillary Zone Electrophoresis After 17 days of culture 1times 108 cells were collected and assessed by capillary zone elec-trophoresis Capillary zone electrophoresis was performed asdescribed previously using the Sebia Capillary system (SebiaNorcross GA) [13] Differentiated erythrocytes (5 times 107cells)were centrifuged at 5000 rpm for 5 minutes Thereafter the
culture medium was removed and the erythrocyte pellet wasvortexed for 5 s Electrophoresis was performed in alkalinebuffer (pH 94) provided by the manufacturer (Sebia) withseparation primarily due to the pH of the solution andendosmosis The hemoglobin was measured at a wavelengthof 415 nm Electrophoretograms were recorded with thelocation of specific hemoglobin in specific zones
28 Statistical Analysis Studentrsquos 119905-test was performed usingExcel (Microsoft) 119875 values less than 005 were consideredstatistically significant
3 Results
31 In Vitro Culture Supports the Differentiation of Ery-throcytes The number of cell divisions observed signifi-cantly increased during the second phase of the cultureperiod Compared to mPB-CD34+ cells CB-CD34+ cellshave greater proliferative capacity during days 10ndash21of culture(Figure 1(a)) This difference led to CB cultures achievinga greater total number of cells than that of mPB cultures(636038 plusmn 182817 versus 83256 plusmn 8858) Cell growth in CBcell cultures exceeded that of mPB cell cultures in the secondphase and early third phase of culture (Figure 1(b)) Followingerythropoietic differentiation decreased cell size nuclearcondensation and nuclear extrusion were confirmed byWright-Giemsa staining Although there were no significantdifferences found our results show that in the blood typecomposition count the CB HSCs have more multipotencywhile mPB-CD34+ cells show earlier differentiation intomature erythrocytes (Figure 2(a)) These results demonstratethatmPB- andCB-derived CD34+HSCs have similar growthpatterns andmorphological characteristics butmPB-derivedCD34+ cells show faster maturation than CB-derived CD34+cells (Figure 2(b))
4 BioMed Research International
mPB
CB
0d 7d 10d 14d 17d 21d
(a)
00
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896
95
586
338
66
727
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440
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106
229
497
159
82
208
430
270
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611
85
636
277
CB
mPB CB
mPB CB
mPB CB
mPB
Orthochromatic erythroblastPolychromatic erythroblastLate basophilic erythroblast
Early basophilic erythroblastProerythroblast
7d 10d 14d 17d
(b)
Figure 2 Comparison of cell morphological changes in mPB- and CB-derived CD34+ cell cultures CD34+ cells selected from mPB andCB were cultured for 21 days in vitro Morphological changes in differentiated cells from both sources were similar No remarkably differentpatterns were found in the Giemsa staining photos (a) Based on the erythrocyte cell type counting at the end of each phase the mPB-derivedcells matured more rapidly than the cells derived from CB even though cells from both sources have similar maturation patterns (b)
32 Similar Immunophenotypic Patterns CD34 and CD45marker dramatically decreased and finally disappeared fromthe cells during differentiation while GPA expressionincreased during the 21 days of culture Although CD71expression increased until early in the third phase of cultureit gradually decreased following final maturation (Figure 3)From the immunophenotypic data we found no significantdifferences between mPB- and CB-derived CD34+ cellsduring erythroid cell maturation
33 Different mRNA Expression Levels From our data wecan see different patterns in the differentiation of mPB- andCB-derived CD34+ cells Our data clearly show that GATA-1expression gradually increases during erythrocyte differenti-ation especially in CB cells while GATA-2 expression grad-ually decreases following cell maturation The erythrocyte-specific isoforms ALAS and SCLTall are upregulated duringerythrocyte differentiation and in particular show higher
levels inmPB-derived cells than inCB-derived cells Only onefactor EKLF which is a 120573-globin gene transcription factorincreased during erythrocyte differentiation inmPB cells butin contrast decreased during differentiation of CB-derivedcells (Figure 4) These results clearly demonstrate that mPB-derived CD34+ cells differentiate faster into erythrocyteswith Hb-120573 production than CB cells At the same timeunder these in vitro culture conditions CB-derived CD34+hematopoietic cells exhibit higher multipotency than mPBcells
34 Different Hemoglobin Type Development in mPB- andCB-Derived CD34+ Cells Over 80 of the hemoglobin pro-duced by CB-derived CD34+ cells was hemoglobin subtypeHbF while only 175 was subtype HbA Over 95 of thehemoglobin generated by mPB-derived cells was subtypeHbA (Figure 5)
BioMed Research International 5
64
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CD34
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CD71
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-1CD
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71G
PA-1
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CD34
CD71
GPA
-1
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CD34
CD71
GPA
-1
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-1
GPA
-1
GPA
-1
mPB
CB
0d 7d 14d 21d
Figure 3 Phenotypic markers in erythrocytes differentiated from mPB- and CB-derived CD34+ cells Cells (3 times 105) from the end of eachphase of culture were stained and hematopoietic and erythropoietic markers were measured Flow cytometry results show similar patternsin the cultured cells from both sources and no significant differences
6 BioMed Research International
00
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0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
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Figure 4 Erythrocyte-specific gene expression in mPB and CB cells At the end of each phase of culture cells were harvested and totalRNA was extracted for quantitative PCRThe expression of GATA-1 GATA-2 eALAS EKLF and SCLTal1 was measured by real-time PCRThe results show an increasing pattern for the GATA-1 transcript and a decreasing pattern for GATA-2 which did not significantly differbetween mPB and CB cells EKLF eALAs and SCLTall expression levels increased during differentiation but were significantly greater inmPB-derived erythrocytes than in those from CB
BioMed Research International 7
Z15
Z14
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Z8
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1Z(A)
Z(D
)
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(F)
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)
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)Z
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)
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Control
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Hb F
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Cord blood derived CD34+ cells
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Hb FHb A2
G-CSF mobilized peripheral blood derived CD34+ cells
Figure 5 Electrophoretic determination of main hemoglobin subtypes in differentiated mPB and CB cells Capillary zone electrophoresisshows distinct patterns of hemoglobin subtype expression between differentiated mPB and CB cells About 80 of the hemoglobin producedby CB-CD34-derived erythrocytes is HbF while only 175 is HbA However 955 of the hemoglobin produced by mPB-CD34-derivederythrocytes is HbA
35 Similar Hemoglobin Dissociation Curve with MatureRBCs from mPB and CB Cells While erythrocytes derivedfrom each source have different combinations of HbA andHbF similar hemoglobin dissociation curve was observed(Figure 6) Apart from this hemoglobin subtype variationthis result clearly shows that in vitro cultured RBCs canproduce hemoglobin with oxygen binding and dissociationabilities equivalent to red blood cells produced in vivo
4 Discussion
The shortage of blood supply and the ever-growing demandfor blood transfusion represent a significant emerging issue in
transfusionmedicine Shortage of donated blood and the riskof infection have created limitations in the availability of redblood cells available for transfusion More recently isolatedCD34+ cells from human umbilical CB obtained from dis-carded maternity products and G-CSF-mPB obtained fromhealthy volunteers through leukapheresis have emerged aspotential alternative sources of HSCs The HSCs collectedfrom both sources have high rates of proliferation andcapacities for differentiation and create mature RBCs underthe proper culture conditions through three phases of invitro culture We note that though both types of stemcells show similar characteristics in general growth patternswith morphological and immunophenotypic changes they
8 BioMed Research International
0
0 50 150
0
0 50 150
21-day cultured RBCs from mPB 21-day cultured RBCs from CB
Figure 6 Hemox analysis in mPB and CB cells cultured for 21 days Differentiated erythrocytes were harvested at the end of the 21 daysof culture and oxygen equilibria were measured by Hemox-Analyzer Erythrocytes derived from mPB and from CB both show greateroxygenation abilities compared with normal human blood There are no significant differences between differentiated mPB and CB cells
show different characteristics in gene expression levels andhemoglobin subtype production
Our results show that bothmPB- and CB-derived CD34+cells began to proliferate extremely quickly during 7ndash14 daysof culture However both cell sources produce similar totalcell numbers after 21 days of culture And morphologicalchanges in differentiated cells from both sources were similarand no remarkable differences were found during the cultureexcept when comparing cell differentiation rates The mPBcells seemed to mature earlier than CB cells These resultsshow that mPB cells from circulating blood possess moreprogenitor cells than CB and have the ability of rapiddifferentiation into the mature RBCs Following cell growthat the end of each cell culture phase mPB- and CB-derivedCD34+ GPA and CD70 (transferrin receptor) expressionshowed similar patterns in flow cytometry analyses Thesedata are consistent with previous results regarding erythro-cyte induction mechanisms [8 14] To confirm whether theinitial exposition of cells is due to different sources deriveddifferentiation gene expression profiles were analyzed byquantitative RT-PCR GATA-1 is a transcription factor thatdetermines erythroid differentiation survival and 120573-globingene expression [15] GATA-2 inhibits GATA-1 functionGATA-2 expression exhibited a downregulation followingerythropoietin stimulation and its levels were higher incultured mPB cells This can be explained in earlier dif-ferentiation into mature RBCs in mPB source cells thanin CB This finding is also consistent with the result ofdifferential counts that mPB showed faster shift from pronor-moblasts to orthochromatic normoblasts in comparison toCB Ikonomi et al have previously shown that GATA-2preferentially increases 120574-globin gene expression indicatingthat the prolonged expression of GATA-2 contributes to theearly increase in 120574-globin in CB [16] EKLF binds specificallyto the 120573-globin promoter and is critical in establishingchromatin structure for high-level 120573-globin transcription via
its acetylation by CREB binding protein [17] SCLTall isrequired for the progression of erythroid differentiation andenforced expression of SCLTall increases 120573-globin expres-sion and BFU-E and CFU-E production [18] Because thesetranscription factors are closely related to increased 120573-globingene expression changes in their expression may accountfor the delay and reduction of 120573-globin expression in CB-derived differentiated cells The HbA- and HbF-related geneexpression tests exhibit different expression levels dependingon the cell sourceWe culturedmPB- andCB-derived CD34+cells through three phases harvested them after 17 daysof culture and analyzed them by hemoglobin type testingThese results clearly demonstrated different subtypes ofhemoglobin expressed bymPB- andCB-derivedmature cellsCB-derived cells mostly express HbF and mPB-derived cellsmainly express HbA but based on these data the originalsources of these cells appear to possess different propensitiesfor hemoglobin production patterns These results demon-strate that hemoglobin subtypes are not related to cultureconditions and culture time but are strongly affected by thesource material To evaluate the function of the differentiatedmPB and CB cells CD34+ cells derived from each sourcewere expanded and differentiated to large cell numbers ofup to a total of 5 times 107 cells and oxygen equilibria weremeasured by Hemox-Analyzer From the result the oxygendissociation curves indicate that the cells from both sourcesdo not significantly differ from one another with respectto hemoglobin function Although the types of hemoglobinexpression differed between CB- and mPB-derived maturecells the oxygen binding and dissociation curves may besimilar due to variation among adult type hemoglobin fetaltype hemoglobin or mixture of both types in vitro cultureprocessing after 17 days of culture The cultured cells indeedhave slightly greater deoxygenation functionalities comparedwith normal cells which showed the immaturity in shift toleft
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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GenomicsInternational Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Microbiology
2 BioMed Research International
widely researched as a potential alternate source for stemcell procurement However this has not been a widespreadstandard of therapy and the characteristics of mature redblood cells derived from HSCs after mass production are notyet well known Our study focuses on comparing CB- andmPB-derived stem cells with respect to their characteristicsand function after differentiation
2 Materials and Methods
21 CD34+ HSC Isolation Culture and Erythropoietic Dif-ferentiation CB samples from normal full-term deliveries(119899 = 7) were collected in a bag (Green Cross CorpYong-in Korea) containing 245mL of citrate phosphatedextrose A (CPDA-1) Five milliliters of G-CSF-mPB wasobtained (119899 = 7) with the written informed consent ofnormal voluntary allogeneic HSC donors This study wasapproved by Severance Hospital IRB (IRB number 4-2011-0081)TheCD34+ cells from both sources were isolated usinga MACS isolation kit (density 1077 Pharmacia BiotechUppsala Sweden) using an antibody against CD34 accordingto the manufacturerrsquos instructions And the sorted CD34+cells were cultured at a density of 1 times 105 cellsmL in astroma-free condition for 17ndash21 days as described previously[8 9] Briefly from day 0 to 7 sorted CD34+ cells werecontinually cultured in serum-free conditioned erythrocyteculture medium with 100 ngmL SCF (Peprotech RehovotIsrael) 10 ngmL IL-3 (Peprotech) and 6 IUmL recombinantEPO (Recormon Epoetin beta Roche) with a half-volumemedium change twice a week Serum-free culture mediumconsisted of StemPro-34 SFM Complete Medium (GibcoGrad Island NY) supplemented with 1 bovine serum albu-min (Sigma) 150 120583gmL iron-saturated human transferrin(Sigma) 50120583gmL insulin (Sigma) 90 ngmL ferrous nitrate(Sigma) 2mMolL L-glutamine (Sigma) 16 times 10minus4molLmonothioglycerol (Sigma) 308 120583ML vitamin C (Sigma)2 120583gmL cholesterol (Sigma) and 1 penicillin-streptomycinsolution (Gibco) In the second 7-day period of culture themedium was replaced with serum-free conditioned mediumwith 3 IUmL of recombinant EPO 50 ngmL of SCF and10 ngmL of IL-3 for expansion and differentiation Duringdays 15ndash18 of culture only one cytokine (EPO at 2 IUmL)was used for erythrocyte differentiation and poloxamer 188(Pluronic F68 (F68) Sigma MW 8400) was added at aconcentration of 005No cytokineswere added during days19ndash21 of culture and only poloxamer 188 was added duringthis period At the end of each phase cultured cells werecounted using a hemocytometer The trypan blue stain wasused in all cell counts and only viable cells are included in thefold expansion results All cultures were maintained at 37∘Cin a humidified atmosphere of 5 CO
2
22 Assessment of Cell Morphology Cell morphology wasassessed using slides prepared by Cytospin using a cyto-centrifuge (Cytospin 3 Shandon Scientific Tokyo Japan)at 800 rpm for 4min followed by Wright-Giemsa stainingPictures of the stained cells were taken with a digital camera(DP70 Olympus Tokyo Japan) at 400x magnification
23 Differential Counting of Cultured Erythroblasts Fivedifferential countings were enumerated as proerythroblastsearly and late basophilic erythroblasts polychromatic ery-throblasts and orthochromatic erythroblasts at 1000x mag-nification
24 Flow Cytometric Analyses of Erythroid Markers For flowcytometric analyses of cell surface antigens a total of 1 times 105cells were stained with phycoerythrin- (PE-) or fluoresceinisothiocyanate- (FITC-) conjugated mouse anti-human anti-bodies against CD45 CD34 CD71 and glycophorin A (GpA)for 15min washed resuspended in FACS buffer and analyzedusing aCell LabQuanta SC (BeckmanCoulter Fullerton CAUSA) using a 488 nm wavelength laser Cells were analyzedusing two-color flow cytometry through WinMDI 29 Theantibody combinations usedwere CD45-FITCCD34-PE andCD71-FITCGpA-PE using G1-FITCG1-PE as a control Allfluorescent conjugated monoclonal antibodies used werepurchased from BD Biosciences (San Jose CA)
25 Quantitative Real-Time Polymerase Chain Reaction Toevaluate gene expression levels during erythrocyte differen-tiation from different sources we harvested over 1 times 106cells from cultured erythrocytes at 7 10 14 and 17 days andisolated total RNA for quantitative polymerase chain reaction(PCR) Gene expression levels were quantified using the LightCycler 480 Real-time PCR System (Roche Applied Science)Quantitative real-time polymerase chain reaction (qPCR)was performed using Light Cycler 480 SYBR Green I Mastermix (Roche Applied Science) according to themanufacturerrsquosinstructions Primers were designed [10 11] and generatedby Bioneer (Korea) (Table 1) Total RNA (800 ng) was usedto generate first-strand cDNA using the Maxime RT PremixKit (Intron Biotech) Differences between the Cp (crossingpoint) values of actin and target mRNAs for each samplewere used to calculate ΔCp values The ΔCp values derivedfrom the isolated undifferentiated CD34+ cells were usedas control ΔCp values Relative expression levels betweensamples and controls were determined using the formularelative expression level = 2minus(119878ΔCpminus119862ΔCp) Comparative real-time PCR with primers specific for GATA1 GATA2 EKLFeALAS and SCLTall (Table 1) was performed in triplicateReactions were performed at 95∘C for 10min followed by 45cycles of 95∘C for 30 s 60∘C for 30 s and 72∘C for 30 s
26 Functional Analysis of Hemoglobin We used a Hemox-Analyzer (TCS Medical Products Division SouthamptonPA) to measure the oxygen binding and dissociation abilitiesof the hemoglobin produced in mature erythrocytes derivedfrom the mPB and cord blood Hemox-Analyzer is an auto-matic system for recording blood oxygen equilibrium curvesand related phenomena [12] The operating principle of theHemox-Analyzer is based on dual-wavelength spectropho-tometry for the measurement of the optical properties ofhemoglobin and a Clark electrode for measuring the oxygenpartial pressure in millimeters of mercury The resultingsignals from both measuring systems are fed to the 119883-119884recorder Both the P
50value and observation of the fine
BioMed Research International 3
1
10
100
1000
10000
100000
1000000
Cel
l num
bers
mPBCB
1010
5022
411481
11087 98386 498114 83256218277 177284 259210 636038
Comparison of cultivative cell growth
0d 4d 7d 10d 14d 17d 21d
(a)
mPB 100 502 820 2695 887 506 167CB 10 35 137 380 97 146 25
000
1000
2000
3000
4000
5000
6000
Fold
indu
ctio
n
Comparison of cell fold induction
0d 4d 7d 10d 14d 17d 21d
(b)
Figure 1 Comparison of cell growth in PB- and CB-derived CD34+ cell cultures Stem cells were cultured for 21 days and counted at the endof each phase (a) Erythroid cell amplification (mean plusmn standard deviation) of mPB- and CB-derived CD34+ cells (b) Numbers of total cellsexpanded from cultures of mPB- and CB-derived CD34+ cells CB-CD34+ cells exhibit higher amplification efficiency than mPB cells lowast119875 lt0001
Table 1 Real-time polymerase chain reaction primers
Gene Primer sequence120573-actin
Forward primerReverse primer
51015840-ATTGGCAATGAGCGGTTC-3101584051015840-GGATGCCACAGGACTCCAT-31015840
GATA-1Forward primerReverse primer
51015840-CACTGAGCTTGCCACATCC-3101584051015840-ATGGAGCCTCTGGGGATTA-31015840
GATA-2Forward primerReverse primer
51015840-GGCAGAACCGACCACTCATC-3101584051015840-TCTGACAATTTGCACAACAGGTG-31015840
eALASForward primerReverse primer
51015840-GATGTGAAGGCTTTCAAGACAGA-3101584051015840-GGAAAATGGCTTCCTTAGGC-31015840
EKLFForward primerReverse primer
51015840-ATCGAGTGAAGAGGAGACCTTCC-3101584051015840-TGAAGATACGCCGCACAACTT-31015840
SCLTal1Forward primerReverse primer
51015840-ACACACAGGATGACTTCCTC-3101584051015840-CCCATGTCCTGCGC-31015840
structure of the curve can furnish information about thedelivery of oxygen to tissues CD34+ cells derived from CBand mPB that were cultured for 17 days in three separatephases were analyzed using this system Normal red bloodcells were used as a control
27 Capillary Zone Electrophoresis After 17 days of culture 1times 108 cells were collected and assessed by capillary zone elec-trophoresis Capillary zone electrophoresis was performed asdescribed previously using the Sebia Capillary system (SebiaNorcross GA) [13] Differentiated erythrocytes (5 times 107cells)were centrifuged at 5000 rpm for 5 minutes Thereafter the
culture medium was removed and the erythrocyte pellet wasvortexed for 5 s Electrophoresis was performed in alkalinebuffer (pH 94) provided by the manufacturer (Sebia) withseparation primarily due to the pH of the solution andendosmosis The hemoglobin was measured at a wavelengthof 415 nm Electrophoretograms were recorded with thelocation of specific hemoglobin in specific zones
28 Statistical Analysis Studentrsquos 119905-test was performed usingExcel (Microsoft) 119875 values less than 005 were consideredstatistically significant
3 Results
31 In Vitro Culture Supports the Differentiation of Ery-throcytes The number of cell divisions observed signifi-cantly increased during the second phase of the cultureperiod Compared to mPB-CD34+ cells CB-CD34+ cellshave greater proliferative capacity during days 10ndash21of culture(Figure 1(a)) This difference led to CB cultures achievinga greater total number of cells than that of mPB cultures(636038 plusmn 182817 versus 83256 plusmn 8858) Cell growth in CBcell cultures exceeded that of mPB cell cultures in the secondphase and early third phase of culture (Figure 1(b)) Followingerythropoietic differentiation decreased cell size nuclearcondensation and nuclear extrusion were confirmed byWright-Giemsa staining Although there were no significantdifferences found our results show that in the blood typecomposition count the CB HSCs have more multipotencywhile mPB-CD34+ cells show earlier differentiation intomature erythrocytes (Figure 2(a)) These results demonstratethatmPB- andCB-derived CD34+HSCs have similar growthpatterns andmorphological characteristics butmPB-derivedCD34+ cells show faster maturation than CB-derived CD34+cells (Figure 2(b))
4 BioMed Research International
mPB
CB
0d 7d 10d 14d 17d 21d
(a)
00
100
200
300
400
500
600
700
800
900
1000
896
95
586
338
66
727
257
440
401
141
106
229
497
159
82
208
430
270
356
611
85
636
277
CB
mPB CB
mPB CB
mPB CB
mPB
Orthochromatic erythroblastPolychromatic erythroblastLate basophilic erythroblast
Early basophilic erythroblastProerythroblast
7d 10d 14d 17d
(b)
Figure 2 Comparison of cell morphological changes in mPB- and CB-derived CD34+ cell cultures CD34+ cells selected from mPB andCB were cultured for 21 days in vitro Morphological changes in differentiated cells from both sources were similar No remarkably differentpatterns were found in the Giemsa staining photos (a) Based on the erythrocyte cell type counting at the end of each phase the mPB-derivedcells matured more rapidly than the cells derived from CB even though cells from both sources have similar maturation patterns (b)
32 Similar Immunophenotypic Patterns CD34 and CD45marker dramatically decreased and finally disappeared fromthe cells during differentiation while GPA expressionincreased during the 21 days of culture Although CD71expression increased until early in the third phase of cultureit gradually decreased following final maturation (Figure 3)From the immunophenotypic data we found no significantdifferences between mPB- and CB-derived CD34+ cellsduring erythroid cell maturation
33 Different mRNA Expression Levels From our data wecan see different patterns in the differentiation of mPB- andCB-derived CD34+ cells Our data clearly show that GATA-1expression gradually increases during erythrocyte differenti-ation especially in CB cells while GATA-2 expression grad-ually decreases following cell maturation The erythrocyte-specific isoforms ALAS and SCLTall are upregulated duringerythrocyte differentiation and in particular show higher
levels inmPB-derived cells than inCB-derived cells Only onefactor EKLF which is a 120573-globin gene transcription factorincreased during erythrocyte differentiation inmPB cells butin contrast decreased during differentiation of CB-derivedcells (Figure 4) These results clearly demonstrate that mPB-derived CD34+ cells differentiate faster into erythrocyteswith Hb-120573 production than CB cells At the same timeunder these in vitro culture conditions CB-derived CD34+hematopoietic cells exhibit higher multipotency than mPBcells
34 Different Hemoglobin Type Development in mPB- andCB-Derived CD34+ Cells Over 80 of the hemoglobin pro-duced by CB-derived CD34+ cells was hemoglobin subtypeHbF while only 175 was subtype HbA Over 95 of thehemoglobin generated by mPB-derived cells was subtypeHbA (Figure 5)
BioMed Research International 5
64
010
010
110
210
310
410
5
64
0
100
101
102
103
104
105
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
50
80
100
101
102
103
104
105
100
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5
64 64
010
010
110
210
310
410
5010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
010
010
110
210
310
410
5
100
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
100
101
102
103
104
105
200
0
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5010
010
110
210
310
410
5
128
64
0
100
101
102
103
104
105
100
101
102
103
104
105
200
0
80
0
100
101
102
103
104
105
0
100
101
102
103
104
105
128
0
100
101
102
103
104
105
128 64
0
100
101
102
103
104
105
0
100
101
102
103
104
105
128 64
0
100
101
102
103
104
105
100
101
102
103
104
105
0
128
10010
110
210
310
410
5
64
0
100
101
102
103
104
105
0
128 80
0
100
101
102
103
104
105
CD45
CD45
CD45
CD45
CD34
CD34
CD34
CD34
CD71
CD71
CD71
CD71
GPA
-1CD
45CD
34CD
71G
PA-1
CD45
CD34
CD71
GPA
-1
CD45
CD34
CD71
GPA
-1
CD45
CD34
CD71
GPA
-1
GPA
-1
GPA
-1
GPA
-1
mPB
CB
0d 7d 14d 21d
Figure 3 Phenotypic markers in erythrocytes differentiated from mPB- and CB-derived CD34+ cells Cells (3 times 105) from the end of eachphase of culture were stained and hematopoietic and erythropoietic markers were measured Flow cytometry results show similar patternsin the cultured cells from both sources and no significant differences
6 BioMed Research International
00
2000
4000
6000
8000 eALAS
00
20
40
60
80SCLTal1
00
10
20
30
40 GATA-1
GATA-2
00
05
10
15
PBCB
00
100
200
300 EKLF
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d
Figure 4 Erythrocyte-specific gene expression in mPB and CB cells At the end of each phase of culture cells were harvested and totalRNA was extracted for quantitative PCRThe expression of GATA-1 GATA-2 eALAS EKLF and SCLTal1 was measured by real-time PCRThe results show an increasing pattern for the GATA-1 transcript and a decreasing pattern for GATA-2 which did not significantly differbetween mPB and CB cells EKLF eALAs and SCLTall expression levels increased during differentiation but were significantly greater inmPB-derived erythrocytes than in those from CB
BioMed Research International 7
Z15
Z14
Z13
Z12
Z11
Z8
Z10 Z
1Z(A)
Z(D
)
Hb A
Hb F
Hb A2
Hb S
Hb CZ
(F)
Z(S
)
Z(E
)Z
(A2)
Z(C
)
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Control
Denatured Hb AHb A
Hb F
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Cord blood derived CD34+ cells
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Hb A
Hb FHb A2
G-CSF mobilized peripheral blood derived CD34+ cells
Figure 5 Electrophoretic determination of main hemoglobin subtypes in differentiated mPB and CB cells Capillary zone electrophoresisshows distinct patterns of hemoglobin subtype expression between differentiated mPB and CB cells About 80 of the hemoglobin producedby CB-CD34-derived erythrocytes is HbF while only 175 is HbA However 955 of the hemoglobin produced by mPB-CD34-derivederythrocytes is HbA
35 Similar Hemoglobin Dissociation Curve with MatureRBCs from mPB and CB Cells While erythrocytes derivedfrom each source have different combinations of HbA andHbF similar hemoglobin dissociation curve was observed(Figure 6) Apart from this hemoglobin subtype variationthis result clearly shows that in vitro cultured RBCs canproduce hemoglobin with oxygen binding and dissociationabilities equivalent to red blood cells produced in vivo
4 Discussion
The shortage of blood supply and the ever-growing demandfor blood transfusion represent a significant emerging issue in
transfusionmedicine Shortage of donated blood and the riskof infection have created limitations in the availability of redblood cells available for transfusion More recently isolatedCD34+ cells from human umbilical CB obtained from dis-carded maternity products and G-CSF-mPB obtained fromhealthy volunteers through leukapheresis have emerged aspotential alternative sources of HSCs The HSCs collectedfrom both sources have high rates of proliferation andcapacities for differentiation and create mature RBCs underthe proper culture conditions through three phases of invitro culture We note that though both types of stemcells show similar characteristics in general growth patternswith morphological and immunophenotypic changes they
8 BioMed Research International
0
0 50 150
0
0 50 150
21-day cultured RBCs from mPB 21-day cultured RBCs from CB
Figure 6 Hemox analysis in mPB and CB cells cultured for 21 days Differentiated erythrocytes were harvested at the end of the 21 daysof culture and oxygen equilibria were measured by Hemox-Analyzer Erythrocytes derived from mPB and from CB both show greateroxygenation abilities compared with normal human blood There are no significant differences between differentiated mPB and CB cells
show different characteristics in gene expression levels andhemoglobin subtype production
Our results show that bothmPB- and CB-derived CD34+cells began to proliferate extremely quickly during 7ndash14 daysof culture However both cell sources produce similar totalcell numbers after 21 days of culture And morphologicalchanges in differentiated cells from both sources were similarand no remarkable differences were found during the cultureexcept when comparing cell differentiation rates The mPBcells seemed to mature earlier than CB cells These resultsshow that mPB cells from circulating blood possess moreprogenitor cells than CB and have the ability of rapiddifferentiation into the mature RBCs Following cell growthat the end of each cell culture phase mPB- and CB-derivedCD34+ GPA and CD70 (transferrin receptor) expressionshowed similar patterns in flow cytometry analyses Thesedata are consistent with previous results regarding erythro-cyte induction mechanisms [8 14] To confirm whether theinitial exposition of cells is due to different sources deriveddifferentiation gene expression profiles were analyzed byquantitative RT-PCR GATA-1 is a transcription factor thatdetermines erythroid differentiation survival and 120573-globingene expression [15] GATA-2 inhibits GATA-1 functionGATA-2 expression exhibited a downregulation followingerythropoietin stimulation and its levels were higher incultured mPB cells This can be explained in earlier dif-ferentiation into mature RBCs in mPB source cells thanin CB This finding is also consistent with the result ofdifferential counts that mPB showed faster shift from pronor-moblasts to orthochromatic normoblasts in comparison toCB Ikonomi et al have previously shown that GATA-2preferentially increases 120574-globin gene expression indicatingthat the prolonged expression of GATA-2 contributes to theearly increase in 120574-globin in CB [16] EKLF binds specificallyto the 120573-globin promoter and is critical in establishingchromatin structure for high-level 120573-globin transcription via
its acetylation by CREB binding protein [17] SCLTall isrequired for the progression of erythroid differentiation andenforced expression of SCLTall increases 120573-globin expres-sion and BFU-E and CFU-E production [18] Because thesetranscription factors are closely related to increased 120573-globingene expression changes in their expression may accountfor the delay and reduction of 120573-globin expression in CB-derived differentiated cells The HbA- and HbF-related geneexpression tests exhibit different expression levels dependingon the cell sourceWe culturedmPB- andCB-derived CD34+cells through three phases harvested them after 17 daysof culture and analyzed them by hemoglobin type testingThese results clearly demonstrated different subtypes ofhemoglobin expressed bymPB- andCB-derivedmature cellsCB-derived cells mostly express HbF and mPB-derived cellsmainly express HbA but based on these data the originalsources of these cells appear to possess different propensitiesfor hemoglobin production patterns These results demon-strate that hemoglobin subtypes are not related to cultureconditions and culture time but are strongly affected by thesource material To evaluate the function of the differentiatedmPB and CB cells CD34+ cells derived from each sourcewere expanded and differentiated to large cell numbers ofup to a total of 5 times 107 cells and oxygen equilibria weremeasured by Hemox-Analyzer From the result the oxygendissociation curves indicate that the cells from both sourcesdo not significantly differ from one another with respectto hemoglobin function Although the types of hemoglobinexpression differed between CB- and mPB-derived maturecells the oxygen binding and dissociation curves may besimilar due to variation among adult type hemoglobin fetaltype hemoglobin or mixture of both types in vitro cultureprocessing after 17 days of culture The cultured cells indeedhave slightly greater deoxygenation functionalities comparedwith normal cells which showed the immaturity in shift toleft
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 3
1
10
100
1000
10000
100000
1000000
Cel
l num
bers
mPBCB
1010
5022
411481
11087 98386 498114 83256218277 177284 259210 636038
Comparison of cultivative cell growth
0d 4d 7d 10d 14d 17d 21d
(a)
mPB 100 502 820 2695 887 506 167CB 10 35 137 380 97 146 25
000
1000
2000
3000
4000
5000
6000
Fold
indu
ctio
n
Comparison of cell fold induction
0d 4d 7d 10d 14d 17d 21d
(b)
Figure 1 Comparison of cell growth in PB- and CB-derived CD34+ cell cultures Stem cells were cultured for 21 days and counted at the endof each phase (a) Erythroid cell amplification (mean plusmn standard deviation) of mPB- and CB-derived CD34+ cells (b) Numbers of total cellsexpanded from cultures of mPB- and CB-derived CD34+ cells CB-CD34+ cells exhibit higher amplification efficiency than mPB cells lowast119875 lt0001
Table 1 Real-time polymerase chain reaction primers
Gene Primer sequence120573-actin
Forward primerReverse primer
51015840-ATTGGCAATGAGCGGTTC-3101584051015840-GGATGCCACAGGACTCCAT-31015840
GATA-1Forward primerReverse primer
51015840-CACTGAGCTTGCCACATCC-3101584051015840-ATGGAGCCTCTGGGGATTA-31015840
GATA-2Forward primerReverse primer
51015840-GGCAGAACCGACCACTCATC-3101584051015840-TCTGACAATTTGCACAACAGGTG-31015840
eALASForward primerReverse primer
51015840-GATGTGAAGGCTTTCAAGACAGA-3101584051015840-GGAAAATGGCTTCCTTAGGC-31015840
EKLFForward primerReverse primer
51015840-ATCGAGTGAAGAGGAGACCTTCC-3101584051015840-TGAAGATACGCCGCACAACTT-31015840
SCLTal1Forward primerReverse primer
51015840-ACACACAGGATGACTTCCTC-3101584051015840-CCCATGTCCTGCGC-31015840
structure of the curve can furnish information about thedelivery of oxygen to tissues CD34+ cells derived from CBand mPB that were cultured for 17 days in three separatephases were analyzed using this system Normal red bloodcells were used as a control
27 Capillary Zone Electrophoresis After 17 days of culture 1times 108 cells were collected and assessed by capillary zone elec-trophoresis Capillary zone electrophoresis was performed asdescribed previously using the Sebia Capillary system (SebiaNorcross GA) [13] Differentiated erythrocytes (5 times 107cells)were centrifuged at 5000 rpm for 5 minutes Thereafter the
culture medium was removed and the erythrocyte pellet wasvortexed for 5 s Electrophoresis was performed in alkalinebuffer (pH 94) provided by the manufacturer (Sebia) withseparation primarily due to the pH of the solution andendosmosis The hemoglobin was measured at a wavelengthof 415 nm Electrophoretograms were recorded with thelocation of specific hemoglobin in specific zones
28 Statistical Analysis Studentrsquos 119905-test was performed usingExcel (Microsoft) 119875 values less than 005 were consideredstatistically significant
3 Results
31 In Vitro Culture Supports the Differentiation of Ery-throcytes The number of cell divisions observed signifi-cantly increased during the second phase of the cultureperiod Compared to mPB-CD34+ cells CB-CD34+ cellshave greater proliferative capacity during days 10ndash21of culture(Figure 1(a)) This difference led to CB cultures achievinga greater total number of cells than that of mPB cultures(636038 plusmn 182817 versus 83256 plusmn 8858) Cell growth in CBcell cultures exceeded that of mPB cell cultures in the secondphase and early third phase of culture (Figure 1(b)) Followingerythropoietic differentiation decreased cell size nuclearcondensation and nuclear extrusion were confirmed byWright-Giemsa staining Although there were no significantdifferences found our results show that in the blood typecomposition count the CB HSCs have more multipotencywhile mPB-CD34+ cells show earlier differentiation intomature erythrocytes (Figure 2(a)) These results demonstratethatmPB- andCB-derived CD34+HSCs have similar growthpatterns andmorphological characteristics butmPB-derivedCD34+ cells show faster maturation than CB-derived CD34+cells (Figure 2(b))
4 BioMed Research International
mPB
CB
0d 7d 10d 14d 17d 21d
(a)
00
100
200
300
400
500
600
700
800
900
1000
896
95
586
338
66
727
257
440
401
141
106
229
497
159
82
208
430
270
356
611
85
636
277
CB
mPB CB
mPB CB
mPB CB
mPB
Orthochromatic erythroblastPolychromatic erythroblastLate basophilic erythroblast
Early basophilic erythroblastProerythroblast
7d 10d 14d 17d
(b)
Figure 2 Comparison of cell morphological changes in mPB- and CB-derived CD34+ cell cultures CD34+ cells selected from mPB andCB were cultured for 21 days in vitro Morphological changes in differentiated cells from both sources were similar No remarkably differentpatterns were found in the Giemsa staining photos (a) Based on the erythrocyte cell type counting at the end of each phase the mPB-derivedcells matured more rapidly than the cells derived from CB even though cells from both sources have similar maturation patterns (b)
32 Similar Immunophenotypic Patterns CD34 and CD45marker dramatically decreased and finally disappeared fromthe cells during differentiation while GPA expressionincreased during the 21 days of culture Although CD71expression increased until early in the third phase of cultureit gradually decreased following final maturation (Figure 3)From the immunophenotypic data we found no significantdifferences between mPB- and CB-derived CD34+ cellsduring erythroid cell maturation
33 Different mRNA Expression Levels From our data wecan see different patterns in the differentiation of mPB- andCB-derived CD34+ cells Our data clearly show that GATA-1expression gradually increases during erythrocyte differenti-ation especially in CB cells while GATA-2 expression grad-ually decreases following cell maturation The erythrocyte-specific isoforms ALAS and SCLTall are upregulated duringerythrocyte differentiation and in particular show higher
levels inmPB-derived cells than inCB-derived cells Only onefactor EKLF which is a 120573-globin gene transcription factorincreased during erythrocyte differentiation inmPB cells butin contrast decreased during differentiation of CB-derivedcells (Figure 4) These results clearly demonstrate that mPB-derived CD34+ cells differentiate faster into erythrocyteswith Hb-120573 production than CB cells At the same timeunder these in vitro culture conditions CB-derived CD34+hematopoietic cells exhibit higher multipotency than mPBcells
34 Different Hemoglobin Type Development in mPB- andCB-Derived CD34+ Cells Over 80 of the hemoglobin pro-duced by CB-derived CD34+ cells was hemoglobin subtypeHbF while only 175 was subtype HbA Over 95 of thehemoglobin generated by mPB-derived cells was subtypeHbA (Figure 5)
BioMed Research International 5
64
010
010
110
210
310
410
5
64
0
100
101
102
103
104
105
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
50
80
100
101
102
103
104
105
100
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5
64 64
010
010
110
210
310
410
5010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
010
010
110
210
310
410
5
100
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
100
101
102
103
104
105
200
0
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5010
010
110
210
310
410
5
128
64
0
100
101
102
103
104
105
100
101
102
103
104
105
200
0
80
0
100
101
102
103
104
105
0
100
101
102
103
104
105
128
0
100
101
102
103
104
105
128 64
0
100
101
102
103
104
105
0
100
101
102
103
104
105
128 64
0
100
101
102
103
104
105
100
101
102
103
104
105
0
128
10010
110
210
310
410
5
64
0
100
101
102
103
104
105
0
128 80
0
100
101
102
103
104
105
CD45
CD45
CD45
CD45
CD34
CD34
CD34
CD34
CD71
CD71
CD71
CD71
GPA
-1CD
45CD
34CD
71G
PA-1
CD45
CD34
CD71
GPA
-1
CD45
CD34
CD71
GPA
-1
CD45
CD34
CD71
GPA
-1
GPA
-1
GPA
-1
GPA
-1
mPB
CB
0d 7d 14d 21d
Figure 3 Phenotypic markers in erythrocytes differentiated from mPB- and CB-derived CD34+ cells Cells (3 times 105) from the end of eachphase of culture were stained and hematopoietic and erythropoietic markers were measured Flow cytometry results show similar patternsin the cultured cells from both sources and no significant differences
6 BioMed Research International
00
2000
4000
6000
8000 eALAS
00
20
40
60
80SCLTal1
00
10
20
30
40 GATA-1
GATA-2
00
05
10
15
PBCB
00
100
200
300 EKLF
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d
Figure 4 Erythrocyte-specific gene expression in mPB and CB cells At the end of each phase of culture cells were harvested and totalRNA was extracted for quantitative PCRThe expression of GATA-1 GATA-2 eALAS EKLF and SCLTal1 was measured by real-time PCRThe results show an increasing pattern for the GATA-1 transcript and a decreasing pattern for GATA-2 which did not significantly differbetween mPB and CB cells EKLF eALAs and SCLTall expression levels increased during differentiation but were significantly greater inmPB-derived erythrocytes than in those from CB
BioMed Research International 7
Z15
Z14
Z13
Z12
Z11
Z8
Z10 Z
1Z(A)
Z(D
)
Hb A
Hb F
Hb A2
Hb S
Hb CZ
(F)
Z(S
)
Z(E
)Z
(A2)
Z(C
)
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Control
Denatured Hb AHb A
Hb F
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Cord blood derived CD34+ cells
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Hb A
Hb FHb A2
G-CSF mobilized peripheral blood derived CD34+ cells
Figure 5 Electrophoretic determination of main hemoglobin subtypes in differentiated mPB and CB cells Capillary zone electrophoresisshows distinct patterns of hemoglobin subtype expression between differentiated mPB and CB cells About 80 of the hemoglobin producedby CB-CD34-derived erythrocytes is HbF while only 175 is HbA However 955 of the hemoglobin produced by mPB-CD34-derivederythrocytes is HbA
35 Similar Hemoglobin Dissociation Curve with MatureRBCs from mPB and CB Cells While erythrocytes derivedfrom each source have different combinations of HbA andHbF similar hemoglobin dissociation curve was observed(Figure 6) Apart from this hemoglobin subtype variationthis result clearly shows that in vitro cultured RBCs canproduce hemoglobin with oxygen binding and dissociationabilities equivalent to red blood cells produced in vivo
4 Discussion
The shortage of blood supply and the ever-growing demandfor blood transfusion represent a significant emerging issue in
transfusionmedicine Shortage of donated blood and the riskof infection have created limitations in the availability of redblood cells available for transfusion More recently isolatedCD34+ cells from human umbilical CB obtained from dis-carded maternity products and G-CSF-mPB obtained fromhealthy volunteers through leukapheresis have emerged aspotential alternative sources of HSCs The HSCs collectedfrom both sources have high rates of proliferation andcapacities for differentiation and create mature RBCs underthe proper culture conditions through three phases of invitro culture We note that though both types of stemcells show similar characteristics in general growth patternswith morphological and immunophenotypic changes they
8 BioMed Research International
0
0 50 150
0
0 50 150
21-day cultured RBCs from mPB 21-day cultured RBCs from CB
Figure 6 Hemox analysis in mPB and CB cells cultured for 21 days Differentiated erythrocytes were harvested at the end of the 21 daysof culture and oxygen equilibria were measured by Hemox-Analyzer Erythrocytes derived from mPB and from CB both show greateroxygenation abilities compared with normal human blood There are no significant differences between differentiated mPB and CB cells
show different characteristics in gene expression levels andhemoglobin subtype production
Our results show that bothmPB- and CB-derived CD34+cells began to proliferate extremely quickly during 7ndash14 daysof culture However both cell sources produce similar totalcell numbers after 21 days of culture And morphologicalchanges in differentiated cells from both sources were similarand no remarkable differences were found during the cultureexcept when comparing cell differentiation rates The mPBcells seemed to mature earlier than CB cells These resultsshow that mPB cells from circulating blood possess moreprogenitor cells than CB and have the ability of rapiddifferentiation into the mature RBCs Following cell growthat the end of each cell culture phase mPB- and CB-derivedCD34+ GPA and CD70 (transferrin receptor) expressionshowed similar patterns in flow cytometry analyses Thesedata are consistent with previous results regarding erythro-cyte induction mechanisms [8 14] To confirm whether theinitial exposition of cells is due to different sources deriveddifferentiation gene expression profiles were analyzed byquantitative RT-PCR GATA-1 is a transcription factor thatdetermines erythroid differentiation survival and 120573-globingene expression [15] GATA-2 inhibits GATA-1 functionGATA-2 expression exhibited a downregulation followingerythropoietin stimulation and its levels were higher incultured mPB cells This can be explained in earlier dif-ferentiation into mature RBCs in mPB source cells thanin CB This finding is also consistent with the result ofdifferential counts that mPB showed faster shift from pronor-moblasts to orthochromatic normoblasts in comparison toCB Ikonomi et al have previously shown that GATA-2preferentially increases 120574-globin gene expression indicatingthat the prolonged expression of GATA-2 contributes to theearly increase in 120574-globin in CB [16] EKLF binds specificallyto the 120573-globin promoter and is critical in establishingchromatin structure for high-level 120573-globin transcription via
its acetylation by CREB binding protein [17] SCLTall isrequired for the progression of erythroid differentiation andenforced expression of SCLTall increases 120573-globin expres-sion and BFU-E and CFU-E production [18] Because thesetranscription factors are closely related to increased 120573-globingene expression changes in their expression may accountfor the delay and reduction of 120573-globin expression in CB-derived differentiated cells The HbA- and HbF-related geneexpression tests exhibit different expression levels dependingon the cell sourceWe culturedmPB- andCB-derived CD34+cells through three phases harvested them after 17 daysof culture and analyzed them by hemoglobin type testingThese results clearly demonstrated different subtypes ofhemoglobin expressed bymPB- andCB-derivedmature cellsCB-derived cells mostly express HbF and mPB-derived cellsmainly express HbA but based on these data the originalsources of these cells appear to possess different propensitiesfor hemoglobin production patterns These results demon-strate that hemoglobin subtypes are not related to cultureconditions and culture time but are strongly affected by thesource material To evaluate the function of the differentiatedmPB and CB cells CD34+ cells derived from each sourcewere expanded and differentiated to large cell numbers ofup to a total of 5 times 107 cells and oxygen equilibria weremeasured by Hemox-Analyzer From the result the oxygendissociation curves indicate that the cells from both sourcesdo not significantly differ from one another with respectto hemoglobin function Although the types of hemoglobinexpression differed between CB- and mPB-derived maturecells the oxygen binding and dissociation curves may besimilar due to variation among adult type hemoglobin fetaltype hemoglobin or mixture of both types in vitro cultureprocessing after 17 days of culture The cultured cells indeedhave slightly greater deoxygenation functionalities comparedwith normal cells which showed the immaturity in shift toleft
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 BioMed Research International
mPB
CB
0d 7d 10d 14d 17d 21d
(a)
00
100
200
300
400
500
600
700
800
900
1000
896
95
586
338
66
727
257
440
401
141
106
229
497
159
82
208
430
270
356
611
85
636
277
CB
mPB CB
mPB CB
mPB CB
mPB
Orthochromatic erythroblastPolychromatic erythroblastLate basophilic erythroblast
Early basophilic erythroblastProerythroblast
7d 10d 14d 17d
(b)
Figure 2 Comparison of cell morphological changes in mPB- and CB-derived CD34+ cell cultures CD34+ cells selected from mPB andCB were cultured for 21 days in vitro Morphological changes in differentiated cells from both sources were similar No remarkably differentpatterns were found in the Giemsa staining photos (a) Based on the erythrocyte cell type counting at the end of each phase the mPB-derivedcells matured more rapidly than the cells derived from CB even though cells from both sources have similar maturation patterns (b)
32 Similar Immunophenotypic Patterns CD34 and CD45marker dramatically decreased and finally disappeared fromthe cells during differentiation while GPA expressionincreased during the 21 days of culture Although CD71expression increased until early in the third phase of cultureit gradually decreased following final maturation (Figure 3)From the immunophenotypic data we found no significantdifferences between mPB- and CB-derived CD34+ cellsduring erythroid cell maturation
33 Different mRNA Expression Levels From our data wecan see different patterns in the differentiation of mPB- andCB-derived CD34+ cells Our data clearly show that GATA-1expression gradually increases during erythrocyte differenti-ation especially in CB cells while GATA-2 expression grad-ually decreases following cell maturation The erythrocyte-specific isoforms ALAS and SCLTall are upregulated duringerythrocyte differentiation and in particular show higher
levels inmPB-derived cells than inCB-derived cells Only onefactor EKLF which is a 120573-globin gene transcription factorincreased during erythrocyte differentiation inmPB cells butin contrast decreased during differentiation of CB-derivedcells (Figure 4) These results clearly demonstrate that mPB-derived CD34+ cells differentiate faster into erythrocyteswith Hb-120573 production than CB cells At the same timeunder these in vitro culture conditions CB-derived CD34+hematopoietic cells exhibit higher multipotency than mPBcells
34 Different Hemoglobin Type Development in mPB- andCB-Derived CD34+ Cells Over 80 of the hemoglobin pro-duced by CB-derived CD34+ cells was hemoglobin subtypeHbF while only 175 was subtype HbA Over 95 of thehemoglobin generated by mPB-derived cells was subtypeHbA (Figure 5)
BioMed Research International 5
64
010
010
110
210
310
410
5
64
0
100
101
102
103
104
105
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
50
80
100
101
102
103
104
105
100
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5
64 64
010
010
110
210
310
410
5010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
010
010
110
210
310
410
5
100
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
100
101
102
103
104
105
200
0
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5010
010
110
210
310
410
5
128
64
0
100
101
102
103
104
105
100
101
102
103
104
105
200
0
80
0
100
101
102
103
104
105
0
100
101
102
103
104
105
128
0
100
101
102
103
104
105
128 64
0
100
101
102
103
104
105
0
100
101
102
103
104
105
128 64
0
100
101
102
103
104
105
100
101
102
103
104
105
0
128
10010
110
210
310
410
5
64
0
100
101
102
103
104
105
0
128 80
0
100
101
102
103
104
105
CD45
CD45
CD45
CD45
CD34
CD34
CD34
CD34
CD71
CD71
CD71
CD71
GPA
-1CD
45CD
34CD
71G
PA-1
CD45
CD34
CD71
GPA
-1
CD45
CD34
CD71
GPA
-1
CD45
CD34
CD71
GPA
-1
GPA
-1
GPA
-1
GPA
-1
mPB
CB
0d 7d 14d 21d
Figure 3 Phenotypic markers in erythrocytes differentiated from mPB- and CB-derived CD34+ cells Cells (3 times 105) from the end of eachphase of culture were stained and hematopoietic and erythropoietic markers were measured Flow cytometry results show similar patternsin the cultured cells from both sources and no significant differences
6 BioMed Research International
00
2000
4000
6000
8000 eALAS
00
20
40
60
80SCLTal1
00
10
20
30
40 GATA-1
GATA-2
00
05
10
15
PBCB
00
100
200
300 EKLF
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d
Figure 4 Erythrocyte-specific gene expression in mPB and CB cells At the end of each phase of culture cells were harvested and totalRNA was extracted for quantitative PCRThe expression of GATA-1 GATA-2 eALAS EKLF and SCLTal1 was measured by real-time PCRThe results show an increasing pattern for the GATA-1 transcript and a decreasing pattern for GATA-2 which did not significantly differbetween mPB and CB cells EKLF eALAs and SCLTall expression levels increased during differentiation but were significantly greater inmPB-derived erythrocytes than in those from CB
BioMed Research International 7
Z15
Z14
Z13
Z12
Z11
Z8
Z10 Z
1Z(A)
Z(D
)
Hb A
Hb F
Hb A2
Hb S
Hb CZ
(F)
Z(S
)
Z(E
)Z
(A2)
Z(C
)
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Control
Denatured Hb AHb A
Hb F
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Cord blood derived CD34+ cells
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Hb A
Hb FHb A2
G-CSF mobilized peripheral blood derived CD34+ cells
Figure 5 Electrophoretic determination of main hemoglobin subtypes in differentiated mPB and CB cells Capillary zone electrophoresisshows distinct patterns of hemoglobin subtype expression between differentiated mPB and CB cells About 80 of the hemoglobin producedby CB-CD34-derived erythrocytes is HbF while only 175 is HbA However 955 of the hemoglobin produced by mPB-CD34-derivederythrocytes is HbA
35 Similar Hemoglobin Dissociation Curve with MatureRBCs from mPB and CB Cells While erythrocytes derivedfrom each source have different combinations of HbA andHbF similar hemoglobin dissociation curve was observed(Figure 6) Apart from this hemoglobin subtype variationthis result clearly shows that in vitro cultured RBCs canproduce hemoglobin with oxygen binding and dissociationabilities equivalent to red blood cells produced in vivo
4 Discussion
The shortage of blood supply and the ever-growing demandfor blood transfusion represent a significant emerging issue in
transfusionmedicine Shortage of donated blood and the riskof infection have created limitations in the availability of redblood cells available for transfusion More recently isolatedCD34+ cells from human umbilical CB obtained from dis-carded maternity products and G-CSF-mPB obtained fromhealthy volunteers through leukapheresis have emerged aspotential alternative sources of HSCs The HSCs collectedfrom both sources have high rates of proliferation andcapacities for differentiation and create mature RBCs underthe proper culture conditions through three phases of invitro culture We note that though both types of stemcells show similar characteristics in general growth patternswith morphological and immunophenotypic changes they
8 BioMed Research International
0
0 50 150
0
0 50 150
21-day cultured RBCs from mPB 21-day cultured RBCs from CB
Figure 6 Hemox analysis in mPB and CB cells cultured for 21 days Differentiated erythrocytes were harvested at the end of the 21 daysof culture and oxygen equilibria were measured by Hemox-Analyzer Erythrocytes derived from mPB and from CB both show greateroxygenation abilities compared with normal human blood There are no significant differences between differentiated mPB and CB cells
show different characteristics in gene expression levels andhemoglobin subtype production
Our results show that bothmPB- and CB-derived CD34+cells began to proliferate extremely quickly during 7ndash14 daysof culture However both cell sources produce similar totalcell numbers after 21 days of culture And morphologicalchanges in differentiated cells from both sources were similarand no remarkable differences were found during the cultureexcept when comparing cell differentiation rates The mPBcells seemed to mature earlier than CB cells These resultsshow that mPB cells from circulating blood possess moreprogenitor cells than CB and have the ability of rapiddifferentiation into the mature RBCs Following cell growthat the end of each cell culture phase mPB- and CB-derivedCD34+ GPA and CD70 (transferrin receptor) expressionshowed similar patterns in flow cytometry analyses Thesedata are consistent with previous results regarding erythro-cyte induction mechanisms [8 14] To confirm whether theinitial exposition of cells is due to different sources deriveddifferentiation gene expression profiles were analyzed byquantitative RT-PCR GATA-1 is a transcription factor thatdetermines erythroid differentiation survival and 120573-globingene expression [15] GATA-2 inhibits GATA-1 functionGATA-2 expression exhibited a downregulation followingerythropoietin stimulation and its levels were higher incultured mPB cells This can be explained in earlier dif-ferentiation into mature RBCs in mPB source cells thanin CB This finding is also consistent with the result ofdifferential counts that mPB showed faster shift from pronor-moblasts to orthochromatic normoblasts in comparison toCB Ikonomi et al have previously shown that GATA-2preferentially increases 120574-globin gene expression indicatingthat the prolonged expression of GATA-2 contributes to theearly increase in 120574-globin in CB [16] EKLF binds specificallyto the 120573-globin promoter and is critical in establishingchromatin structure for high-level 120573-globin transcription via
its acetylation by CREB binding protein [17] SCLTall isrequired for the progression of erythroid differentiation andenforced expression of SCLTall increases 120573-globin expres-sion and BFU-E and CFU-E production [18] Because thesetranscription factors are closely related to increased 120573-globingene expression changes in their expression may accountfor the delay and reduction of 120573-globin expression in CB-derived differentiated cells The HbA- and HbF-related geneexpression tests exhibit different expression levels dependingon the cell sourceWe culturedmPB- andCB-derived CD34+cells through three phases harvested them after 17 daysof culture and analyzed them by hemoglobin type testingThese results clearly demonstrated different subtypes ofhemoglobin expressed bymPB- andCB-derivedmature cellsCB-derived cells mostly express HbF and mPB-derived cellsmainly express HbA but based on these data the originalsources of these cells appear to possess different propensitiesfor hemoglobin production patterns These results demon-strate that hemoglobin subtypes are not related to cultureconditions and culture time but are strongly affected by thesource material To evaluate the function of the differentiatedmPB and CB cells CD34+ cells derived from each sourcewere expanded and differentiated to large cell numbers ofup to a total of 5 times 107 cells and oxygen equilibria weremeasured by Hemox-Analyzer From the result the oxygendissociation curves indicate that the cells from both sourcesdo not significantly differ from one another with respectto hemoglobin function Although the types of hemoglobinexpression differed between CB- and mPB-derived maturecells the oxygen binding and dissociation curves may besimilar due to variation among adult type hemoglobin fetaltype hemoglobin or mixture of both types in vitro cultureprocessing after 17 days of culture The cultured cells indeedhave slightly greater deoxygenation functionalities comparedwith normal cells which showed the immaturity in shift toleft
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 5
64
010
010
110
210
310
410
5
64
0
100
101
102
103
104
105
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
50
80
100
101
102
103
104
105
100
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5
64 64
010
010
110
210
310
410
5010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
010
010
110
210
310
410
5
100
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
100
010
010
110
210
310
410
5
100
101
102
103
104
105
200
0
64
010
010
110
210
310
410
5
64
010
010
110
210
310
410
5010
010
110
210
310
410
5
128
64
0
100
101
102
103
104
105
100
101
102
103
104
105
200
0
80
0
100
101
102
103
104
105
0
100
101
102
103
104
105
128
0
100
101
102
103
104
105
128 64
0
100
101
102
103
104
105
0
100
101
102
103
104
105
128 64
0
100
101
102
103
104
105
100
101
102
103
104
105
0
128
10010
110
210
310
410
5
64
0
100
101
102
103
104
105
0
128 80
0
100
101
102
103
104
105
CD45
CD45
CD45
CD45
CD34
CD34
CD34
CD34
CD71
CD71
CD71
CD71
GPA
-1CD
45CD
34CD
71G
PA-1
CD45
CD34
CD71
GPA
-1
CD45
CD34
CD71
GPA
-1
CD45
CD34
CD71
GPA
-1
GPA
-1
GPA
-1
GPA
-1
mPB
CB
0d 7d 14d 21d
Figure 3 Phenotypic markers in erythrocytes differentiated from mPB- and CB-derived CD34+ cells Cells (3 times 105) from the end of eachphase of culture were stained and hematopoietic and erythropoietic markers were measured Flow cytometry results show similar patternsin the cultured cells from both sources and no significant differences
6 BioMed Research International
00
2000
4000
6000
8000 eALAS
00
20
40
60
80SCLTal1
00
10
20
30
40 GATA-1
GATA-2
00
05
10
15
PBCB
00
100
200
300 EKLF
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d
Figure 4 Erythrocyte-specific gene expression in mPB and CB cells At the end of each phase of culture cells were harvested and totalRNA was extracted for quantitative PCRThe expression of GATA-1 GATA-2 eALAS EKLF and SCLTal1 was measured by real-time PCRThe results show an increasing pattern for the GATA-1 transcript and a decreasing pattern for GATA-2 which did not significantly differbetween mPB and CB cells EKLF eALAs and SCLTall expression levels increased during differentiation but were significantly greater inmPB-derived erythrocytes than in those from CB
BioMed Research International 7
Z15
Z14
Z13
Z12
Z11
Z8
Z10 Z
1Z(A)
Z(D
)
Hb A
Hb F
Hb A2
Hb S
Hb CZ
(F)
Z(S
)
Z(E
)Z
(A2)
Z(C
)
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Control
Denatured Hb AHb A
Hb F
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Cord blood derived CD34+ cells
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Hb A
Hb FHb A2
G-CSF mobilized peripheral blood derived CD34+ cells
Figure 5 Electrophoretic determination of main hemoglobin subtypes in differentiated mPB and CB cells Capillary zone electrophoresisshows distinct patterns of hemoglobin subtype expression between differentiated mPB and CB cells About 80 of the hemoglobin producedby CB-CD34-derived erythrocytes is HbF while only 175 is HbA However 955 of the hemoglobin produced by mPB-CD34-derivederythrocytes is HbA
35 Similar Hemoglobin Dissociation Curve with MatureRBCs from mPB and CB Cells While erythrocytes derivedfrom each source have different combinations of HbA andHbF similar hemoglobin dissociation curve was observed(Figure 6) Apart from this hemoglobin subtype variationthis result clearly shows that in vitro cultured RBCs canproduce hemoglobin with oxygen binding and dissociationabilities equivalent to red blood cells produced in vivo
4 Discussion
The shortage of blood supply and the ever-growing demandfor blood transfusion represent a significant emerging issue in
transfusionmedicine Shortage of donated blood and the riskof infection have created limitations in the availability of redblood cells available for transfusion More recently isolatedCD34+ cells from human umbilical CB obtained from dis-carded maternity products and G-CSF-mPB obtained fromhealthy volunteers through leukapheresis have emerged aspotential alternative sources of HSCs The HSCs collectedfrom both sources have high rates of proliferation andcapacities for differentiation and create mature RBCs underthe proper culture conditions through three phases of invitro culture We note that though both types of stemcells show similar characteristics in general growth patternswith morphological and immunophenotypic changes they
8 BioMed Research International
0
0 50 150
0
0 50 150
21-day cultured RBCs from mPB 21-day cultured RBCs from CB
Figure 6 Hemox analysis in mPB and CB cells cultured for 21 days Differentiated erythrocytes were harvested at the end of the 21 daysof culture and oxygen equilibria were measured by Hemox-Analyzer Erythrocytes derived from mPB and from CB both show greateroxygenation abilities compared with normal human blood There are no significant differences between differentiated mPB and CB cells
show different characteristics in gene expression levels andhemoglobin subtype production
Our results show that bothmPB- and CB-derived CD34+cells began to proliferate extremely quickly during 7ndash14 daysof culture However both cell sources produce similar totalcell numbers after 21 days of culture And morphologicalchanges in differentiated cells from both sources were similarand no remarkable differences were found during the cultureexcept when comparing cell differentiation rates The mPBcells seemed to mature earlier than CB cells These resultsshow that mPB cells from circulating blood possess moreprogenitor cells than CB and have the ability of rapiddifferentiation into the mature RBCs Following cell growthat the end of each cell culture phase mPB- and CB-derivedCD34+ GPA and CD70 (transferrin receptor) expressionshowed similar patterns in flow cytometry analyses Thesedata are consistent with previous results regarding erythro-cyte induction mechanisms [8 14] To confirm whether theinitial exposition of cells is due to different sources deriveddifferentiation gene expression profiles were analyzed byquantitative RT-PCR GATA-1 is a transcription factor thatdetermines erythroid differentiation survival and 120573-globingene expression [15] GATA-2 inhibits GATA-1 functionGATA-2 expression exhibited a downregulation followingerythropoietin stimulation and its levels were higher incultured mPB cells This can be explained in earlier dif-ferentiation into mature RBCs in mPB source cells thanin CB This finding is also consistent with the result ofdifferential counts that mPB showed faster shift from pronor-moblasts to orthochromatic normoblasts in comparison toCB Ikonomi et al have previously shown that GATA-2preferentially increases 120574-globin gene expression indicatingthat the prolonged expression of GATA-2 contributes to theearly increase in 120574-globin in CB [16] EKLF binds specificallyto the 120573-globin promoter and is critical in establishingchromatin structure for high-level 120573-globin transcription via
its acetylation by CREB binding protein [17] SCLTall isrequired for the progression of erythroid differentiation andenforced expression of SCLTall increases 120573-globin expres-sion and BFU-E and CFU-E production [18] Because thesetranscription factors are closely related to increased 120573-globingene expression changes in their expression may accountfor the delay and reduction of 120573-globin expression in CB-derived differentiated cells The HbA- and HbF-related geneexpression tests exhibit different expression levels dependingon the cell sourceWe culturedmPB- andCB-derived CD34+cells through three phases harvested them after 17 daysof culture and analyzed them by hemoglobin type testingThese results clearly demonstrated different subtypes ofhemoglobin expressed bymPB- andCB-derivedmature cellsCB-derived cells mostly express HbF and mPB-derived cellsmainly express HbA but based on these data the originalsources of these cells appear to possess different propensitiesfor hemoglobin production patterns These results demon-strate that hemoglobin subtypes are not related to cultureconditions and culture time but are strongly affected by thesource material To evaluate the function of the differentiatedmPB and CB cells CD34+ cells derived from each sourcewere expanded and differentiated to large cell numbers ofup to a total of 5 times 107 cells and oxygen equilibria weremeasured by Hemox-Analyzer From the result the oxygendissociation curves indicate that the cells from both sourcesdo not significantly differ from one another with respectto hemoglobin function Although the types of hemoglobinexpression differed between CB- and mPB-derived maturecells the oxygen binding and dissociation curves may besimilar due to variation among adult type hemoglobin fetaltype hemoglobin or mixture of both types in vitro cultureprocessing after 17 days of culture The cultured cells indeedhave slightly greater deoxygenation functionalities comparedwith normal cells which showed the immaturity in shift toleft
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 BioMed Research International
00
2000
4000
6000
8000 eALAS
00
20
40
60
80SCLTal1
00
10
20
30
40 GATA-1
GATA-2
00
05
10
15
PBCB
00
100
200
300 EKLF
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d 0d 7d 10d 14d 17d
0d 7d 10d 14d 17d
Figure 4 Erythrocyte-specific gene expression in mPB and CB cells At the end of each phase of culture cells were harvested and totalRNA was extracted for quantitative PCRThe expression of GATA-1 GATA-2 eALAS EKLF and SCLTal1 was measured by real-time PCRThe results show an increasing pattern for the GATA-1 transcript and a decreasing pattern for GATA-2 which did not significantly differbetween mPB and CB cells EKLF eALAs and SCLTall expression levels increased during differentiation but were significantly greater inmPB-derived erythrocytes than in those from CB
BioMed Research International 7
Z15
Z14
Z13
Z12
Z11
Z8
Z10 Z
1Z(A)
Z(D
)
Hb A
Hb F
Hb A2
Hb S
Hb CZ
(F)
Z(S
)
Z(E
)Z
(A2)
Z(C
)
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Control
Denatured Hb AHb A
Hb F
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Cord blood derived CD34+ cells
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Hb A
Hb FHb A2
G-CSF mobilized peripheral blood derived CD34+ cells
Figure 5 Electrophoretic determination of main hemoglobin subtypes in differentiated mPB and CB cells Capillary zone electrophoresisshows distinct patterns of hemoglobin subtype expression between differentiated mPB and CB cells About 80 of the hemoglobin producedby CB-CD34-derived erythrocytes is HbF while only 175 is HbA However 955 of the hemoglobin produced by mPB-CD34-derivederythrocytes is HbA
35 Similar Hemoglobin Dissociation Curve with MatureRBCs from mPB and CB Cells While erythrocytes derivedfrom each source have different combinations of HbA andHbF similar hemoglobin dissociation curve was observed(Figure 6) Apart from this hemoglobin subtype variationthis result clearly shows that in vitro cultured RBCs canproduce hemoglobin with oxygen binding and dissociationabilities equivalent to red blood cells produced in vivo
4 Discussion
The shortage of blood supply and the ever-growing demandfor blood transfusion represent a significant emerging issue in
transfusionmedicine Shortage of donated blood and the riskof infection have created limitations in the availability of redblood cells available for transfusion More recently isolatedCD34+ cells from human umbilical CB obtained from dis-carded maternity products and G-CSF-mPB obtained fromhealthy volunteers through leukapheresis have emerged aspotential alternative sources of HSCs The HSCs collectedfrom both sources have high rates of proliferation andcapacities for differentiation and create mature RBCs underthe proper culture conditions through three phases of invitro culture We note that though both types of stemcells show similar characteristics in general growth patternswith morphological and immunophenotypic changes they
8 BioMed Research International
0
0 50 150
0
0 50 150
21-day cultured RBCs from mPB 21-day cultured RBCs from CB
Figure 6 Hemox analysis in mPB and CB cells cultured for 21 days Differentiated erythrocytes were harvested at the end of the 21 daysof culture and oxygen equilibria were measured by Hemox-Analyzer Erythrocytes derived from mPB and from CB both show greateroxygenation abilities compared with normal human blood There are no significant differences between differentiated mPB and CB cells
show different characteristics in gene expression levels andhemoglobin subtype production
Our results show that bothmPB- and CB-derived CD34+cells began to proliferate extremely quickly during 7ndash14 daysof culture However both cell sources produce similar totalcell numbers after 21 days of culture And morphologicalchanges in differentiated cells from both sources were similarand no remarkable differences were found during the cultureexcept when comparing cell differentiation rates The mPBcells seemed to mature earlier than CB cells These resultsshow that mPB cells from circulating blood possess moreprogenitor cells than CB and have the ability of rapiddifferentiation into the mature RBCs Following cell growthat the end of each cell culture phase mPB- and CB-derivedCD34+ GPA and CD70 (transferrin receptor) expressionshowed similar patterns in flow cytometry analyses Thesedata are consistent with previous results regarding erythro-cyte induction mechanisms [8 14] To confirm whether theinitial exposition of cells is due to different sources deriveddifferentiation gene expression profiles were analyzed byquantitative RT-PCR GATA-1 is a transcription factor thatdetermines erythroid differentiation survival and 120573-globingene expression [15] GATA-2 inhibits GATA-1 functionGATA-2 expression exhibited a downregulation followingerythropoietin stimulation and its levels were higher incultured mPB cells This can be explained in earlier dif-ferentiation into mature RBCs in mPB source cells thanin CB This finding is also consistent with the result ofdifferential counts that mPB showed faster shift from pronor-moblasts to orthochromatic normoblasts in comparison toCB Ikonomi et al have previously shown that GATA-2preferentially increases 120574-globin gene expression indicatingthat the prolonged expression of GATA-2 contributes to theearly increase in 120574-globin in CB [16] EKLF binds specificallyto the 120573-globin promoter and is critical in establishingchromatin structure for high-level 120573-globin transcription via
its acetylation by CREB binding protein [17] SCLTall isrequired for the progression of erythroid differentiation andenforced expression of SCLTall increases 120573-globin expres-sion and BFU-E and CFU-E production [18] Because thesetranscription factors are closely related to increased 120573-globingene expression changes in their expression may accountfor the delay and reduction of 120573-globin expression in CB-derived differentiated cells The HbA- and HbF-related geneexpression tests exhibit different expression levels dependingon the cell sourceWe culturedmPB- andCB-derived CD34+cells through three phases harvested them after 17 daysof culture and analyzed them by hemoglobin type testingThese results clearly demonstrated different subtypes ofhemoglobin expressed bymPB- andCB-derivedmature cellsCB-derived cells mostly express HbF and mPB-derived cellsmainly express HbA but based on these data the originalsources of these cells appear to possess different propensitiesfor hemoglobin production patterns These results demon-strate that hemoglobin subtypes are not related to cultureconditions and culture time but are strongly affected by thesource material To evaluate the function of the differentiatedmPB and CB cells CD34+ cells derived from each sourcewere expanded and differentiated to large cell numbers ofup to a total of 5 times 107 cells and oxygen equilibria weremeasured by Hemox-Analyzer From the result the oxygendissociation curves indicate that the cells from both sourcesdo not significantly differ from one another with respectto hemoglobin function Although the types of hemoglobinexpression differed between CB- and mPB-derived maturecells the oxygen binding and dissociation curves may besimilar due to variation among adult type hemoglobin fetaltype hemoglobin or mixture of both types in vitro cultureprocessing after 17 days of culture The cultured cells indeedhave slightly greater deoxygenation functionalities comparedwith normal cells which showed the immaturity in shift toleft
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 7
Z15
Z14
Z13
Z12
Z11
Z8
Z10 Z
1Z(A)
Z(D
)
Hb A
Hb F
Hb A2
Hb S
Hb CZ
(F)
Z(S
)
Z(E
)Z
(A2)
Z(C
)
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Control
Denatured Hb AHb A
Hb F
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Cord blood derived CD34+ cells
0 20 40 60 80 100
120
140
160
180
200
220
240
260
280
300
Hemoglobin electrophoresis
Hb A
Hb FHb A2
G-CSF mobilized peripheral blood derived CD34+ cells
Figure 5 Electrophoretic determination of main hemoglobin subtypes in differentiated mPB and CB cells Capillary zone electrophoresisshows distinct patterns of hemoglobin subtype expression between differentiated mPB and CB cells About 80 of the hemoglobin producedby CB-CD34-derived erythrocytes is HbF while only 175 is HbA However 955 of the hemoglobin produced by mPB-CD34-derivederythrocytes is HbA
35 Similar Hemoglobin Dissociation Curve with MatureRBCs from mPB and CB Cells While erythrocytes derivedfrom each source have different combinations of HbA andHbF similar hemoglobin dissociation curve was observed(Figure 6) Apart from this hemoglobin subtype variationthis result clearly shows that in vitro cultured RBCs canproduce hemoglobin with oxygen binding and dissociationabilities equivalent to red blood cells produced in vivo
4 Discussion
The shortage of blood supply and the ever-growing demandfor blood transfusion represent a significant emerging issue in
transfusionmedicine Shortage of donated blood and the riskof infection have created limitations in the availability of redblood cells available for transfusion More recently isolatedCD34+ cells from human umbilical CB obtained from dis-carded maternity products and G-CSF-mPB obtained fromhealthy volunteers through leukapheresis have emerged aspotential alternative sources of HSCs The HSCs collectedfrom both sources have high rates of proliferation andcapacities for differentiation and create mature RBCs underthe proper culture conditions through three phases of invitro culture We note that though both types of stemcells show similar characteristics in general growth patternswith morphological and immunophenotypic changes they
8 BioMed Research International
0
0 50 150
0
0 50 150
21-day cultured RBCs from mPB 21-day cultured RBCs from CB
Figure 6 Hemox analysis in mPB and CB cells cultured for 21 days Differentiated erythrocytes were harvested at the end of the 21 daysof culture and oxygen equilibria were measured by Hemox-Analyzer Erythrocytes derived from mPB and from CB both show greateroxygenation abilities compared with normal human blood There are no significant differences between differentiated mPB and CB cells
show different characteristics in gene expression levels andhemoglobin subtype production
Our results show that bothmPB- and CB-derived CD34+cells began to proliferate extremely quickly during 7ndash14 daysof culture However both cell sources produce similar totalcell numbers after 21 days of culture And morphologicalchanges in differentiated cells from both sources were similarand no remarkable differences were found during the cultureexcept when comparing cell differentiation rates The mPBcells seemed to mature earlier than CB cells These resultsshow that mPB cells from circulating blood possess moreprogenitor cells than CB and have the ability of rapiddifferentiation into the mature RBCs Following cell growthat the end of each cell culture phase mPB- and CB-derivedCD34+ GPA and CD70 (transferrin receptor) expressionshowed similar patterns in flow cytometry analyses Thesedata are consistent with previous results regarding erythro-cyte induction mechanisms [8 14] To confirm whether theinitial exposition of cells is due to different sources deriveddifferentiation gene expression profiles were analyzed byquantitative RT-PCR GATA-1 is a transcription factor thatdetermines erythroid differentiation survival and 120573-globingene expression [15] GATA-2 inhibits GATA-1 functionGATA-2 expression exhibited a downregulation followingerythropoietin stimulation and its levels were higher incultured mPB cells This can be explained in earlier dif-ferentiation into mature RBCs in mPB source cells thanin CB This finding is also consistent with the result ofdifferential counts that mPB showed faster shift from pronor-moblasts to orthochromatic normoblasts in comparison toCB Ikonomi et al have previously shown that GATA-2preferentially increases 120574-globin gene expression indicatingthat the prolonged expression of GATA-2 contributes to theearly increase in 120574-globin in CB [16] EKLF binds specificallyto the 120573-globin promoter and is critical in establishingchromatin structure for high-level 120573-globin transcription via
its acetylation by CREB binding protein [17] SCLTall isrequired for the progression of erythroid differentiation andenforced expression of SCLTall increases 120573-globin expres-sion and BFU-E and CFU-E production [18] Because thesetranscription factors are closely related to increased 120573-globingene expression changes in their expression may accountfor the delay and reduction of 120573-globin expression in CB-derived differentiated cells The HbA- and HbF-related geneexpression tests exhibit different expression levels dependingon the cell sourceWe culturedmPB- andCB-derived CD34+cells through three phases harvested them after 17 daysof culture and analyzed them by hemoglobin type testingThese results clearly demonstrated different subtypes ofhemoglobin expressed bymPB- andCB-derivedmature cellsCB-derived cells mostly express HbF and mPB-derived cellsmainly express HbA but based on these data the originalsources of these cells appear to possess different propensitiesfor hemoglobin production patterns These results demon-strate that hemoglobin subtypes are not related to cultureconditions and culture time but are strongly affected by thesource material To evaluate the function of the differentiatedmPB and CB cells CD34+ cells derived from each sourcewere expanded and differentiated to large cell numbers ofup to a total of 5 times 107 cells and oxygen equilibria weremeasured by Hemox-Analyzer From the result the oxygendissociation curves indicate that the cells from both sourcesdo not significantly differ from one another with respectto hemoglobin function Although the types of hemoglobinexpression differed between CB- and mPB-derived maturecells the oxygen binding and dissociation curves may besimilar due to variation among adult type hemoglobin fetaltype hemoglobin or mixture of both types in vitro cultureprocessing after 17 days of culture The cultured cells indeedhave slightly greater deoxygenation functionalities comparedwith normal cells which showed the immaturity in shift toleft
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 BioMed Research International
0
0 50 150
0
0 50 150
21-day cultured RBCs from mPB 21-day cultured RBCs from CB
Figure 6 Hemox analysis in mPB and CB cells cultured for 21 days Differentiated erythrocytes were harvested at the end of the 21 daysof culture and oxygen equilibria were measured by Hemox-Analyzer Erythrocytes derived from mPB and from CB both show greateroxygenation abilities compared with normal human blood There are no significant differences between differentiated mPB and CB cells
show different characteristics in gene expression levels andhemoglobin subtype production
Our results show that bothmPB- and CB-derived CD34+cells began to proliferate extremely quickly during 7ndash14 daysof culture However both cell sources produce similar totalcell numbers after 21 days of culture And morphologicalchanges in differentiated cells from both sources were similarand no remarkable differences were found during the cultureexcept when comparing cell differentiation rates The mPBcells seemed to mature earlier than CB cells These resultsshow that mPB cells from circulating blood possess moreprogenitor cells than CB and have the ability of rapiddifferentiation into the mature RBCs Following cell growthat the end of each cell culture phase mPB- and CB-derivedCD34+ GPA and CD70 (transferrin receptor) expressionshowed similar patterns in flow cytometry analyses Thesedata are consistent with previous results regarding erythro-cyte induction mechanisms [8 14] To confirm whether theinitial exposition of cells is due to different sources deriveddifferentiation gene expression profiles were analyzed byquantitative RT-PCR GATA-1 is a transcription factor thatdetermines erythroid differentiation survival and 120573-globingene expression [15] GATA-2 inhibits GATA-1 functionGATA-2 expression exhibited a downregulation followingerythropoietin stimulation and its levels were higher incultured mPB cells This can be explained in earlier dif-ferentiation into mature RBCs in mPB source cells thanin CB This finding is also consistent with the result ofdifferential counts that mPB showed faster shift from pronor-moblasts to orthochromatic normoblasts in comparison toCB Ikonomi et al have previously shown that GATA-2preferentially increases 120574-globin gene expression indicatingthat the prolonged expression of GATA-2 contributes to theearly increase in 120574-globin in CB [16] EKLF binds specificallyto the 120573-globin promoter and is critical in establishingchromatin structure for high-level 120573-globin transcription via
its acetylation by CREB binding protein [17] SCLTall isrequired for the progression of erythroid differentiation andenforced expression of SCLTall increases 120573-globin expres-sion and BFU-E and CFU-E production [18] Because thesetranscription factors are closely related to increased 120573-globingene expression changes in their expression may accountfor the delay and reduction of 120573-globin expression in CB-derived differentiated cells The HbA- and HbF-related geneexpression tests exhibit different expression levels dependingon the cell sourceWe culturedmPB- andCB-derived CD34+cells through three phases harvested them after 17 daysof culture and analyzed them by hemoglobin type testingThese results clearly demonstrated different subtypes ofhemoglobin expressed bymPB- andCB-derivedmature cellsCB-derived cells mostly express HbF and mPB-derived cellsmainly express HbA but based on these data the originalsources of these cells appear to possess different propensitiesfor hemoglobin production patterns These results demon-strate that hemoglobin subtypes are not related to cultureconditions and culture time but are strongly affected by thesource material To evaluate the function of the differentiatedmPB and CB cells CD34+ cells derived from each sourcewere expanded and differentiated to large cell numbers ofup to a total of 5 times 107 cells and oxygen equilibria weremeasured by Hemox-Analyzer From the result the oxygendissociation curves indicate that the cells from both sourcesdo not significantly differ from one another with respectto hemoglobin function Although the types of hemoglobinexpression differed between CB- and mPB-derived maturecells the oxygen binding and dissociation curves may besimilar due to variation among adult type hemoglobin fetaltype hemoglobin or mixture of both types in vitro cultureprocessing after 17 days of culture The cultured cells indeedhave slightly greater deoxygenation functionalities comparedwith normal cells which showed the immaturity in shift toleft
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 9
In summary mPB and CB are undoubtedly excellentsources for mature RBC production and may be key incontributing to a solution for the RBC supply shortageproblem Our study shows that despite similar phenotypesand functionalities following erythrocytematuration the twoare discrete in that they show different Hb types and geneexpression levels This study demonstrates distinctions thatshould be taken into account when choosing the source ofHSCs for artificial mature RBC production form stem cells
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This study was supported by a Grant from the KoreanHealthcare Technology RampD Project Ministry of Health andWelfare Republic of Korea (no HI10C1740)
References
[1] World Health Organization ldquoGlobal Database on Blood Safety(GDBS)rdquo Summary Report 2011 2011
[2] Y Ebihara F Ma and K Tsuji ldquoGeneration of red bloodcells from human embryonicinduced pluripotent stem cells forblood transfusionrdquo International Journal of Hematology vol 95pp 610ndash616 2012
[3] CMazurier L Douay andH Lapillonne ldquoRed blood cells frominduced pluripotent stem cells hurdles and developmentsrdquoCurrent Opinion in Hematology vol 18 no 4 pp 249ndash253 2011
[4] M-CGiarratana L KobariH Lapillonne et al ldquoEx vivo gener-ation of fully mature human red blood cells from hematopoieticstem cellsrdquoNature Biotechnology vol 23 no 1 pp 69ndash74 2005
[5] K Miharada T Hiroyama K Sudo T Nagasawa and YNakamura ldquoEfficient enucleation of erythroblasts differentiatedin vitro from hematopoietic stem and progenitor cellsrdquo NatureBiotechnology vol 24 no 10 pp 1255ndash1256 2006
[6] E J Baek H-S Kim S Kim H Jin T-Y Choi and H OKim ldquoIn vitro clinical-grade generation of red blood cells fromhuman umbilical cord blood CD34+ cellsrdquo Transfusion vol 48no 10 pp 2235ndash2245 2008
[7] D Boehm W G Murphy and M Al-Rubeai ldquoThe potential ofhuman peripheral blood derived CD34+ cells for ex vivo redblood cell productionrdquo Journal of Biotechnology vol 144 no 2pp 127ndash134 2009
[8] E J Baek H-S Kim J-H Kim N J Kim and H O KimldquoStroma-free mass production of clinical-grade red blood cells(RBCs) by using poloxamer 188 as an RBC survival enhancerrdquoTransfusion vol 49 no 11 pp 2285ndash2295 2009
[9] H O Kim ldquoIn-vitro stem cell derived red blood cells fortransfusion are we there yetrdquo Yonsei Medical Journal vol 55pp 304ndash309 2014
[10] M Vlaski X Lafarge J Chevaleyre P Duchez J-M Boironand Z Ivanovic ldquoLow oxygen concentration as a generalphysiologic regulator of erythropoiesis beyond the EPO-relateddownstream tuning and a tool for the optimization of red bloodcell production ex vivordquo Experimental Hematology vol 37 no5 pp 573ndash584 2009
[11] H M Rogers X Yu J Wen R Smith E Fibach and C TNoguchi ldquoHypoxia alters progression of the erythroid pro-gramrdquo Experimental Hematology vol 36 no 1 pp 17ndash27 2008
[12] D L Vanhille R H Nussenzveig C Glezos S Perkins and AM Agarwal ldquoBest practices for use of the HEMOX analyzerin the clinical laboratory quality control determination andchoice of anticoagulantrdquo Laboratory Hematology vol 18 pp 17ndash19 2012
[13] D F Keren D Hedstrom R Gulbranson C-N Ou and RBak ldquoComparison of Sebia Capillarys capillary electrophoresiswith the Primus high-pressure liquid chromatography in theevaluation of hemoglobinopathiesrdquo American Journal of Clin-ical Pathology vol 130 no 5 pp 824ndash831 2008
[14] H O Kim and E J Baek ldquoRed blood cell engineering in stromaand serumplasma-free conditions and long term storagerdquoTissue Engineering A vol 18 no 1-2 pp 117ndash126 2012
[15] R Ferreira K Ohneda M Yamamoto and S PhilipsenldquoGATA1 function a paradigm for transcription factors inhematopoiesisrdquo Molecular and Cellular Biology vol 25 no 4pp 1215ndash1227 2005
[16] P Ikonomi C T Noguchi W Miller H Kassahun R Hardi-son and A N Schechter ldquoLevels of GATA-1GATA-2 tran-scription factors modulate expression of embryonic and fetalhemoglobinsrdquo Gene vol 261 no 2 pp 277ndash287 2000
[17] W Zhang S Kadam B M Emerson and J J Bieker ldquoSite-specific acetylation by p300 or CREB binding protein regulateserythroid Kruppel-like factor transcriptional activity via itsinteraction with the SWI-SNF complexrdquoMolecular and CellularBiology vol 21 no 7 pp 2413ndash2422 2001
[18] E Ravet D Reynaud M Titeux et al ldquoCharacterizationof DNA-binding-dependent and -independent functions ofSCLTAL1 during human erythropoiesisrdquo Blood vol 103 no 9pp 3326ndash3335 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
Hindawi Publishing Corporationhttpwwwhindawicom
GenomicsInternational Journal of
Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
