Study on the Dynamic Biological Characteristics of Sca-1 ...€¦ · 2 StemCellsInternational...

Research ArticleStudy on the Dynamic Biological Characteristics ofSca-1+ Hematopoietic Stem and Progenitor Cell Senescence

Shan Geng1 Xin-Yi Mu2 Xiong-Bin Chen2 Ji-Ying Hou2 Dao-Yong Jia2

Chun-Yan Xu2 and Ya-Ping Wang1

1Laboratory of Stem Cells and Tissue Engineering Department of Histology and Embryology Chongqing Medical UniversityChongqing 400016 China2Chongqing Medical University Chongqing 400016 China

Correspondence should be addressed to Ya-Ping Wang ypwangcqaliyuncom

Received 7 March 2015 Revised 19 April 2015 Accepted 5 May 2015

Academic Editor Jia Xie

Copyright copy 2015 Shan Geng 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

The researches in the dynamic changes of the progress of HSCs aging are very limited and necessary In this study male C57BL6mice were divided into 5 groups by age We found that the superoxide damage of HSPCs started to increase from the middle age(6 months old) with notably reduced antioxidation ability In accordance with that the senescence of HSPCs also started fromthe middle age since the self-renewal and differentiation ability remarkably decreased and senescence-associated markers SA-120573-GAL increased in the 6-month-old and the older groups Interestingly the telomere length and telomerase activity increased to acertain degree in the 6-month-old group It suggested an intrinsic spontaneous ability of HSPCs against aging It may provide atheoretical and experimental foundation for better understanding the senescence progress of HSPCs And the dynamic biologicalcharacteristics of HSPCs senescence may also contribute to the clinical optimal time for antiaging drug intervention

1 Introduction

With abilities of self-renewing multipotent differentiationcross-differentiation and reconstruction of long-term hem-atopoietic potential hematopoietic stem cells (HSCs) candifferentiate into all hematopoietic lineages throughout thelifetime of an organism Blood cells are continually producedfrom the HSCs that reside in the bone marrow (BM)Throughout the lifespan of the organism the HSC reservoirsustains life A major property of stem cells is their abilityto self-renew which is important to maintain the HSC poolfor the lifespan Notably age-related changes in the HSCcompartment correlate with organismal aging which appearsas anemia increased propensity for myeloproliferative neo-plasms (MPNs) decreased immune function and increasedcancer incidence [1ndash4] Upon aging the number of HSCs inthe BM increases while their repopulation potential declinesMoreover aged HSCs exhibit lineage bias in reconstitutionexperiments with an inclination toward myeloid at theexpense of lymphoid potential

Superoxide damage theory is considered to be one ofthe key mechanisms of cellular aging the theory holds thatthe body has sound oxygen-free radicals generation andscavenging balance system [5] Since the antioxidant enzymesof body such as superoxide dismutase (SOD) and glutathione(GSH) in the environment of cell survival fall causing theaccumulation of oxygen radicals in cells cells graduallydamage and eventually senescence occur [6] Although thebasic components of the hematopoietic system in aging bodyare maintained in the process the quantity and function ofHSCs decrease gradually [7]

Stem cell antigen-1 (Sca-1) is the most common markerused to enrich adult murine hematopoietic stem cells (HSCs)and can be used to isolate a nearly pure HSC populationwhen used in conjunction with additional markers [8]Our recent studies have shown that compared with Sca-1+hematopoietic stem and progenitor cells (HSPCs) fromyoungmice those from agedmice showed significantly fewerSca-1+ HSPCs higher expression of p53 P16Ink4a P19Arf andP21Cip1 and decreased telomeric activity Adhesion ability

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 954120 10 pageshttpdxdoiorg1011552015954120

2 Stem Cells International

Table 1 The unique PCR primers

Primers Forward (51015840-31015840) Reverse (51015840-31015840) Length (bp)p16INK4a TCCGCTGCAGACAGACTGGCCAG CATCGCGCACATCCAGCCGAGC 295p19Arf AAGAAGTCTGCGTCGGCGAC AGTACCGGAGGCATCTTGGACA 215p53 CACGTACTCTCCTCCCCTCAA GGCTCATAAGGTACCACCACG 294p21Cip1Waf1 ATTCCTGGTGATGTCCGACC AAAGTTCCACCGTTCTCGG 144GAPDH GTGCTGAGTATGTCGTGGAGTCT GAGTGGGAGTTGCTGTTGAAGT 602

and CFU-Mix potency were also reduced in Sca-1+ HSPCsfrom aged mice indicating an aging-related impairment [79 10]

This studywas intended to explore the dynamic biologicalchanges of HSCs aging characteristics Sca-1+ HSPCs wereseparated and purified from mice at different ages by themagnetic absorptive cell sorting (MACS)We investigated theroles of the superoxide damage and antioxidant capacity inthe natural aging process of HSCs providing experimentalevidence and theoretical basis in HSPCs aging mechanismand antiaging drug development

2 Materials and Methods

21 Ethics Statement and Animal Treatment The maleC57BL6J mice were purchased from the Medical and Lab-oratory Animal Center of Chongqing (Animal certificatenumber SCXK(yu)2007-0001) and housed in a temperature-and light-controlled room with free access to water andfood All experiments were performed in accordance withinstitutional and national guidelines and regulations andwere approved by the ChongqingMedical University AnimalCare and Use Committee All surgery was performed undersodium pentobarbital anesthesia and all the efforts weremade to minimize suffering

Animals were divided into 5 groups Group A (3-4 weeksold 13ndash16 g in body weight) Group B (8-9 weeks old 20ndash25 gin body weight) Group C (6 months old 35ndash40 g in bodyweight) Group D (12 months old 39ndash43 g in body weight)Group E (18 months old 37ndash45 g in body weight)

22 Isolation and Purification of Sca-1+ HSPCs in Mouse BoneMarrow Bone marrow was collected from the femurs andtibiasMononuclear cells were obtained fromBMaspirates bydensity gradient separation and then subjected to immuno-magnetic enrichment of Sca-1+ HSPCs (Miltenyi BiotecBergisch Gladbach Germany) as described previously [5ndash7]

23 Flow Cytometric Analysis All flow cytometric analyseswere performed using FACSCalibur (BD Biosciences Hei-delberg Germany) and data were analyzed with FCS ExpressV3 software (De Novo Software Los Angeles CA USA)

24 Detection of Oxidation-Associated Biomarkers After theisolation Sca-1+ HSPCs were collected and washed by PBSGSH content superoxide content SOD activity and MDAcontent were detected by chemical colorimetric analysis

according to themanufacturerrsquos instructions (Beyotime Insti-tute of Biotechnology Shanghai China)

25 Cell-Cycle Analysis of Sca-1+ HSPCs Cell-cycle analysiswas performed with the FITC BrdU Flow Kit (BD Bios-ciences Heidelberg Germany) according to the manufac-turerrsquos instructions Flow cytometric analyses were per-formed using FACSCalibur (BD Biosciences HeidelbergGermany) and Cell-cycle data were analyzed with Multicyclesoftware (PHENIX Japan)

26 The CFU-Mix Formation In Vitro Initially 4 times 104 Sca-1+ HSPCs were cultivated in 24-well plates (Costar Corn-ing Incorporated Corning NY USA) with the clonogenicmethylcellulose medium H3434 (Stem Cell TechnologiesVancouver Canada) and cells were cultured in a humidifiedatmosphere with 5 CO

2at 37∘C for 2 weeks Finally the

numbers of CFU-Mix formations were counted under a lightmicroscope

27 Senescence-Associated 120573-Galactosidase CytochemicalStaining The senescence-associated 120573-galactosidase (SA-120573-gal) activity was determined in Sca-1+ HSPCs using theCellular Senescence Detection Kit (Biolabs San Diego CAUSA) according to the manufacturer In brief 1 times 105 Sca-1+HSPCs were stained by staining solution for 24 hours at 37∘Cwithout CO

2in 6-well plates Cells were counted and the frac-

tion of senescent cells (positive for SA-120573-gal) was assessed

28 RNA Extraction and RT-PCR 6 times 106 Sca-1+ HSPCsof each group were collected Total mRNA was extractedaccording to the manufacturerrsquos protocol using TRIZOLReagent (TaKaRA Japan) OD260OD280 of RNA 18sim20First-strand cDNAwas created byAMV reverse transcriptionKit (Invitrogen USA) The unique PCR primers were shownin Table 1 DNA was amplified by an initial incubation at94∘C for 5min followed by 40 cycles of 94∘C denaturationfor 15 sec annealing at 60∘C for 60 sec and 72∘C extensionfor 1min PCR product was subjected to 1 agarose gelelectrophoresis Optical intensities were quantified usingQuantity One (Bio-Rad) The relative expression levels werecalculated as the ratio of the optical intensity of targeted geneto that of the internal reference

29 Western Blotting Analysis To examine the expression ofP53 P19 P16INK4a P21Cip1Waf1 cyclinD1 CDK4 CDK2 and

Stem Cells International 3

CyclinE which are markers of cell senescence in freshly iso-lated Sca-1+ HSPCs from each group we performed westernblot analysis Cells were lysed in buffer [20mmolL Tris-HClpH 75 150mmolLNaCl 1TritonX-100 and complete pro-tease inhibitormixture tablets (Beyotime Institute of Biotech-nology Shanghai China)] and centrifuged at 1000 g for 5minat 4∘C Protein concentrations of the cell were determinedby a BCA protein assay reagent kit (Beyotime Institute ofBiotechnology Shanghai China)The cell lysates (40120583gwell)were subjected to SDS-PAGE and transferred onto a PVDFmembrane (Millipore Bedford MA USA) The blottedmembranes were incubated with primary antibodies to p53(Abcam Cambridge UK) P19 (Abcam Cambridge UK)P16INK4a (ANBOUSA) P21Cip1Waf1 (Ptglab USA) cyclinD1(ANBO USA) CDK4 (Ptglab USA) CDK2 (ANBO USA)and CyclinE (Abcam Cambridge UK) or ACTB (PtglabUSA) Corresponding horseradish peroxidase-conjugatedantibodies were used as the second antibody (ZSGB-BIOBeijing China) The membranes were visualized using theenhanced chemiluminescence detection system (Pierce)Thelevel of 120573-actin was used as an internal control Relativeintensities were quantified using Quantity One (Bio-Rad)

210Measurement of Telomere Length by Southern Blot Telo-mere lengths were measured from the Sca-1+ HSPCs fromeach group according to the previously described method[11] In brief after extraction DNA was inspected forintegrity digested resolved by gel electrophoresis transferredto a membrane hybridized with labeled probes and exposedto X-ray film using DAB The telomere length was measuredby Western Biotechnology Corporation

211 Detection Activity of Telomerase by Silver Staining TRAP-PCR The Sca-1+ HSPCs were homogenized and lysed onthe ice and the supernatant was collected after centrifuga-tion The concentrations were measured The PCR reactionmixture contained 5120583L 10X TRAP buffer 1 120583L dNTPs 1 120583LTaq-DNA polymerase 1 120583L TS primer and 2 120583L extract oftelomerase and 39 120583L DEPC water was incubated for 30minat 23∘C for telomerase-mediated extension of the TS primerThe reaction mixture was added 1 120583L CX primer to amplifyat 94∘C for 5min and then subjected to 35 cycles at 94∘C for30 s 50∘C for 30 s and 72∘C for 90 s TRAP reaction productswere separated by 10 polyacrylamide gel electrophoresisand detected by SYBR green (Gene Inc) staining The ratioof enzyme activity = the fluorescence intensitythe proteinconcentration Telomerase activity is proportional to the ratioof enzyme activity The telomerase activity was measured byWestern Biotechnology Corporation

212 Statistical Analysis Data were analyzed by 119905-test andANOVA using SPSS Version 170 software and represented as(119909 plusmn 119904) Differences were considered significant at 119875 lt 005

3 Results

31 The Purification of Sca-1+ HSCHPC Flow cytometryshowed that the percentage of Sca-1+ HSPCs in collected

MNCs was only (102 plusmn 019) before isolation but the purityof separated Sca-1+ HSCHPS was (9366 plusmn 083) afterMACS The differences in five groups were not significant instatistics (Figure 1)

32 The Superoxide Damage and the Antisuperoxide Ability inSca-1+ HSPCs From group A to E the activities of SOD ofeach sample were 2751 plusmn 111 Ug 3823 plusmn 350Ug 2885 plusmn065Ug 1752plusmn 127Ug and 1267plusmn 089Ug respectively Itwas significantly correlated with changes of age (119903

2= 08819

119875 lt 001) (Figure 2) Glutathione contents in each groupwere2878 plusmn 077 120583M 3148 plusmn 203 120583M 4762 plusmn 493 120583M 3910 plusmn219 120583M and 2563 plusmn 123 120583M respectively A significantcorrelation was found between GSH contents and ages (119903

2=

08754 119875 lt 001) (Figure 2) From group A to E theabsorbance value of each group which is proportional tosuperoxide was 0114 plusmn 0005 0109 plusmn 0005 0133 plusmn 00020141 plusmn 0005 and 0151 plusmn 0009 respectively The changesof superoxide levels reflecting absorbance value were founda significant correlation with the ages (119903

2= 09464 119875 lt

005) (Figure 2) MDA contents of Sca-1+ HSPCs in eachgroup were 206 plusmn 054 nmolmg 081 plusmn 035 nmolmg269 plusmn 062 nmolmg 706 plusmn 095 nmolmg and 1144 plusmn119 nmolmg respectively The MDA contents were foundto have a positive correlation with the ages (119903

2= 09552

119875 lt 001) (Figure 2)

33 The Changes of the SA-120573-Gal Staining in Sca-1+ HSPCsby Aging In SA-120573-gal staining aged cells were stained inblue with blue granules in the cytoplasm SA-120573-gal positivecells from groups A to E gradually increased significantly(Figure 3)

34 The Cell Cycle of Sca-1+ HSPCs by Aging From GroupsA to E Sca-1+ HSPCs of each group displayed significantincreased cell-cycle arrests in G1 phase as the proportionof G0G1 phase increased and the proportion of S phasereduced significantly (Figure 4 119875 lt 005)

35The Ratio of CFU-Mix Formation In Vitro of Sca-1+ HSPCsby Aging The numbers of CFU-Mix formations of Sca-1+HSPCs were reduced gradually from group A to group Eillustrating that the ability of colony formation of Sca-1+HSPCs gradually decreased by aging (Figure 5)

36 The Effect of Aging on the Expression of Senescence-Asso-ciated Genes P21Cip1 P16Ink4a P19Arf and P53 mRNA on Sca-1+ HSPCs The results of PCR showed that the expression ofp16INK4a p19Arf p53 p21Cip1Waf1 and CyclinD1 mRNA inSca-1+ HSPCs from groups A to E was increased graduallyIn group E the mRNA levels of these genes were significantlyhigher than the other groups (Figure 6 119875 lt 005)

37 The Effect of Aging on the Expression of Senescence-Asso-ciated Proteins P53 P19 P16Ink4a P21Cip1Waf1 CyclinD1 CDK4CDK2 and CyclinE on Sca-1+ HSPCs By western blottingthe protein levels of P16INK4a P21Cip1Waf1 CyclinD1 P53


Before purificationAfter purification

Group A Group B Group C Group EGroup D

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atio

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File ctrl001Acquisition date 09-Nov-11

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Mean3573371738

File B+001Acquisition date 09-Nov-11

MarkerAllM1M2

Gated ()100006629340

Mean1069854311416

X parameter FITC (Log) X parameter FITC (Log)

(b)

Figure 1The ratio of sca-1+ HSPCs before and after MACS purification (a)The ratio of sca-1+ HSPCs in all groups ( 119909plusmn 119904 119899 = 10) GroupA 3-4-week-old mice 13ndash16 g in body weight Group B 8-9-week-old mice 20ndash25 g in body weight Group C 6-month-old mice 35ndash40 gin body weight Group D 12-month-old mice 39ndash43 g in body weight Group E 18-month-old mice 37ndash45 g in body weight (b) Result offlow cytometry (A) The ratio of sca-1+ HSPCs before MACS purification (B) The ratio of sca-1+ HSPCs after MACS purification lowast119875 lt 001compared with other groups

and P19 increased gradually in accordance with the mRNAexpression Meanwhile the protein levels of CDK4 CDK2and CyclinE reduced gradually with the aging (Figure 7)

38The Effect of Aging on the Telomere Length and TelomeraseActivity in Sca-1+HSPCs FromgroupsA to E the telomeraseactivity ratios of Sca-1+ HSPCs were 1424 1492 0925 1235and 0570 respectively (Figure 8) while the telomere length

was 246 kb 255 kb 223 kb 238 kb and 195 kb respectively(Figure 9)

4 Discussion

Theoretically from the point of view of organs development3-4-week-old mice are equivalent to 1-2-year-old human 2-month-old mice are equivalent to young adults at the age of


Group A Group B Group C Group E

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The t

otal

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0Tota

l GSH

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ent o

f Sca

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Supe

roxi

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-1+

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15

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0MD

A co

nten

t of S

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Figure 2 The superoxide damage and the antisuperoxide ability of Sca-1+ HSPCs by aging (a) The activities of SOD of each sample (Ug119909 plusmn 119904 119899 = 10) (b) Glutathione contents in each group (120583M 119909 plusmn 119904 119899 = 10) (c) The absorbance value of each group which is proportional tosuperoxide (OD 119909 plusmn 119904 119899 = 10) (d) MDA contents of Sca-1+ HSPCs in each group (nmolmg 119909 plusmn 119904 119899 = 10) Group A 3-4-week-old mice13ndash16 g in body weight Group B 8-9-week-old mice 20ndash25 g in body weight Group C 6-month-old mice 35ndash40 g in body weight GroupD 12-month-old mice 39ndash43 g in body weight Group E 18-month-old mice 37ndash45 g in body weight (andashd) represent a significant differenceof these columns (119875 lt 005)

15 to 20 years 6-month-old mice are equivalent to middle-aged human at the age of 40 to 50 years 12-month-old miceare equivalent to middle-aged human about 60 years old and18-month-old mice were equivalent to aged human over 70years old [12]

As HSCs age the abilities of self-renewal and differen-tiation decline It results in reduced number of HSCs withimpaired abilities of proliferation and differentiation Sincethe lineages of mature blood cells are differentiated fromHSCs the whole peripheral blood cells decrease and therisk of aplastic anemia grows It is shown that HSCs agingand senile leukemia are closely related and for the elderlypeople the risk of acute myeloid leukemia (AML) growswith years [10] Superoxide dismutase (SOD) is an importantantioxidant enzymes in vivo which can catalyze the dis-proportionation of superoxide anion and generate hydrogenperoxide (H

2O2) and oxygen (O

2) [13] In this studywe found

on that SOD contents of Sca-1+ HSPCs decreased with agefollowing a general trend throughout lifespan from juvenileto young adulthood it increased rapidly and reached thepeak and then gradually decreased The trend is consistentwith the trend of Sca-1+ HSPCs development and agingwhich indicates that the aging-related decline of antioxidantcapacity begins frommiddle age Glutathione is an importantantioxidant providing mercapto group to the majority ofliving cells in vivo The mercapto group can keep proteinsat an appropriate redox state In the present study we foundthat with increasing age the overall trend of total glutathionecontent was similar to the trends of Sca-1+ HSPCs formationand development after reaching the peak at themiddle age itdecreased slowly afterwards It suggests that the aging-relateddecline of antioxidant capacity begins frommiddle-age stage

Superoxide usually refers to superoxide anionO2

minus whichis a radical of oxygen molecular and a strong oxidizer It


a

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in S

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Group A

Group B Group C

Group EGroup D

(b)

Figure 3 The changes of the SA-120573-Gal staining in Sca-1+ HSPCs by aging (a) The percentage of SA-120573-gal positive cells in Sca-1+ HSPCs( 119909 plusmn 119904 119899 = 10) Group A 3-4-week-old mice 13ndash16 g in body weight Group B 8-9-week-old mice 20ndash25 g in body weight Group C6-month-old mice 35ndash40 g in body weight Group D 12-month-old mice 39ndash43 g in body weight Group E 18-month-old mice 37ndash45 gin body weight (andashd) represent a significant difference of these columns (119875 lt 005) (b) Representative micrographs depict morphology ofSA-120573-gal positive cells and SA-120573-gal negative cells in Sca-1+ HSPCs (times400) SA-120573-gal positive cells are dyed in blue Scale bars indicate 10120583m


hi

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cle o

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)

Figure 4 Distribution of cell cycle of Sca-1+ HSPCs ( 119909 plusmn 119904119899 = 10) GroupA 3-4-week-oldmice 13ndash16 g in bodyweight GroupB 8-9-week-old mice 20ndash25 g in body weight Group C 6-month-oldmice 35ndash40 g in body weight Group D 12-month-oldmice 39ndash43 g in body weight Group E 18-month-old mice 37ndash45 g in bodyweight (andashk) represent a significant difference of these columns(119875 lt 005)

aa

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(10

4Sc

a-1+

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Cs)

Num

ber o

f CFU

-Mix

form

atio

ns

Figure 5 Number of CFU-Mix formations by Sca-1+HSPCs GroupA 3-4-week-old mice 13ndash16 g in body weight Group B 8-9-week-old mice 20ndash25 g in body weight Group C 6-month-old mice 35ndash40 g in body weight Group D 12-month-old mice 39ndash43 g in bodyweight Group E 18-month-old mice 37ndash45 g in body weight (andashc)represent a significant difference of these columns (119875 lt 005)

can be produced by stimulated white blood cells againstmicrobial infection Superoxide can cause superoxide damageand is closely related with many diseases In this study wefound that superoxide content increased with aging fromjuvenile to young adult it went down and reached the lowestpoint which indicated the stronger antioxidant capacity andslowly increased afterwards This tendency suggests thatthe superoxide accumulates from the middle age which iscoincident with the Sca-1+ HSPCs aging

MDA is a lipid peroxide produced when radicals attackpolyunsaturated fatty acids in the biomembrane The pro-duction of MDA is used as a biomarker to measure the levelof superoxide stress in an organism [14] We found that thecontents of MDA in Sca-1+ HSPCs stay at low levels for thefirst two months of the life maintain stable levels for the nextfourmonths and increase dramatically after the sixthmonthThis result demonstrated that superoxide damage of Sca-1+HSPCs was slight at the juvenile and young adult stage withthe similar levels but increased significantly from themiddle-aged stage

Aging cells usually appear as enlarged size and increaselevels of 120573-gal In this study we observed that the 120573-galactivity in Sca-1+ HSPCs gradually increased While thecolony forming ability decreased by aging indicating thegradual senescence and function loss of Sca-1+ HSPCs

From the above results we speculate that in the process ofaging because of accumulation of risk factors such as the lackof vitamins metabolic disorders of trace elements endocrineeffects ultraviolet radiation or potential threat of ionizingradiation the antioxidant ability of Sca-1+HSPCs remarkablydecreases It breaks the homeostasis of cellular oxidationand antioxidation which leads to the increase of superoxidedamage The accumulated superoxide damage may be heldresponsible for the senescence of Sca-1+ HSPCs and thepotential risk for senile leukemia and aplastic anemia Recentstudy has shown that although the basic components of thehematopoietic systemmaintain during the aging process theself-renewal and differentiation potentials of Sca-1+ HSPCsdecrease suggesting the senescence of Sca-1+ HSPCs [15]

In this study the cell-cycle analysis by flow cytometryshowed that Sca-1+ HSPCs displayed G1 phase arrests withaging with significant increase in proportion of G0G1phase and decrease in the proportion of S phase In thisstudy RT-PCR and western Blot showed that the mRNAexpressions of p16INK4a p19Arf p53 and p21Cip1Waf1 of Sca-1+ HSPCs gradually increased with age and protein levelsof P53 P19 P16INK4a P21Cip1Waf1 and cyclinD1 graduallyincreased while CDK4 CDK2 and CyclinE decreasedThesechanges were consistent with the cell-cycle analysis Thisresult suggested roles of p16INK4a-Rb and p19Arf -Mdm2-p53-p21Cip1Waf1 signaling pathways in the natural agingof Sca-1+ HSPCs Telomere length and telomerase activityassay showed the same trend by aging that it increasedat first (from the childhood to youth) and then decreasedsignificantly afterwards Interestingly telomere length andtelomerase activity have a rebound at the elder age butdecreased dramatically after that It suggested that althoughthe senescence of Sca-1+ HSPCs starts at middle age HSCsshows intrinsic antiaging ability to get recovery at thebeginning of senescence However the antiaging ability waslost gradually when the body got older Thus it implied theexistence of a spontaneous antiaging ability in the early agingstage And it also enlightened us that maybe we can improvethis antiaging capability by drug intervention or whether theearly aging stage is the optimal time for antiaging These arewhat we are most interested in and what we want to continueto discuss and research in the future


M A B C D E

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10

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Figure 6 The mRNA expression of p16INK4a p19Arf p53 and p21Cip1Waf1 in Sca-1+ HSPCs (a) Result of RT-PCR M DNAmark A GroupA (3-4-week-old mice 13ndash16 g in body weight) B Group B (8-9-week-old mice 20ndash25 g in body weight) C Group C (6-month-old mice35ndash40 g in body weight) D Group D (12-month-old mice 39ndash43 g in body weight) and E Group E (18-month-old mice 37ndash45 g in bodyweight) (b) Ratio of mRNA to GAPDH (andashl) represent a significant difference of these columns (119875 lt 005)

CyclinD1

p19

CDK2

CyclinE1

CDK4

120573-actin

p53

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A B C D E

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Group EGroup D

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Figure 7 Protein expression of P16INK4a P53 P21Cip1Waf1 CyclinD1 P19 CDK2 CyclinE and CDK4 in Sca-1+ HSPCs (a) Result of westernblot A Group A (3-4-week-old mice 13ndash16 g in body weight) B Group B (8-9-week-old mice 20ndash25 g in body weight) C Group C (6-month-old mice 35ndash40 g in body weight) D Group D (12-month-old mice 39ndash43 g in body weight) and E Group E (18-month-old mice37ndash45 g in body weight) (b) Ratio of protein to 120573-actin (andashd) represent a significant difference of these columns (119875 lt 001)


M A B C D E

330bp

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(a)


b

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abaa

20

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The r

atio

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elom

eras

e act

ivity

(b)

Figure 8 Telomerase activity in the Sca-1+ HSPCs (a) Result of TRAP-PCR (b) Telomerase activity M DNAmark A Group A (3-4-week-old mice 13ndash16 g in body weight) B Group B (8-9-week-old mice 20ndash25 g in body weight) C Group C (6-month-old mice 35ndash40 g inbody weight) D Group D (12-month-old mice 39ndash43 g in body weight) and E Group E (18-month-old mice 37ndash45 g in body weight) (andashc)represent a significant difference of these columns (119875 lt 001)

A B C D E

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bp)

(b)

Figure 9 Telomere length of Sca-1+ HSPCs (a) Result of Southern blotting A Group A (3-4-week-old mice 13ndash16 g in body weight) BGroup B (8-9-week-old mice 20ndash25 g in body weight) C Group C (6-month-old mice 35ndash40 g in body weight) D Group D (12-month-oldmice 39ndash43 g in body weight) and E Group E (18-month-oldmice 37ndash45 g in body weight) (b) Telomere length (andashc) represent a significantdifference of these columns (119875 lt 005)

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to Grants from the NationalNatural Science Foundation of China (no 30973818) and

the Science Foundation of Ministry of Education of China(no 20125503110006)

References

[1] S J Morrison A M Wandycz K Akashi A Globerson andI L Weissman ldquoThe aging of hematopoietic stem cellsrdquoNatureMedicine vol 2 no 9 pp 1011ndash1016 1996


[2] K Sudo H Ema Y Morita and H Nakauchi ldquoAge-associatedcharacteristics of murine hematopoietic stem cellsrdquo Journal ofExperimental Medicine vol 192 no 9 pp 1273ndash1280 2000

[3] D J Rossi D Bryder J M Zahn et al ldquoCell intrinsic alterationsunderlie hematopoietic stem cell agingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 26 pp 9194ndash9199 2005

[4] S M Chambers C A Shaw C Gatza C J Fisk L ADonehower and M A Goodell ldquoAging hematopoietic stemcells decline in function and exhibit epigenetic dysregulationrdquoPLoS Biology vol 5 article e201 2007

[5] Y Zhou B Yang X Yao and Y Wang ldquoEstablishment ofan aging model of Sca-1+ hematopoietic stem cell and studieson its relative biological mechanismsrdquo In Vitro Cellular andDevelopmental BiologymdashAnimal vol 47 no 2 pp 149ndash156 2011

[6] E Lagasse H Connors M Al-Dhalimy et al ldquoPurifiedhematopoietic stem cells can differentiate into hepatocytes invivordquo Nature Medicine vol 6 no 11 pp 1229ndash1234 2000

[7] I Bellantuono ldquoHaemopoietic stem cellsrdquo International Journalof Biochemistry and Cell Biology vol 36 no 4 pp 607ndash6202004

[8] C Holmes and W L Stanford ldquoConcise review stem cellantigen-1 Expression function and enigmardquo StemCells vol 25no 6 pp 1339ndash1347 2007

[9] A C Berardi A Wang J D Levine P Lopez and D TScadden ldquoFunctional isolation and characterization of humanhematopoietic stem cellsrdquo Science vol 267 no 5195 pp 104ndash108 1995

[10] G J Spangrude S Heimfeld and I L Weissman ldquoPurificationand characterization of mouse hematopoietic stem cellsrdquo Sci-ence vol 241 no 4861 pp 58ndash62 1988

[11] A JWagers ldquoThe stem cell niche in regenerativemedicinerdquoCellStem Cell vol 10 no 4 pp 362ndash369 2012

[12] X Y Fang Medical Laboratory Zoology Peoplersquos Medical Pub-lishing House 1st edition 1995

[13] H R Warner ldquoSuperoxide dismutase aging and degenerativediseaserdquo Free Radical Biology and Medicine vol 17 no 3 pp249ndash258 1994

[14] Y Lu K-H Zhao L Li R Wang and L Zhang ldquoLipidperoxidation and antioxidation in lens of aging ratsrdquo RecentAdvances in Ophthalmology vol 28 no 4 pp 241ndash244 2008

[15] W Wagner S Bork P Horn et al ldquoAging and replicativesenescence have related effects on human stem and progenitorcellsrdquo PLoS ONE vol 4 no 6 Article ID e5846 2009


Table 1 The unique PCR primers

Primers Forward (51015840-31015840) Reverse (51015840-31015840) Length (bp)p16INK4a TCCGCTGCAGACAGACTGGCCAG CATCGCGCACATCCAGCCGAGC 295p19Arf AAGAAGTCTGCGTCGGCGAC AGTACCGGAGGCATCTTGGACA 215p53 CACGTACTCTCCTCCCCTCAA GGCTCATAAGGTACCACCACG 294p21Cip1Waf1 ATTCCTGGTGATGTCCGACC AAAGTTCCACCGTTCTCGG 144GAPDH GTGCTGAGTATGTCGTGGAGTCT GAGTGGGAGTTGCTGTTGAAGT 602

and CFU-Mix potency were also reduced in Sca-1+ HSPCsfrom aged mice indicating an aging-related impairment [79 10]

This studywas intended to explore the dynamic biologicalchanges of HSCs aging characteristics Sca-1+ HSPCs wereseparated and purified from mice at different ages by themagnetic absorptive cell sorting (MACS)We investigated theroles of the superoxide damage and antioxidant capacity inthe natural aging process of HSCs providing experimentalevidence and theoretical basis in HSPCs aging mechanismand antiaging drug development

2 Materials and Methods

21 Ethics Statement and Animal Treatment The maleC57BL6J mice were purchased from the Medical and Lab-oratory Animal Center of Chongqing (Animal certificatenumber SCXK(yu)2007-0001) and housed in a temperature-and light-controlled room with free access to water andfood All experiments were performed in accordance withinstitutional and national guidelines and regulations andwere approved by the ChongqingMedical University AnimalCare and Use Committee All surgery was performed undersodium pentobarbital anesthesia and all the efforts weremade to minimize suffering

Animals were divided into 5 groups Group A (3-4 weeksold 13ndash16 g in body weight) Group B (8-9 weeks old 20ndash25 gin body weight) Group C (6 months old 35ndash40 g in bodyweight) Group D (12 months old 39ndash43 g in body weight)Group E (18 months old 37ndash45 g in body weight)

22 Isolation and Purification of Sca-1+ HSPCs in Mouse BoneMarrow Bone marrow was collected from the femurs andtibiasMononuclear cells were obtained fromBMaspirates bydensity gradient separation and then subjected to immuno-magnetic enrichment of Sca-1+ HSPCs (Miltenyi BiotecBergisch Gladbach Germany) as described previously [5ndash7]

23 Flow Cytometric Analysis All flow cytometric analyseswere performed using FACSCalibur (BD Biosciences Hei-delberg Germany) and data were analyzed with FCS ExpressV3 software (De Novo Software Los Angeles CA USA)

24 Detection of Oxidation-Associated Biomarkers After theisolation Sca-1+ HSPCs were collected and washed by PBSGSH content superoxide content SOD activity and MDAcontent were detected by chemical colorimetric analysis

according to themanufacturerrsquos instructions (Beyotime Insti-tute of Biotechnology Shanghai China)

25 Cell-Cycle Analysis of Sca-1+ HSPCs Cell-cycle analysiswas performed with the FITC BrdU Flow Kit (BD Bios-ciences Heidelberg Germany) according to the manufac-turerrsquos instructions Flow cytometric analyses were per-formed using FACSCalibur (BD Biosciences HeidelbergGermany) and Cell-cycle data were analyzed with Multicyclesoftware (PHENIX Japan)

26 The CFU-Mix Formation In Vitro Initially 4 times 104 Sca-1+ HSPCs were cultivated in 24-well plates (Costar Corn-ing Incorporated Corning NY USA) with the clonogenicmethylcellulose medium H3434 (Stem Cell TechnologiesVancouver Canada) and cells were cultured in a humidifiedatmosphere with 5 CO

2at 37∘C for 2 weeks Finally the

numbers of CFU-Mix formations were counted under a lightmicroscope

27 Senescence-Associated 120573-Galactosidase CytochemicalStaining The senescence-associated 120573-galactosidase (SA-120573-gal) activity was determined in Sca-1+ HSPCs using theCellular Senescence Detection Kit (Biolabs San Diego CAUSA) according to the manufacturer In brief 1 times 105 Sca-1+HSPCs were stained by staining solution for 24 hours at 37∘Cwithout CO

2in 6-well plates Cells were counted and the frac-

tion of senescent cells (positive for SA-120573-gal) was assessed

28 RNA Extraction and RT-PCR 6 times 106 Sca-1+ HSPCsof each group were collected Total mRNA was extractedaccording to the manufacturerrsquos protocol using TRIZOLReagent (TaKaRA Japan) OD260OD280 of RNA 18sim20First-strand cDNAwas created byAMV reverse transcriptionKit (Invitrogen USA) The unique PCR primers were shownin Table 1 DNA was amplified by an initial incubation at94∘C for 5min followed by 40 cycles of 94∘C denaturationfor 15 sec annealing at 60∘C for 60 sec and 72∘C extensionfor 1min PCR product was subjected to 1 agarose gelelectrophoresis Optical intensities were quantified usingQuantity One (Bio-Rad) The relative expression levels werecalculated as the ratio of the optical intensity of targeted geneto that of the internal reference

29 Western Blotting Analysis To examine the expression ofP53 P19 P16INK4a P21Cip1Waf1 cyclinD1 CDK4 CDK2 and






3 Results




2= 08819


2=


2= 09464 119875 lt


2= 09552

119875 lt 001) (Figure 2)









150

100

50

0

The r

atio

of S

ca-1+

HSP

Cs (

)


(a)

Cou

nts

100 101 102 103 104

FITC

(A) (B)

100 101 102 103 104

FITC

Ctrl001

M1

M2

B+001150

120

90

60

30

0

Cou

nts

M1

M2

150

120

90

60

30

0


MarkerAllM1M2

Gated ()100009864141

Mean3573371738


MarkerAllM1M2

Gated ()100006629340

Mean1069854311416


(b)





4 Discussion




a

Group D

b

c c

d

50

40

30

20

10

0

The t

otal

SO

D o

f Sca

-1+

HSP

Cs (U

g)

(a)

a


b

c

abab

60

40

20

0Tota

l GSH

cont

ent o

f Sca

-1+

HSP

Cs (120583

M)

(b)

a


b

c

ab

bc

020

015

010

005

000

Supe

roxi

de o

f Sca

-1+

HSP

Cs (O

D)

(c)


ab

b

c

d

15

10

5

0MD

A co

nten

t of S

ca-1+

HSP

Cs (n

mol

mg)

(d)




2O2) and oxygen (O






a

b

c

d

bc


40

30

20

10

0SA-120573

-gal

pos

itive

cells

in S

ca-1+

HSP

Cs (

)

(a)

Group A

Group B Group C

Group EGroup D

(b)



hi

g g

g

gk

ccdb

e e ef

a


G0G1G2M

S

100

80

60

40

20

0

Dist

ribut

ion

of ce

ll cy

cle o

f Sca

-1+

HSP

Cs (

)


aa

b

cc

15

10

5


(10

4Sc

a-1+

HSP

Cs)

Num

ber o

f CFU

-Mix

form

atio

ns








M A B C D E

M A B C D E

750bp500bp

250bp

750bp500bp

250bp

750bp

500bp

250bp

100 bp

750bp500bp

250bp100 bp

GAPDH

GAPDH

GAPDH

GAPDH

p53

p16INK4a

p19Arf

p21Cip1Waf1

(a)


Group EGroup D

h

I I

g g

c

d d

b

e e e

f ff

ab b b

a

Ratio

of m

RNA

to G

APD

H

p16IN

K4a

GA

PDH

p19A

rfG

APD

H

p53

GA

PDH

p21Ci

p1W

af1

GA

PDH

10

08

06

04

02

00

(b)


CyclinD1

p19

CDK2

CyclinE1

CDK4

120573-actin

p53

p16INK4a

p21Cip1Waf1

A B C D E

43kDa

19kDa

36kDa

21kDa

53kDa

16kDa

34kDa

33kDa

50kDa

(a)

bbb

bb bb

b

c cc c c c

c c c

ab ab abab

cdcdcd

cdcd cd

dd

d dd

d d

aaaa

a a

p53

p16IN

K4a

p19

p21Ci

p1W

af1

Cycli

nD1

CDK2

Cycli

nE

CDK4


Group EGroup D

15

10

05

00Ratio

of p

rote

in to

120573-a

ctin

(b)



M A B C D E

330bp


50bp

40bp

30bp

(a)


b

bc

abaa

20

15

10

05

00

The r

atio

of t

elom

eras

e act

ivity

(b)


A B C D E

50kb

38kb

23kb

10kb

(a)


bc

ab aba

30

20

10

0

TRF

leng

th o

f Sca

-1+

HSP

Cs (k

bp)

(b)




Acknowledgments



References






















3 Results




2= 08819


2=


2= 09464 119875 lt


2= 09552

119875 lt 001) (Figure 2)









150

100

50

0

The r

atio

of S

ca-1+

HSP

Cs (

)


(a)

Cou

nts

100 101 102 103 104

FITC

(A) (B)

100 101 102 103 104

FITC

Ctrl001

M1

M2

B+001150

120

90

60

30

0

Cou

nts

M1

M2

150

120

90

60

30

0


MarkerAllM1M2

Gated ()100009864141

Mean3573371738


MarkerAllM1M2

Gated ()100006629340

Mean1069854311416


(b)





4 Discussion




a

Group D

b

c c

d

50

40

30

20

10

0

The t

otal

SO

D o

f Sca

-1+

HSP

Cs (U

g)

(a)

a


b

c

abab

60

40

20

0Tota

l GSH

cont

ent o

f Sca

-1+

HSP

Cs (120583

M)

(b)

a


b

c

ab

bc

020

015

010

005

000

Supe

roxi

de o

f Sca

-1+

HSP

Cs (O

D)

(c)


ab

b

c

d

15

10

5

0MD

A co

nten

t of S

ca-1+

HSP

Cs (n

mol

mg)

(d)




2O2) and oxygen (O






a

b

c

d

bc


40

30

20

10

0SA-120573

-gal

pos

itive

cells

in S

ca-1+

HSP

Cs (

)

(a)

Group A

Group B Group C

Group EGroup D

(b)



hi

g g

g

gk

ccdb

e e ef

a


G0G1G2M

S

100

80

60

40

20

0

Dist

ribut

ion

of ce

ll cy

cle o

f Sca

-1+

HSP

Cs (

)


aa

b

cc

15

10

5


(10

4Sc

a-1+

HSP

Cs)

Num

ber o

f CFU

-Mix

form

atio

ns








M A B C D E

M A B C D E

750bp500bp

250bp

750bp500bp

250bp

750bp

500bp

250bp

100 bp

750bp500bp

250bp100 bp

GAPDH

GAPDH

GAPDH

GAPDH

p53

p16INK4a

p19Arf

p21Cip1Waf1

(a)


Group EGroup D

h

I I

g g

c

d d

b

e e e

f ff

ab b b

a

Ratio

of m

RNA

to G

APD

H

p16IN

K4a

GA

PDH

p19A

rfG

APD

H

p53

GA

PDH

p21Ci

p1W

af1

GA

PDH

10

08

06

04

02

00

(b)


CyclinD1

p19

CDK2

CyclinE1

CDK4

120573-actin

p53

p16INK4a

p21Cip1Waf1

A B C D E

43kDa

19kDa

36kDa

21kDa

53kDa

16kDa

34kDa

33kDa

50kDa

(a)

bbb

bb bb

b

c cc c c c

c c c

ab ab abab

cdcdcd

cdcd cd

dd

d dd

d d

aaaa

a a

p53

p16IN

K4a

p19

p21Ci

p1W

af1

Cycli

nD1

CDK2

Cycli

nE

CDK4


Group EGroup D

15

10

05

00Ratio

of p

rote

in to

120573-a

ctin

(b)



M A B C D E

330bp


50bp

40bp

30bp

(a)


b

bc

abaa

20

15

10

05

00

The r

atio

of t

elom

eras

e act

ivity

(b)


A B C D E

50kb

38kb

23kb

10kb

(a)


bc

ab aba

30

20

10

0

TRF

leng

th o

f Sca

-1+

HSP

Cs (k

bp)

(b)




Acknowledgments



References




















150

100

50

0

The r

atio

of S

ca-1+

HSP

Cs (

)


(a)

Cou

nts

100 101 102 103 104

FITC

(A) (B)

100 101 102 103 104

FITC

Ctrl001

M1

M2

B+001150

120

90

60

30

0

Cou

nts

M1

M2

150

120

90

60

30

0


MarkerAllM1M2

Gated ()100009864141

Mean3573371738


MarkerAllM1M2

Gated ()100006629340

Mean1069854311416


(b)





4 Discussion




a

Group D

b

c c

d

50

40

30

20

10

0

The t

otal

SO

D o

f Sca

-1+

HSP

Cs (U

g)

(a)

a


b

c

abab

60

40

20

0Tota

l GSH

cont

ent o

f Sca

-1+

HSP

Cs (120583

M)

(b)

a


b

c

ab

bc

020

015

010

005

000

Supe

roxi

de o

f Sca

-1+

HSP

Cs (O

D)

(c)


ab

b

c

d

15

10

5

0MD

A co

nten

t of S

ca-1+

HSP

Cs (n

mol

mg)

(d)




2O2) and oxygen (O






a

b

c

d

bc


40

30

20

10

0SA-120573

-gal

pos

itive

cells

in S

ca-1+

HSP

Cs (

)

(a)

Group A

Group B Group C

Group EGroup D

(b)



hi

g g

g

gk

ccdb

e e ef

a


G0G1G2M

S

100

80

60

40

20

0

Dist

ribut

ion

of ce

ll cy

cle o

f Sca

-1+

HSP

Cs (

)


aa

b

cc

15

10

5


(10

4Sc

a-1+

HSP

Cs)

Num

ber o

f CFU

-Mix

form

atio

ns








M A B C D E

M A B C D E

750bp500bp

250bp

750bp500bp

250bp

750bp

500bp

250bp

100 bp

750bp500bp

250bp100 bp

GAPDH

GAPDH

GAPDH

GAPDH

p53

p16INK4a

p19Arf

p21Cip1Waf1

(a)


Group EGroup D

h

I I

g g

c

d d

b

e e e

f ff

ab b b

a

Ratio

of m

RNA

to G

APD

H

p16IN

K4a

GA

PDH

p19A

rfG

APD

H

p53

GA

PDH

p21Ci

p1W

af1

GA

PDH

10

08

06

04

02

00

(b)


CyclinD1

p19

CDK2

CyclinE1

CDK4

120573-actin

p53

p16INK4a

p21Cip1Waf1

A B C D E

43kDa

19kDa

36kDa

21kDa

53kDa

16kDa

34kDa

33kDa

50kDa

(a)

bbb

bb bb

b

c cc c c c

c c c

ab ab abab

cdcdcd

cdcd cd

dd

d dd

d d

aaaa

a a

p53

p16IN

K4a

p19

p21Ci

p1W

af1

Cycli

nD1

CDK2

Cycli

nE

CDK4


Group EGroup D

15

10

05

00Ratio

of p

rote

in to

120573-a

ctin

(b)



M A B C D E

330bp


50bp

40bp

30bp

(a)


b

bc

abaa

20

15

10

05

00

The r

atio

of t

elom

eras

e act

ivity

(b)


A B C D E

50kb

38kb

23kb

10kb

(a)


bc

ab aba

30

20

10

0

TRF

leng

th o

f Sca

-1+

HSP

Cs (k

bp)

(b)




Acknowledgments



References



















a

Group D

b

c c

d

50

40

30

20

10

0

The t

otal

SO

D o

f Sca

-1+

HSP

Cs (U

g)

(a)

a


b

c

abab

60

40

20

0Tota

l GSH

cont

ent o

f Sca

-1+

HSP

Cs (120583

M)

(b)

a


b

c

ab

bc

020

015

010

005

000

Supe

roxi

de o

f Sca

-1+

HSP

Cs (O

D)

(c)


ab

b

c

d

15

10

5

0MD

A co

nten

t of S

ca-1+

HSP

Cs (n

mol

mg)

(d)




2O2) and oxygen (O






a

b

c

d

bc


40

30

20

10

0SA-120573

-gal

pos

itive

cells

in S

ca-1+

HSP

Cs (

)

(a)

Group A

Group B Group C

Group EGroup D

(b)



hi

g g

g

gk

ccdb

e e ef

a


G0G1G2M

S

100

80

60

40

20

0

Dist

ribut

ion

of ce

ll cy

cle o

f Sca

-1+

HSP

Cs (

)


aa

b

cc

15

10

5


(10

4Sc

a-1+

HSP

Cs)

Num

ber o

f CFU

-Mix

form

atio

ns








M A B C D E

M A B C D E

750bp500bp

250bp

750bp500bp

250bp

750bp

500bp

250bp

100 bp

750bp500bp

250bp100 bp

GAPDH

GAPDH

GAPDH

GAPDH

p53

p16INK4a

p19Arf

p21Cip1Waf1

(a)


Group EGroup D

h

I I

g g

c

d d

b

e e e

f ff

ab b b

a

Ratio

of m

RNA

to G

APD

H

p16IN

K4a

GA

PDH

p19A

rfG

APD

H

p53

GA

PDH

p21Ci

p1W

af1

GA

PDH

10

08

06

04

02

00

(b)


CyclinD1

p19

CDK2

CyclinE1

CDK4

120573-actin

p53

p16INK4a

p21Cip1Waf1

A B C D E

43kDa

19kDa

36kDa

21kDa

53kDa

16kDa

34kDa

33kDa

50kDa

(a)

bbb

bb bb

b

c cc c c c

c c c

ab ab abab

cdcdcd

cdcd cd

dd

d dd

d d

aaaa

a a

p53

p16IN

K4a

p19

p21Ci

p1W

af1

Cycli

nD1

CDK2

Cycli

nE

CDK4


Group EGroup D

15

10

05

00Ratio

of p

rote

in to

120573-a

ctin

(b)



M A B C D E

330bp


50bp

40bp

30bp

(a)


b

bc

abaa

20

15

10

05

00

The r

atio

of t

elom

eras

e act

ivity

(b)


A B C D E

50kb

38kb

23kb

10kb

(a)


bc

ab aba

30

20

10

0

TRF

leng

th o

f Sca

-1+

HSP

Cs (k

bp)

(b)




Acknowledgments



References


















a

b

c

d

bc


40

30

20

10

0SA-120573

-gal

pos

itive

cells

in S

ca-1+

HSP

Cs (

)

(a)

Group A

Group B Group C

Group EGroup D

(b)



hi

g g

g

gk

ccdb

e e ef

a


G0G1G2M

S

100

80

60

40

20

0

Dist

ribut

ion

of ce

ll cy

cle o

f Sca

-1+

HSP

Cs (

)


aa

b

cc

15

10

5


(10

4Sc

a-1+

HSP

Cs)

Num

ber o

f CFU

-Mix

form

atio

ns








M A B C D E

M A B C D E

750bp500bp

250bp

750bp500bp

250bp

750bp

500bp

250bp

100 bp

750bp500bp

250bp100 bp

GAPDH

GAPDH

GAPDH

GAPDH

p53

p16INK4a

p19Arf

p21Cip1Waf1

(a)


Group EGroup D

h

I I

g g

c

d d

b

e e e

f ff

ab b b

a

Ratio

of m

RNA

to G

APD

H

p16IN

K4a

GA

PDH

p19A

rfG

APD

H

p53

GA

PDH

p21Ci

p1W

af1

GA

PDH

10

08

06

04

02

00

(b)


CyclinD1

p19

CDK2

CyclinE1

CDK4

120573-actin

p53

p16INK4a

p21Cip1Waf1

A B C D E

43kDa

19kDa

36kDa

21kDa

53kDa

16kDa

34kDa

33kDa

50kDa

(a)

bbb

bb bb

b

c cc c c c

c c c

ab ab abab

cdcdcd

cdcd cd

dd

d dd

d d

aaaa

a a

p53

p16IN

K4a

p19

p21Ci

p1W

af1

Cycli

nD1

CDK2

Cycli

nE

CDK4


Group EGroup D

15

10

05

00Ratio

of p

rote

in to

120573-a

ctin

(b)



M A B C D E

330bp


50bp

40bp

30bp

(a)


b

bc

abaa

20

15

10

05

00

The r

atio

of t

elom

eras

e act

ivity

(b)


A B C D E

50kb

38kb

23kb

10kb

(a)


bc

ab aba

30

20

10

0

TRF

leng

th o

f Sca

-1+

HSP

Cs (k

bp)

(b)




Acknowledgments



References


















hi

g g

g

gk

ccdb

e e ef

a


G0G1G2M

S

100

80

60

40

20

0

Dist

ribut

ion

of ce

ll cy

cle o

f Sca

-1+

HSP

Cs (

)


aa

b

cc

15

10

5


(10

4Sc

a-1+

HSP

Cs)

Num

ber o

f CFU

-Mix

form

atio

ns








M A B C D E

M A B C D E

750bp500bp

250bp

750bp500bp

250bp

750bp

500bp

250bp

100 bp

750bp500bp

250bp100 bp

GAPDH

GAPDH

GAPDH

GAPDH

p53

p16INK4a

p19Arf

p21Cip1Waf1

(a)


Group EGroup D

h

I I

g g

c

d d

b

e e e

f ff

ab b b

a

Ratio

of m

RNA

to G

APD

H

p16IN

K4a

GA

PDH

p19A

rfG

APD

H

p53

GA

PDH

p21Ci

p1W

af1

GA

PDH

10

08

06

04

02

00

(b)


CyclinD1

p19

CDK2

CyclinE1

CDK4

120573-actin

p53

p16INK4a

p21Cip1Waf1

A B C D E

43kDa

19kDa

36kDa

21kDa

53kDa

16kDa

34kDa

33kDa

50kDa

(a)

bbb

bb bb

b

c cc c c c

c c c

ab ab abab

cdcdcd

cdcd cd

dd

d dd

d d

aaaa

a a

p53

p16IN

K4a

p19

p21Ci

p1W

af1

Cycli

nD1

CDK2

Cycli

nE

CDK4


Group EGroup D

15

10

05

00Ratio

of p

rote

in to

120573-a

ctin

(b)



M A B C D E

330bp


50bp

40bp

30bp

(a)


b

bc

abaa

20

15

10

05

00

The r

atio

of t

elom

eras

e act

ivity

(b)


A B C D E

50kb

38kb

23kb

10kb

(a)


bc

ab aba

30

20

10

0

TRF

leng

th o

f Sca

-1+

HSP

Cs (k

bp)

(b)




Acknowledgments



References


















M A B C D E

M A B C D E

750bp500bp

250bp

750bp500bp

250bp

750bp

500bp

250bp

100 bp

750bp500bp

250bp100 bp

GAPDH

GAPDH

GAPDH

GAPDH

p53

p16INK4a

p19Arf

p21Cip1Waf1

(a)


Group EGroup D

h

I I

g g

c

d d

b

e e e

f ff

ab b b

a

Ratio

of m

RNA

to G

APD

H

p16IN

K4a

GA

PDH

p19A

rfG

APD

H

p53

GA

PDH

p21Ci

p1W

af1

GA

PDH

10

08

06

04

02

00

(b)


CyclinD1

p19

CDK2

CyclinE1

CDK4

120573-actin

p53

p16INK4a

p21Cip1Waf1

A B C D E

43kDa

19kDa

36kDa

21kDa

53kDa

16kDa

34kDa

33kDa

50kDa

(a)

bbb

bb bb

b

c cc c c c

c c c

ab ab abab

cdcdcd

cdcd cd

dd

d dd

d d

aaaa

a a

p53

p16IN

K4a

p19

p21Ci

p1W

af1

Cycli

nD1

CDK2

Cycli

nE

CDK4


Group EGroup D

15

10

05

00Ratio

of p

rote

in to

120573-a

ctin

(b)



M A B C D E

330bp


50bp

40bp

30bp

(a)


b

bc

abaa

20

15

10

05

00

The r

atio

of t

elom

eras

e act

ivity

(b)


A B C D E

50kb

38kb

23kb

10kb

(a)


bc

ab aba

30

20

10

0

TRF

leng

th o

f Sca

-1+

HSP

Cs (k

bp)

(b)




Acknowledgments



References


















M A B C D E

330bp


50bp

40bp

30bp

(a)


b

bc

abaa

20

15

10

05

00

The r

atio

of t

elom

eras

e act

ivity

(b)


A B C D E

50kb

38kb

23kb

10kb

(a)


bc

ab aba

30

20

10

0

TRF

leng

th o

f Sca

-1+

HSP

Cs (k

bp)

(b)




Acknowledgments



References

















Study on the Dynamic Biological Characteristics of Sca-1 ...€¦ · 2 StemCellsInternational...

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Transcript of Study on the Dynamic Biological Characteristics of Sca-1 ...€¦ · 2 StemCellsInternational...