Functionality of Human DD Mesenchymal Stem...
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Departme nt of Biotech nology a nd Chemical Tech nology, Aalto U niversity F unct io nal it y o f H uman Me se nch ymal Stem Cell s Asse sse d by Ene rgy Met abol ism and Immunomo dul at ive Capac it y Pietil ä Mika DOCTORAL DISSERTATIONS
Transcript of Functionality of Human DD Mesenchymal Stem...
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ISBN 978-952-60-4889-5 ISBN 978-952-60-4890-1 (pdf) ISSN-L 1799-4934 ISSN 1799-4934 ISSN 1799-4942 (pdf) Aalto University School of Chemical Technology Department of Biotechnology and Chemical Technology, Aalto University
BUSINESS + ECONOMY ART + DESIGN + ARCHITECTURE SCIENCE + TECHNOLOGY CROSSOVER DOCTORAL DISSERTATIONS
Aalto-D
D 161
/2012
Pietilä M
ika F
unctionality of Hum
an Mesenchym
al Stem C
ells Assessed by E
nergy Metabolism
and Imm
unomodulative
Capacity
Aalto
Unive
rsity
Department of Biotechnology and Chemical Technology, Aalto University
Functionality of Human Mesenchymal Stem Cells Assessed by Energy Metabolism and Immunomodulative Capacity
Pietilä Mika
DOCTORAL DISSERTATIONS
Aalto University publication series DOCTORAL DISSERTATIONS 161/2012
Functionality of Human Mesenchymal Stem Cells Assessed by Energy Metabolism and Immunomodulative Capacity
Pietilä Mika
Doctoral dissertation for the degree of Doctor of Philosophy to be presented with due permission of the School of Chemical Technology for public examination and debate in Auditorium Puu2 (Forest Products Building 2) at the Aalto University School of Chemical Technology (Espoo, Finland) on the 5th of December, 2012, at 12 noon.
Aalto University School of Chemical Technology Department of Biotechnology and Chemical Technology Microbiology Group
Supervising professors Prof. Katrina Nordström, Aalto University, Finland Prof. Petri Lehenkari, University of Oulu, Finland Preliminary examiners Prof. Outi Hovatta, Karolinska Institutet, Sweden Prof. Timo Otonkoski, University of Helsinki, Finland Opponent Docent Susanna Miettinen, University of Tampere, Finland
Aalto University publication series DOCTORAL DISSERTATIONS 161/2012 © Mika Pietilä ISBN 978-952-60-4889-5 (printed) ISBN 978-952-60-4890-1 (pdf) ISSN-L 1799-4934 ISSN 1799-4934 (printed) ISSN 1799-4942 (pdf) http://urn.fi/URN:ISBN:978-952-60-4890-1 Unigrafia Oy Helsinki 2012 Finland
Abstract Aalto University, P.O. Box 11000, FI-00076 Aalto www.aalto.fi
Author Pietilä Mika Name of the doctoral dissertation Functionality of Human Mesenchymal Stem Cells Assessed by Energy Metabolism and Immunomodulative Capacity Publisher School of Chemical Technology Unit Department of Biotechnology and Chemical Technology
Series Aalto University publication series DOCTORAL DISSERTATIONS 161/2012
Field of research Adult Stem Cell Biology
Manuscript submitted 17 August 2012 Date of the defence 5 December 2012
Permission to publish granted (date) 30 November 2012 Language English
Monograph Article dissertation (summary + original articles)
Abstract Human mesenchymal stem cells (hMSCs) are a heterogenous population of multipotent adult
stem cells that can be used to treat many degenerative and immunological diseases. Currently there is a lack of a specific definition for hMSCs. This is a major problem as
standardization of hMSC products is therefore currently impossible. Accordingly, the current thesis focuses on detailed mitochondrial and ultrastructural characterization of hMSC from different origin. The results show that the increase in mitochondrial mass, mitochondrial reactive oxygen species and activation of the antioxidant defense system occurs during early osteogenic differentiation. Therefore these parameters could be utilized in parallel with current immunophenotype characterization to detect and isolate hMSCs with different metabolical status, which may reflect distinct developmental stages of hMSCs.
hMSCs should posses high viability and functionality to mediate the healing effect to the site of injury or inflammation. Therefore sensitive and especially rapid quality control tools are urgently needed. The present thesis demonstrates that monitoring the mitochondrial energy state could be utilized as a viability and potency measure for hMSCs. Specifically, sensitivity of the JC-1 dye for the mitochondrial inner membrane potential to detect oxidative stress and microbial infection induced apoptosis was clearly superior when compared to traditional methods. Moreover, the mitochondrial inner membrane potential correlated with the osteogenic differentiation potency of hMSCs and could be utilized as a potency assay for hMSCs intended for cellular therapies.
The immunosuppressive activity of hMSCs has been demonstrated both in vivo and in vitro. In the present study, a macrophage model was established to study the role of the CD200-CD200r axis in hMSCs mediated immunosuppression of macrophages. hMSC lines with higher positivity for CD200 showed more efficient inhibition of TNF-alpha secretion from THP-1 macrophages when compared to hMSC lines with low CD200 positivity. Moreover, the role of CD200-CD200r axis in hMSC mediated immunosuppression was confirmed by blocking CD200 from the cell surface of hMSCs.
The results from the present thesis suggest that mitochondrial analysis could be used to manufacture pure and viable hMSC products, essential parameters for highly functional hMSCs. These studies also show that hMSC could be used to ameliorate inflammatorial conditions by suppressing the pro-inflammatorial acitivity of macrophages via the CD200-CD200r axis.
Keywords Human mesenchymal stem cells, cellular therapies, mitochondria, immunosuppression, macrophages
ISBN (printed) 978-952-60-4889-5 ISBN (pdf) 978-952-60-4890-1
ISSN-L 1799-4934 ISSN (printed) 1799-4934 ISSN (pdf) 1799-4942
Location of publisher Espoo Location of printing Helsinki Year 2012
Pages 126 urn http://urn.fi/URN:ISBN:978-952-60-4890-1
Tiivistelmä Aalto-yliopisto, PL 11000, 00076 Aalto www.aalto.fi
Tekijä Pietilä Mika Väitöskirjan nimi Ihmisen Mesenkymaalisten Kantasolujen Funktionaalisuus: Energia-aineenvaihdunnan ja Immunomodulatiivisen Toiminnan Määrittäminen Julkaisija Kemian tekniikan korkeakoulu Yksikkö Bioteknologian ja kemian teknologian laitos
Sarja Aalto University publication series DOCTORAL DISSERTATIONS 161/2012
Tutkimusala Aikuisen ihmisen kantasolujen biologia
Käsikirjoituksen pvm 17.08.2012 Väitöspäivä 05.12.2012
Julkaisuluvan myöntämispäivä 30.11.2012 Kieli Englanti
Monografia Yhdistelmäväitöskirja (yhteenveto-osa + erillisartikkelit)
Tiivistelmä Ihmisen mesenkymaaliset kantasolut (MK-solut) edustavat heterogeenistä solujoukkoa, joilla
on multipotentiaalinen erilaistumiskyky. MK-solujen on osoitettu vähentävän tulehdusreaktiota vakavan vaurion yhteydessä tai autoimmuunisairauksissa ja siten parantavan erilaisia rappeumatauteja sekä immunologisia sairauksia.
Tällä hetkellä ei ole olemassa selkeitä ohjeistuksia MK-solujen tunnistamiseen ja eristämiseen mikä aiheuttaa ongelmia soluviljelmien laadun standardoimisessa. Tämän työn yhtenä tarkoituksena oli tutkia MK-solujen aineenvaihduntaa ja mitokondrioiden ominaisuuksia tarkoituksena löytää vaihtoehtoisia mitattavia parametrejä MK-solujen tunnistamiseen. Eri-ikäisiltä potilailta ja eri kudoksista eristetyt MK-solut oli mahdollista erotella mitokondriaalisten ominaisuuksien perusteella. Lisäksi mitokondrioiden määrä, mitokondriaalinen superoksidipitoisuus sekä antioksidatiivnen aktiivisuus nousivat varhaisessa luu erilaistumis vaiheessa. Näin ollen mitokondrion toiminnan analysointi tarjoaa hyödyllisiä ominaisuuksia entistä tarkempaan MK-solujen karakterisointiin.
Toimiakseen tehokkaasti kliinisissä sovelluksissa, MK-solujen tulee olla elinkykyisiä. Tässä työssä osoitetaan, että mitokondrion kalvopotentiaali on herkkä solujen kunnon mittari, joka soveltui hyvin myös mikrobikontaminaation aiheuttaman solutuhon detektoimiseen jo erittäin varhaisessa vaiheessa. Mittaamalla mitokondrion kalvopotentiaalia on mahdollista myös ennustaa MK-solujen luu erilaistumis tehokkuutta. Näin ollen kyseinen menetelmä soveltuu erittäin hyvin MK-solujen laadunvalvontaan.
Ihmisen mesenkymaalisilla kantasoluilla on osoitettu olevan tulehdusta lieventävä vaikutus niin in vitro laboratoriossa kuin myös in vivo kliinisissä kokeissa. Liittyen MK-solujen toiminnallisuuteen tämän työn toisena päätavoitteena oli siten selvittää MK-solujen kykyä vaikuttaa makrofaagien tulehdusaktiivisuuteen. Tässä työssä on ensimäistä kertaa osoitettu CD200 ligandin ja CD200 reseptorin rooli MK-solujen välitteisessä makrofaagien tulehdusaktiivisuuden vähentämisessä. CD200 positiiviset MK-solut vähensivät TNF-alfa eritystä makrofaageista tehokkaammin kuin CD200 negatiiviset MK-solut.
Tämän työn tulokset osoittavat, että mitokondrioiden analysoiminen tarjoaa uusia entistä tarkempia parametrejä, joilla mitata MK-solujen kuntoa ja toimintaa sekä parantaa solutuotteiden puhtautta. MK-soluja voidaan hyödyntää soluterapioissa esimerkiksi tulhedussairauksien hoidoissa, koska MK-solut kykenevät vähentämään makrofaagien tulehdusaktiivisuutta CD200-CD200r välitteisen mekanismin avulla.
Avainsanat Ihmisen mesenkymaaliset kantasolut, soluterapia, mitokondriot, immunosuppressio, makrofaagit
ISBN (painettu) 978-952-60-4889-5 ISBN (pdf) 978-952-60-4890-1
ISSN-L 1799-4934 ISSN (painettu) 1799-4934 ISSN (pdf) 1799-4942
Julkaisupaikka Espoo Painopaikka Espoo Vuosi 2012
Sivumäärä 126 urn http://urn.fi/URN:ISBN:978-952-60-4890-1
PREFACE
This study was carried out in collaboration between the Microbiology Group at Aalto
University and the Stem Cell Group at University of Oulu between 2008 and 2012. This
work was funded by the Academy of Finland (grant number 122456).
I am deeply grateful to my supervisors professor Petri Lehenkari and professor
Katrina Nordström who made this whole project possible. Petri has inspired me with his
infinite enthusiasm towards stem cells research. Katrina’s kindness and endless positivity
in all situations have impressed me and I have got all the support and help whatever I have
planned to do. Especially I want to thank you both that you have made it possible by your
connections to collaborate with many top researchers in Finland and also abroad. This has
given me excellent acquirements to continue research after this Ph.D. project.
I want to thank all the members of the Stem Cell Group in University of Oulu. Siri
Lehtonen, Hannu-Ville Leskelä, Maarit Kellinsalmi, Juha Pesälä, Sami Palomäki, Roni
Pernu, Timo Ruuska, Jonne Tikkanen, Antti Nätynki, Minna Savilampi, Henna Ek.
I also want to thank all the members of the Microbiology Group in Aalto University
but especially Marko Närhi for your contribution.
I have been privileged to have the opportunity to work with many top researchers
and I want to thank the co-authors from their valuable contributions: Emeritus Professor
Ilmo Hassinen, Professor Ari Vepsäläinen, Dr. Raija Sormunen and Dr. Maarit Lönnroth.
I want to thank all the people in Finnish Red Cross and Blood Service. Especially I
want to thank Kari Aranko, Saara Laitinen, Kaarina Lähteenmäki, Johanna Nystedt, Leena
Valmu, Ilja Ritola, Jaana Mättö and Elina Tuovinen for your valuable contributions.
I am fortunate to have good friends outside of the research field who have helped
me to carry through this project. Thanks for all the laughs and the quality time I have got
chance to spend with you. Special thanks for the members of Oulu Huippukahvi group.
I want to thank my parents Anneli and Risto, my brothers Aleksi and Jarno for
their endless support whatever I am doing. It has been priceless all of these years.
Most of all I want to thank my love Katja who has been supporting me all the time.
Your kindness, love, care and especially your humor have helped me a lot during this
project. You give me so much joy and love.
Tsukuba, Japan, May 2012 Mika Pietilä
CONTENTS
ABSTRACT
TIIVISTELMÄ
PREFACE
CONTENTS
LIST OF ORIGINAL PUBLICATIONS
AUTHOR’S CONTRIBUTIONS
ABBREVIATIONS AND SYMBOLS
1. ........................................................................................................ 15 INTRODUCTION2. .............................................................................. 17 REVIEW OF THE LITERATURE
2.1. ............................................................................................................. 17 Stem cells2.1.1. ...................................................... 17 Stem cells and their role in the body2.1.2. ................................................................. 17 Human pluripotent stem cells2.1.3. ............................................................. 18 Human mesenchymal stem cells
2.1.3.1. ................................................. 20 Differentiation potential of hMSCs2.1.3.2. ................................. 21 hMSCs can be isolated from different origin2.1.3.3. ............................................. 24 Cell surface characteristics of hMSCs
2.2. ................................................... 25 The mitochondrion as a regulator of stem cells2.2.1. .................................................................... 25 Mitochondrial bioenergetics2.2.2. .................................... 26 The mitochondrion as a regulator of apoptosis2.2.3. ... 28 Mitochondrial bioenergetics and human mesenchymal stem cells
2.3. ........................................................................ 29 Stem cell-based cellular therapies2.3.1. ............................................................................... 29 Mechanisms of action
2.3.1.1. ..................................................... 30 Regeneration of damaged tissue2.3.1.2. .................. 31 The role of immunosuppression in cellular therapies2.3.1.3. ........................................................ 33 Anti-apoptotic effect of hMSCs
2.3.2. .................................... 35 Current status of stem cell-based clinical trials2.4. ................................ 37 Current status of European legislation for cellular products
2.4.1. ..................................................................... 37 Overview of the regulations2.4.2. ......................................... 39 Quality controls and risk analysis of CBMPs2.4.3. .......................................................................................... 41 Microbial safety
3. ............................................................................ 42 AIMS OF THE PRESENT STUDY4. ................................................................................. 43 MATERIALS AND METHODS
4.1. ......................................................................................................... 43 Cell Cultures4.1.1. . 43 Human bone marrow-derived mesenchymal stem cells (I,II,III,IV)4.1.2. ............ 44 Umbilical cord blood-derived mesenchymal stem cells (I,IV)4.1.3. .. 44 Monocyte culture, differentiation and co-culture with hMSCs (IV)
4.2. .................... 45 Microbial cultivations and bacterial infection of hMSCs (III)4.3. ................................................ 46 Induction of osteogenic differentiation (I,II)
4.4. .......................................................................................46 Flow cytometry analysis4.4.1. .........46 Measurement of mitochondrial inner membrane potential (II)4.4.2. .......................47 Measurement of Annexin V and Propidium iodide (II)4.4.3. ..............................................47 The analysis of cell surface antigens (I,II)4.4.4. ...........................47 Measurement of mitochondrial superoxide levels (I)
4.5. ..................................................................48 Transmission electron microscopy (I)4.6. .................................................................................................48 Micro plate reader
4.6.1. ..............48 Measurement of specific alkaline phosphatase activity (I,II)4.6.2. ...................................................48 Measurement of calcium content (I,II)4.6.3. .......49 Measurement of mitochondrial inner membrane potential (III)4.6.4. .....................................................................49 MTT proliferation assay (II)4.6.5. ..........................50 Enzyme linked immuno sorbent assays (ELISA) (IV)
4.7. .....................................................................................................50 Western blot (I)4.8. ......................................................................................51 Trypan blue staining (III)4.9. ..........................................................................................51 Statistics (I, II, III, IV)
5. .....................................................................................................................52 RESULTS5.1.
...............................................................................52 hMSCs from different origin show identical immunophenotype characteristics
but differ by their ultrastructure (I,II)5.2.
....................................................................................53 Differences in mitochondrial properties between UCB-MSC and BM-MSC
before and after osteoinduction (I)5.3. ...................55 Monitoring of �� : a sensitive quality control tool for hMSCs (II)m
5.4........................................................................................................................56
Monitoring of �� : detection of microbial infection caused cellular stress of hMSCs (III)
m
5.5. ....57 Mesenchymal stem cells showed different expression pattern of CD200 (IV)5.6.
................................................................................................57 The secretion of TNF-� from THP-1 macrophages is inhibited by BM-MSCs in
the presence of IFN-� (IV)5.7.
.....................................................................................................................58 The role of CD200-CD200r axis in hMSC mediated immunosuppression of
macrophages6. ...............................................................................................................60 DISCUSSION
6.1..................................................60
Characterization of hMSCs in accordance with ISCT guideline showed no dramatic differences between UCB-MSC and BM-MSC6.2. ..........61 Mitochondrial properties as indicators of the osteoprogenitors of hMSCs6.3. ..........................................66 Monitoring �� as a quality control tool for hMSCsm6.4.
68 Early detection of microbial contamination induced cellular stress by monitoring
��m6.5. .....................69 CD200 positive BM-MSC as effective regulators of macrophages6.6.
.....................................................................................72 Mitochondrial analyses combined with the cell surface characterizations ensure
highly functional hMSC products7. ..........................................................................................................74 CONCLUSIONS
REFERENCES
ORIGINAL PUBLICATIONS
LIST OF ORIGINAL PUBLICATIONS
This dissertation consists of an overview of the following four publications, which are referred to in the text by their Roman numerals. I Pietilä M, Palomäki S, Lehtonen S, Ritamo I, Valmu L, Nystedt J, Laitinen S,
Leskelä HV, Sormunen R, Pesälä J, Nordström, K, Vepsäläinen A and Lehenkari, P. Mitochondrial function and energy metabolism in umbilical cord blood- and bone marrow-derived mesenchymal stem cells. Stem Cells Dev. 2012; 21(4): 578-588
II Pietilä M, Lehtonen S, Närhi M, Hassinen IE, Leskelä HV, Aranko K, Nordström K,
Vepsäläinen A and Lehenkari P. Mitochondrial function determines the viability and osteogenic potency of human mesenchymal stem cells. Tissue Eng Part C Methods. 2010; 16(3): 435-445
III Pietilä M, Lähteenmäki K, Lehtonen S, Leskelä HV, Närhi M, Lönnroth M, Mättö J,
Lehenkari P and Nordström K. Monitoring mitochondrial inner membrane potential for detecting early changes in viability of bacterium-infected human bone marrow derived mesenchymal stem cells. Submitted to Stem Cell Research & Therapy
IV Pietilä M, Lehtonen S, Tuovinen E, Lähteenmäki K, Laitinen S, Leskelä HV, Nätynki A, Pesälä J, Nordström K and Lehenkari, P. CD200 Positive Human Mesenchymal Stem Cells Suppress TNF-alpha Secretion from CD200 Receptor Positive Macrophage-Like Cells. PloS One. 2012; 7(2): e31671
AUTHOR’S CONTRIBUTIONS
I Mika Pietilä performed the experiments and analyzed the data. The collaborating authors contributed to planning of the study. Mika Pietilä wrote the article in collaboration with the other authors.
II Mika Pietilä was responsible for the planning of the study, he also designed and
performed the experiments and analyzed the data. Mika Pietilä wrote the article in collaboration with the other authors.
III Mika Pietilä was responsible for the planning of the study, he also designed and
performed the experiments and analyzed the data. Mika Pietilä wrote the article in collaboration with the other authors. .
IV Mika Pietilä was responsible for the planning of the study, he also designed and
performed the experiments and analyzed the data. Mika Pietilä wrote the article in collaboration with the other authors. .
ABBREVIATIONS AND SYMBOLS
ATMP Advanced therapy medicinal product
AT-MSC Adipose tissue-derived mesenchymal stem cells
ATP Adenosine tri-phosphate
�MEM Alpha minimum essential medium
BM-MSC Bone marrow derived - mesenchymal stem cells
CBMP Cell based medicinal product
CCCP Carbonyl cyanide m-chlorophenylhydrazone
CD Cluster of differentiation
CFU Colony forming unit
CFU-F Colony forming unit fibroblasts
ECM Extracellular matrix
ELISA Enzyme linked immunosorbent assay
EMEA European Medicines Agency
FDA US Food and Drug Administration
FSC Forward scattering
GMP Good manufacturing practices
GVHD Graft versus host disease
hESCs Human embryonic stem cells
HK Hexokinase
hMSCs Human mesenchymal stem/stromal cells
iPSCs Induced pluripotent stem cells
IDO Indoleamine 2,3-dioxygenase
JC-1 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbocyanine iodide
LDH Lactate dehydrogenase
MI Myocardial infarction
MitoSOX Mitochondrial superoxide sensitive dye
MnSOD Manganese superoxide dismutase
mtTFA Transcription factor A, mitochondrial
OCT4 Octamer-binding transcription factor 4
OXPHOS Oxidative phosphorylation
PBS Phosphate buffer saline
PFK Phosphofructokinase
PGC-1� Peroxisome proliferator-activated receptor gamma coactivator 1-alpha.
PI Propidium Iodide
Pol � DNA polymerase subunit gamma
PS Phosphatidyl serine
rER Rough endoplasmic reticulum
ROS Reactive oxygen species
SOX2 Sex determining region Y-box 2
SSC Side scattering
TEM Transmission electron microscopy
TLR Toll like receptor
TSG-6 TNF-� induced protein-6
UCB-MSC Umbilical cord blood - derived mesenchymal stem cells
��m Mitochondrial inner membrane potential
1.INTRODUCTION
In general, human stem cells can be categorized into pluripotent, multipotent and
unipotent stem cells. Pluripotent stem cells can produce every type of cell in the body
and thus their use in regeneration of damage tissues will offer unique possibilities in
the future. On the other hand, multipotent adult stem cells possess more limited
differentiation potency but are known to have specific paracrine properties by which
they can mediate the healing effect at the site of injury or inflammation. However,
currently, the lack of proper control of in vitro differentiation together with political
and ethical issues of pluripotent stem cells has limited their use in clinical trials,
whereas multipotent adult stem cells have already shown their safety and raised hopes
for offering viable alternatives for the treatment of many severe degenerative and
inflammatory conditions. However, there are still many important issues that need to
be solved before the transfer of these cell based medicinal products from laboratory to
clinics can be successfully achieved.
Human mesenchymal stem/stromal cells (hMSCs) are a heterogeneous population
of multipotent adult stem cells that can be isolated from different tissues. hMSCs have
already been used to alleviate large bone and cartilage defects, myocardial infarction
and several autoimmune diseases. At the moment, hMSCs are one of the most
promising cell sources for cellular therapies. However, the heterogeneous nature of
hMSCs creates certain problems. hMSCs can exist in different developmental states
and hMSC culture may also include other cell types, such as fibroblasts. Therefore
more detailed information on the characteristics of hMSCs is urgently needed to get
more standardized hMSC-based products. Traditionally hMSC-populations have been
distinguished by different cell surface antigens. However, most of such cell surface
antigens are not specific for only hMSCs. On the other hand, recent data on
mitochondrial properties and bioenergetics of hMCS have revealed interesting new
information, which suggests that analysis of mitochondrial properties could be utilized
in the future to isolate and distinguish different subpopulations of hMSC.
The lack of sensitive and rapid tools for assessing the quality of living cells is one of
the current problems in cellular therapies. To mediate the healing effect, the delivered
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stem cells need to possess excellent viability (i.e. they need to be in an active state). In
addition, the European legislation demands detailed and accurate information about
the viability and potency of cell based medicinal products. The short shelf-life of
cellular products sets certain limits for the current viability methods. In addition, one
of the major challenges is also ensuring the microbial safety as viable stem cell-based
products can not undergo terminal sterility procedures prior to implantation and use.
Therefore the development of rapid diagnostic tests is urgently called for to ensure the
viability and potency of stem cell products
In addition to the regeneration of bone or cartilage, hMSCs have shown their
potential to ameliorate and even possibly heal autoimmune diseases and
inflammatorial conditions. The current consensus is that the healing effect of hMSC-
based cellular therapies is mainly mediated by their paracrine properties. By secreting
soluble factors these cells deliver the protective anti-inflammatorial and anti-apoptotic
effect to the site of injury or inflammation. Also cell-cell interactions at a site of injury
have a major role in this process. However, the exact mode of action and the role of
different parallel mechanisms should be determined at a more detailed level in the
future in order to enhance the efficacy and reproducibility of hMSC-based cellular
therapies.
In this thesis, the focus is on hMSC – based cellular therapies with emphasis on the
metabolic characterization and functionality of these cells. Mitochondrial analysis
offers excellent parameters for both determing the viability of the cells but also for the
recognition of native stem cells from heterogeneous hMSC culture. Morever, the
specific functionality is also examined as the ability of hMSCs to mediate
immunosuppression in a macrophage model. In general, this thesis concentrates on
current problems in the field of stem cell therapies with an attempt to highlight new
solutions for providing more efficient and standardized hMSC-based cellular therapies
in the future.
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2.REVIEW OF THE LITERATURE
2.1. Stem cells
2.1.1. Stem cells and their role in the body
Stem cells possess several unique characteristics which distinguish them from other
cell types, such as capability to divide asymmetrically, differentiation potency for
several different cell types and expanded self-renewal capacity when compared to
terminally differentiated cells (Fuchs & Segre. 2000, Thomson et al. 1998, Watt &
Hogan. 2000). Tissues are constantly renewed as cells are removed and replaced by
new ones. The renewing activity and regenerative potency of different tissues varies
markedly. Bone, gut and blood cells are under continuous recycling whereas the heart
posses very narrow and limited regenerative potency. The tissue specific adult stem
cells reside in specific microenvironments called niches and their main role is to
maintain tissue homeostasis (Watt & Hogan. 2000). Adult stem cells are multipotent
as they possess limited differentiation potency capable of producing only a few
different lineage specific cell types (Fuchs & Segre. 2000). The number of adult stem
cells in a specific tissue is extremely low, but these stem cells are suggested to be
present in every tissue of the body (Vaananen. 2005). Pluripotent stem cells, on the
other hand, possess wider differentiation potency and more extensive expansion
potency (Thomson et al. 1998).
2.1.2. Human pluripotent stem cells
During early development the inner cell mass of blastocysts contains pluripotent stem
cells that are able to produce every cell type of the developing individual (Fuchs &
Segre. 2000, Thomson et al. 1998). Embryonic stem cells were isolated from the inner
cell mass of blastocyst first from mouse and then from human (Evans et al. 1981,
Thomson et al. 1998). Since then hESCs have been successfully differentiated into a
wide set of different cell types, such as neurons, cardiomyocytes, hepatocytes, etc. (Ma
et al. 2012, Pekkanen-Mattila et al. 2010, Takayama et al. 2012). Moreover, hESC-like
pluripotent stem cells have been produced by retro-virus transfection of pluripotency-
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associated transcription factors, Sox2, Klf4, C-Myc and Oct3/4, into fibroblasts first in
mouse and then in human cells (Takahashi & Yamanaka. 2006, Takahashi et al. 2007,
Yu et al. 2007). More recently, induced pluripotent stem cells (iPSCs) have been
produced from several different cell types. iPSCs possess pluripotent characteristics
similar to those of hESCs, i.e. teratoma formation, wide differentiation potency and
telomerase activity (Ge et al. 2012, Hanna et al. 2008, Niibe et al. 2011, Takahashi et al.
2007). However, recent studies have shown transcriptional memory of the parental
cells from iPSCs and epigenetic differences between iPSCs and hESCs. Therefore more
detailed molecular level comparison of hESCs and iPSCs is needed in future (Bock et al.
2011, Chin et al. 2009, Deng et al. 2009, Ohi et al. 2011). iPSCs have several
advantages when compared to hESCs, i.e. they are ethically less problematic as the
generation of iPSCs does not necessitate breakage of fertilized eggs and they are
immunologically unreactive as the patient’s own cells can be used to produce iPSCs.
Regardless of all the potential that pluripotent stem cells offer, their use in
treatment of diseases has not been as straightforward as previously was anticipated.
Although pluripotent stem cells possess enormous potential for use in the treatment of
degenerative conditions that can not be alleviated by traditional medicine, their use in
clinical trials has been extremely challenging and only recently clinical trials have been
launched and are ongoing using hESC (Schwartz et al. 2012). In addition, the high risk
of tumor formation of undifferentiated hESCs and iPSC and the concerns about the
integrity of the genome of iPSCs have also hindered the transfer of such technologies
from the laboratory into the clinics (Hussein et al. 2011).
2.1.3. Human mesenchymal stem cells
Human mesenchymal stem cells (hMSCs) were first isolated and cultivated by
Friedenstein and co-workers in the early 70s (Friedenstein et al. 1970, Friedenstein et
al. 1987). It was demonstrated that when cells from bone marrow aspirate were plated
in vitro at low density, fibroblast like cells began to adhere in a plastic culture dish.
Moreover, after a few days in culture, adhered cells formed separate colonies and were
first referred as colony forming unit fibroblasts (CFU-F). In a series of experiments,
Friedenstein et al. also demonstrated the differentiation potential of CFU-F into
different mesenchymal lineages (Friedenstein et al. 1966, Friedenstein et al. 1968,
Friedenstein et al. 1970, Friedenstein et al. 1974a, Friedenstein et al. 1974b,
Friedenstein et al. 1976, Friedenstein et al. 1987). The term human mesenchymal stem
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cells was introduced in the early 1990’s, when the tri-lineage differentiation potential of
bone marrow-derived hMSCs was demonstrated in vitro after which hMSCs started to
gain world wide interest (Caplan. 1991, Pittenger et al. 1999). However, even though
the knowledge of the biology of hMSCs has increased tremendously during the 40 years
following the discovery of these cells, the exact definition and characteristics of hMSCs
is still lacking. Especially, the in vivo localization and the role of hMSCs in the bone
marrow are poorly understood (Reviewed in Quinn & Flake. 2008). It has been
postulated that the bone marrow-derived hMSCs or osteoprogenitors reside in niches
and they support hematopoiesis by secretion of soluble factors (Raaijmakers et al. 2010,
Sacchetti et al. 2007). However, a clear demonstration of the engraftment of implanted
hMSCs into bone marrow and their function in vivo is still missing. In addition, hMSC
bear a striking resemblance to other stromal cells, especially fibroblasts and therefore
the stem cell nature of hMSCs has also been questioned (Covas et al. 2008, Haniffa et
al. 2009). Consequently, the name mesenchymal stem cells may not be appropriate
rather a more precise definition of human mesenchymal stromal cells (hMSCs) has
been introduced.
The heterogeneous nature of hMSC in vitro is also a major problem. Even today,
the most common methods for isolating hMSCs rely simply on their plastic adherent
properties. Therefore hMSC cultures will inherently contain also other stromal cells
such as fibroblasts, myoblasts etc. Moreover, hMSCs are by no means static, rather, in
culture many distinct differentiation levels are evident, from native stem cells to the
more developed and lineage specific progenitor cells (Post et al. 2008, Wagner et al.
2006). Accordingly, different isolated cell lines of hMSCs may show significant
variation in vitro and such variation is also evident between different laboratories,
which set important questions for the quality control and standardization of cell
products as well as for reproducibility of the results from clinical trials (Wagner & Ho.
2007).
19
2.1.3.1. Differentiation potential of hMSCs
hMSCs from different sources can be routinely differentiated into functional
osteoblasts, chondroblasts and adipocytes (Kern et al. 2006, Pittenger et al. 1999).
More recently, an even wider differentiation potency of hMSCs has also been shown as
hepatocyte-like cells, cardiomyocyte-like cells and neuronal-like cells have been
produced under specific culture conditions by several different laboratories (Ayatollahi
et al. 2011, Ma et al. 2011, Siegel et al. 2012, Snykers et al. 2007). On the other hand, it
should be noted that the actual function of the hMSC-derived cardiomyocytes and
neurons has not been properly demonstrated therefore their use in regenerative
medicine may be limited (Ma et al. 2011, Siegel et al. 2012). Accordingly these results
most likely do not represent the true differentiation potency of hMSCs, rather the
adaptation of hMSCs to the in vitro differentiation conditions. However, studies have
shown that hMSC cultured under specific in vitro conditions can produce hepatocytes
that possess cytochrome P450-dependent activity, urea production and albumin
secretion, which indicates relatively mature phenotypes of hepatocytes (Ayatollahi et al.
2011, Snykers et al. 2007). In the future, especially adipose derived hMSCs may thus
become a potential cell source for producing functional hepatocytes in addition to
pluripotent stem cells (Al Battah et al. 2011, Aurich et al. 2009).
Recent progress in cellular reprogramming makes it also possible to produce
functional cell types by introducing cell type specific transcription factors into the
genome of terminally differentiated cells or adult stem cells (Han et al. 2012, Thier et
al. 2012, Vierbuchen et al. 2010). This technique is currently under development but
the manipulation of the genome does raise some questions as to the safety of such cells.
However, this technique will obviously offer wider differentiation potency also for
hMSCs in the future. Moreover, preliminary studies have shown that immature mouse
mesenchymal stem cells can be reprogrammed more efficiently when compared to
terminally differentiated fibroblasts and osteoprogenitor cells (Niibe et al. 2011).
Therefore hMSCs may also serve as an ideal cell source for direct reprogramming and
production of tissue specific cell types for regenerative medicine.
20
2.1.3.2. hMSCs can be isolated from different origin
For a long time hMSCs were isolated and studied only from bone marrow, but
nowadays bone marrow-derived hMSC-like cells have been isolated from several
distinct tissues, i.e. adipose (Zuk et al. 2002), umbilical cord blood (Laitinen et al.
2011), dental pulp (Gronthos et al. 2002), hair follicle (Bajpai et al. 2012) etc. Several
comparative studies have been performed on the properties of hMSCs from different
origin, but the results show great inconsistency, highlighting the current problem with
the lack of standardization of hMSCs cultures (Table 1).
21
Table 1. A summary of comparative studies of hMSCs from different origins
Cell types Proliferation Osteogenic
differentiaion
Adipogenic
differentiatoin
Cartilage
Differentiati
on
Immunop
henotype
Reference
BM-MSC,
AT-MSC,
UCB-MSC
No major
differences
No major
differences
UCB-MSC
showed lower
differentiation
potency
No major
differences
No major
differences
(Rebelatto
et al. 2008)
BM-MSC,
AT-MSC,
UCB-MSC
BM-MSC: lowest
proliferation
span
UC-BMC:
highest
proliferation
span
No major
differences
UCB-MSC
showed lack of
differentiation
No major
differences
No major
differences
(Kern et al.
2006)
Fetal BM-
MSC, Adult
BM-MSC
ND Fetal BM-MSC
showed higher
differentiation
efficiency
No major
differences
ND No major
Differences
(Guillot et
al. 2008)
Placental -
MSC, BM-
MSC
No major
differences
ND ND ND No major
differences
(Miao et al.
2006)
BM-MSC,
Foreskin-
MSC, adult
skin-MSC,
AT-MSC
Foreskin-MSC
showed higher
cell proliferation
rate than AT-
MSC
BM-MSC
showed the
highest
efficiency
AT-MSC showed
the highest
efficiency
ND No major
differences
(Al-
Nbaheen et
al. 2012)
Placental-
MSC, BM-
MSC
Proliferation rate
and span higher
in placental-
MSC
No major
differences
BM-MSC
showed higher
differentiation
efficiency
No major
differences
No major
differences
(Barlow et
al. 2008)
AT-MSC,
BM-MSC
ND BM-MSC
showed higher
efficiency
No major
differences
BM-MSC
showed
slightly higher
effiiciency
No major
differences
(Noel et al.
2008)
BM-MSC,
AT-MSC
Colony forming
potential is
distinctly higher
in AT-MSC
BM-MSC
showed higher
efficiency
AT-MSC showed
higher efficiency
ND No major
differences
(Vishnubala
ji et al.
2012)
BM-MSC,
AT-MSC
ND. No major
differences
AT-MSC showed
higher efficiency
ND. Differences
in
expression
of CD34
(Pachon-
Pena et al.
2011)
Abbreviations: AT-MSC, Adipose tissue mesenchymal stem cells; BM-MSC, bone marrow mesenchymal stem cells;
UCB-MSC, umbilical cord blood mesenchymal stem cells; ND, not done
22
It is evident that most of the hMSCs from different origins show tri-lineage
differentiation capacities but there are some differences in their differentiation efficacy.
There seems to be a lineage specific imprinting in the function of hMSCs depending on
the source since some studies show that BM-MSC have higher osteogenic
differentiation potential whereas AT-MSC have higher adipogenic potency (Al-
Nbaheen et al. 2012, Vishnubalaji et al. 2012). The presence of progenitor cells that
already are committed to the lineage at the site of isolation of the hMSCs may explain
the higher differentiation potency to tissue specific cell types (Post et al. 2008).
Morphologically and immunophenotypically hMSCs seem to be similar, at least
according to the minimum criteria panel of cell surface markers established by the
International Society for Cellular Therapies (ISCT) (Dominici et al. 2006). In addition,
the age of cell donors seems to play a significant role in the quality and function of
bone marrow – derived hMSCs. However, the effect of the age of the donor is not a
straightforward issue. Namely, there seem to be differences in the quality of hMSCs of
young vs. elderly people, but the effect of aging seems to diminish and similar
differences are not as evident when cells from adult and elderly donors are compared
(Fickert et al. 2011, Leskela et al. 2003, Zaim et al. 2012, Zhou et al. 2008).
To rule out the effects of age of the donor on cell quality, the establishment of
umbilical cord blood banks could be a step forward, as UCB-MSCs have shown similar
characteristics to those of BM-MSCs and are therefore a very promising source of age-
independent hMSCs. In addition, the expansion capacity of such cells seems to be
higher when compared to bone marrow-derived hMSCs, which also may render UCB-
MSC more suitable for cellular therapies (Kern et al. 2006). However, the specific
molecular characteristic of hMSCs and standardized culture conditions must first be
established before sound conclusions on the differences in properties of hMSCs from
different origins can be made (Wagner et al. 2006, Wagner et al. 2006, Wagner & Ho.
2007).
23
2.1.3.3. Cell surface characteristics of hMSCs
Several characteristics have been used to define hMSCs: 1.) plastic adherence, 2.)
spindle shaped fibroblastic morphology, 3.) tri-lineage mesenchymal differentiation
potency into osteoblasts, chondroblasts and adipocytes. 4.) Immunophenotype;
minimum criteria panel of cell surface markers for hMSCs has been established by
ISCT (Dominici et al. 2006). hMSCs should show over 95 % positivity for CD105, CD73,
CD90, HLA-ABC and they should be negative (under 2 % positivity) for CD14, CD19,
CD45, CD34 and HLA-DR. However, these characterization criteria are not specific for
hMSCs since in some studies fibroblasts have also shown similar differentiation
potency, cell surface characteristics and also immunosuppressive properties as hMSCs
(Haniffa et al. 2009).
In addition to the minimum criteria panel, hMSCs have been shown to express a
wide variety of different cell surface antigens. Mafi et al. (2011) have summarized the
data from 29 different cell surface characterization studies which fulfilled their
inclusion criteria. The summary included characterization of adult hMSCs from
adipose tissue and bone marrow. The most commonly expressed markers that had
been reported in the 29 studies were: CD105, CD90, CD44, CD73, CD29, CD13, CD34,
CD146, CD106, CD54 and CD166 whereas the most frequently being reported as absent
were CD34, CD14, CD45, CD11b, CD49d, CD106, CD10 and CD31. Evidently, there
seems to be a discrepancy with reference to cell surface markers expressed by adult
hMSCs. Several of these studies indicate that hMSC would be negative for CD34
whereas almost as many reports claim that the cells are positive for CD34. There are
also controversial results with reference to expression of CD44, CD49d and CD106.
One explanation for the discrepancies could be due to the different origins of the
hMSCs. It is evident that the above data highlights the current problem with regards to
arriving at a concensus with reference to the characteristics of hMSCs. This is also
further complicated by variations in hMSC cultures from different laboratories.
24
2.2. The mitochondrion as a regulator of stem cells
2.2.1. Mitochondrial bioenergetics
Mitochondria are “power plants” of mammalian cells as they produce most of cellular
adenosine triphosphate (ATP) that enables many vital cellular functions. In addition,
mitochondria control ion homeostasis, they are the main source for both reactive
oxygen species and anti-oxidant defense system and they are the center for many of the
important metabolic reactions (Cadenas & Davies. 2000, Cali et al. 2012). In the matrix
of the mitochondria, energy rich nutrients are metabolized in consecutive reaction
processes of the citric acid cycle. During this process, highly energized co-enzyme
NADH is produced and transported to the mitochondrial inner membrane where
oxidative phosphorylation (OXPHOS) takes place (Nicholls. 2002). During OXPHOS,
electron flow from NADH to O2 is conducted through the respiratory complexes I, III
and IV which simultaneously pump protons from the matrix into the intermembrane
space of mitochondria producing the proton motive force, �p, a central parameter that
enables all the important functions of the mitochondria (Nicholls. 2002). In addition,
the electron flow from succinate through respiratory complex II serves as an additional
electron source for proton pumping complexes III and IV. Moreover the Q cycle, which
includes sequental oxidation and reduction of the electron carrier ubiquinol-
ubiquinone (Coenzyme Q) at the site of complex III, enhances the net pumping of
protons across the mitochondrial membrane (Trumpower. 1990).
The formed proton gradient enables the production of highly energized ATP
molecules at the site of complex V. The proton motive force consists of the
mitochondrial inner membrane potential (��m) and trans-membrane pH gradient
(Nicholls & Ward. 2000). Since the ��m is the major component of the proton motive
force, many ��m sensitive fluorescent membrane-permeant cationic dyes have been
developed to monitor mitochondrial energy state. The most commonly used dyes are
Rhodamine123, 3,30-diethyloxacarbocyanide iodide and tetramethylrhodamine methyl
ester (Heiskanen et al. 1999, Khodorov et al. 1996). These dyes have been used for
several decades to monitor ��m and their function is based on their quenching
property when used in nano-scale concentrations (Nicholls & Ward. 2000). Another
��m sensitive dye, 5,50,6,60-tetrachloro-1,10,3,30-
tetraethylbenzimidazolcarbocyanine iodide (JC-1), has also been widely used (Salvioli
et al. 1997). The advantage of JC-1 is that the monomer and aggregate forms of JC-1
25
have emission peaks at different wave lenghts (Smiley et al. 1991). In the presence of
high ��m JC-1 is accumulated into the mitochondrial matrix forming J-aggregates
which emit at 590 nm. In the presence of low ��m, JC-1 molecules are released from
the mitochondria into the cytoplasm in a monomer form which emit at 527 nm (Smiley
et al. 1991). This makes JC-1 suitable for end point measurement of ��m and thus
especially in detection of apoptosis whereas Rhodamine derivatives are well suited for
monitoring small changes in ��m.
2.2.2. The mitochondrion as a regulator of apoptosis
In addition to energy production tasks, mitochondria also play an important role in
regulation of apoptosis (Kroemer & Reed 2000). Apoptosis, also known as a
‘‘programmed cell death,’’ has been identified as an intrinsic safety measure of cells
after exposure to critical damage and it is executed by the cell itself. Mitochondria and
cysteinyl aspartate–specific proteinases (caspase) cascades play key roles in the
regulation of apoptosis through two major pathways, extrinsic or intrinsic (Kroemer &
Reed. 2000, Nicholson & Thornberry. 1997). The opening of mitochondrial
permeability transition pore (MPTP) is a well characterized event that leads to
membrane rupture, dissipation of proton motive force, ATP hydrolysis, disruption of
metabolic gradients between mitochondria and the cytoplasm and ultimately to
mitochondrial swelling and destruction (Figure 1; Halestrap et al. 1998, Halestrap et
al. 2004)). The primary trigger for MPTP opening is the increase in Ca2+ concentration
in the matrix but also additional factors such as adenine nucleotide depletion,
mitochondrial depolarization and oxidative stress can cause the opening of MPTP
(Halestrap et al. 1998, Halestrap et al. 2004). The role of oxidative stress as an inducer
of MPTP opening is important especially in vivo during ischemia (Kim et al. 2006). In
addition, Bcl-2 protein family members have a key role in controlling MPTP. Bax-, Bid-
and Bak- proteins have been shown to induce MPTP and mitochondrial swelling
whereas Bcl-2 and Bcl-XL act as inhibitors unless cleaved by caspase-3 (Desagher et al.
1999, Griffiths et al. 1999, Jurgensmeier et al. 1998, Kirsch et al. 1999). The swelling
and disruption of mitochondrial membranes leads to the release of several soluble pro-
apoptotic factors, i.e. cytochrome C, Smac/Diablo, apoptosis induced factor (Aif) etc.
(Du et al. 2000, Liu et al. 1996, Susin et al. 1999).
The role of cytochrome C in activation of apoptotic protease-activating factor-1
(Apaf-1) and its following oligomerization has been established. Active Apaf-1
26
apoptosome recruits caspase-9 and forms the Apaf-1/caspase-9 holo-enzyme that
activates caspase-3 which ultimately leads to the activation of caspase cascades and
apoptotic degradation of the cell. In addition to cytochrome C, other mitochondrial
pro-apoptotic proteins, i.e. Smac/Diablo, regulate the formation of apoptosome (Cain.
2003). However, even though the depolarization of ��m is a hallmark of MPTP
opening there is currently no agreement on whether the depolarization of ��m is a
point of no return event in the process of apoptosis (Bossy-Wetzel et al. 1998,
Heiskanen et al. 1999). Therefore in order to obtain a reliable estimate of the metabolic
functionality and viability, i.e. the condition of cells and/or apoptosis, several aspects
of cellular changes should be monitored simultaneously.
Figure 1. Schematic representation of MPTP opening and mitochondrial regulation of cellular death
(Modifyed from Kroemer & Reed 2000).
27
2.2.3. Mitochondrial bioenergetics and human mesenchymal stem cells
The role of mitochondria in terminally differentiated cells of distinct cell types has been
studied extensively. The mitochondrial number and activity shows marked variation
between different cell types depending on the function of the cell and consequently
their demands for energy. However, pioneering research into the role of mitochondria
and energy metabolism of hMSCs has only been published relatively recently
(Lonergan et al. 2006). Mitochondria in hMSCs are unenergized and the main energy
production is conducted via anaerobic glycolysis whereas during osteogenic
differentiation the shift from anaerobic to aerobic energy production occurs due to
increased energy demand (Chen et al. 2008). Chen et al. 2008 compared several
mitochondrial and metabolic parameters between undifferentiated hMSCs and osteo-
induced hMSCs. A clear change was evident from anaerobic metabolism to aerobic
respiration already after a short period of osteoinduction. This was seen by increased
copy number of mitochondrial DNA (mtDNA), increased expression of respiratory
enzymes, increased oxygen consumption rate and increased expression of genes related
to mitochondrial biogenesis. On the contrary, the osteoinduction of hMSCs decreased
the expression of several important glycolytic enzymes (phosphofructokinase, puryvate
dehydrogenase kinase, glucophosphate isomerase) and also lactate production.
It has also been postulated that the anaerobic nature of undifferentiated stem cells
may protect them for toxic forms of oxygen such as ROS which is linked to the higher
mitochondrial energy state and thus may prevent aging. The role of ROS as a regulator
of stem cell differentiation has also been reported elsewhere with pluripotent stem cells
and hematopoietic stem cells and the regulation of ROS production and scavenging
may be more complicated during development of stem cells than has been thought
(Crespo et al. 2010, Owusu-Ansah & Banerjee. 2009). In the hematopoietic system,
stem cells show low ROS levels which increase dramatically when they develop to the
progenitor cells whereas the ROS levels decreased again when the progenitors
differentiate into mature blood cells. In addition, by manipulating ROS levels it has
been possible to influence the differentiation of hematopoietic progenitor cells into
mature blood cells in e.g. drosophila (Owusu-Ansah & Banerjee. 2009). A similar shift
from anaerobic to aerobic energy production has recently been reported for adipogenic
differentiation of hMSC (Hofmann et al. 2012). In addition to the studies with hMSCs,
identical changes associated with differentiation have also been shown with pluripotent
28
stem cells (Cho et al. 2006, Kondoh et al. 2007, St John et al. 2005). Most recently also
lung cancer stem cells were shown to have higher glycolytic activity and lower
mitochondrial activity when compared to somatic lung cancer cells (Ye et al. 2011).
Moreover, by manipulating mitochondrial activity with uncouplers it has been possible
to affect the stemness of embryonic stem cells and induction of mitochondrial
dormancy has been shown during reprogramming of terminally differentiated cells into
iPSCs (Mandal et al. 2011, Prigione et al. 2010).
Accordingly it seems that hMSCs possess similar kind of metabolic status than
more defined stem cell types, i.e. pluripotent stem cells and hematopoietic stem cells.
However, even though a similar shift from anaerobic to aerobic energy production has
recently been reported for adipogenic differentiation of hMSC, additional studies with
stem cells from different sources and also during differentiation towards different
lineages should be performed to clarify the bioenergetic changes during stem cell
differentiation since some discrepant data has also been reported for chondroblast
differentiation of hMSCs and neuronal differentiation of hESCs (Birket et al. 2011,
Hofmann et al. 2012, Pattappa et al. 2011). Clearly mitochondrial analysis and
manipulation offers great possibilities to characterize and isolate different stem cell
subpopulations but also to manipulate the differentiation efficiency of stem cells.
2.3. Stem cell-based cellular therapies
2.3.1. Mechanisms of action
In 1998, the isolation of pluripotent hESCs led to great expectations for stem cell
therapies. However, controlling the complexity of the differentiation processes has
turned out to be more difficult than expected. Therefore the potential of pluripotent
stem cells for regenerative medicine has not been transferred into clinical use as yet.
Accordingly, a completely different approach has been introduced by using adult
mesenchymal stem cells. A paradigm change has occurred, where the emphasis is on
hMSC as a paracrine mediator of active biomolecules instead of trans-differentiation of
hMSCs into specific cell types at the site of injury and thus achieving regeneration of
damaged tissue. hMSCs have been shown to possess immunosuppressive and anti-
apoptotic properties when undifferentiated hMSCs have been introduced at a site of
injury or in close proximity. The regenerative effect of stem cell-based cellular
29
therapies can be achieved by three different mechanisms which are discussed in more
detail: 1.) regeneration of damaged tissue by new functional tissue specific cell types
derived from stem cells, 2.) modulating the immune response at a site of injury and
thus minimizing the damage and/or 3.) protecting tissue specific cells at a site of injury
by delivering anti-apoptotic factors and enhancing the activity of tissue resident stem
cells.
2.3.1.1. Regeneration of damaged tissue
The regenerative use of in vitro expanded hMSCs has shown promising results in pilot
studies for several bone diseases such as osteogenesis imperfecta, spinal fusion and
osteonecrosis of femoral head (Gan et al. 2008, Gangji et al. 2004, Horwitz et al. 1999,
Horwitz et al. 2002, Marcacci et al. 2007). However, several issues in manufacture of
large bone implants from BM-MSCs need to be solved before these treatments can be
produced in a large scale. First, the number of cells needed is extremely high but
hMSCs are a rare population in the bone marrow. Production of a high number of cells
in vitro is therefore challenging especially since long term in vitro culture of BM-MSC
will cause senescence and alter the differentiation efficiency (Baksh et al. 2004,
Wagner et al. 2008). The second important issue is the vascularization of in vitro
manufactured bone implants. The first issue can be tackled by searching for alternative
sources for BM-MSC such as UCB-MSCs which show higher expansion potency but
similar osteogenic differentiation potency as BM-MSC. The second issue can be solved
by combing hMSCs with angiogenesis promoting growth factors (VEGF) or with
endothelial progenitor cells to ensure the vascularization of the bone implant (Kanczler
& Oreffo. 2008). In addition to bone defects, hMSCs have been successfully used to
regenerate cartilage defects in animal models (Ohyabu et al. 2006, Yoshioka et al.
2007). So far the limited differentiation potency of hMSCs and the lack of knowledge of
differentiation of pluripotent stem cells have hindered the progress of a regenerative
medicine for developing treatments for more complicated tissues, such as nervous
system, liver and heart. However, pluripotent stem cells have been used in several
animal models and they have shown promising results (Caspi et al. 2007, Laflamme et
al. 2007). However, the current differentiation techniques are inefficient and therefore
the production of reasonable number of pure functional cell types from pluripotent
stem cells is challenging.
30
2.3.1.2. The role of immunosuppression in cellular therapies
It has been shown that tissue damage, such as myocardial infarction (MI), induces pro-
inflammatory activity at a site of injury which enhances fibrous scar formation, inhibits
healing and regeneration of the damage tissue (Arslan et al. 2011, Eltzschig & Eckle.
2011, Wrigley et al. 2011). In addition to the complex regulatory system of immune
cells themselves, it has also been demonstarted that hMSCs, as also other stromal cells,
possess immunomodulative properties in vitro and in vivo (Aggarwal & Pittenger.
2005, Gonzalez et al. 2009). hMSCs have been shown to inhibit T-cell proliferation
(Aggarwal & Pittenger. 2005), prevent dendritic cell maturation (Jiang et al. 2005),
inhibit B-cell proliferation (Tabera et al. 2008), suppress natural killer-cell activity
(Giuliani et al. 2011) and to change the polarity of macrophages into an anti-
inflammatory M2 phenotype (Figure 2.) (Kim & Hematti. 2009, Zhang et al. 2010).
However, even though there are also some promising results from the
immunosuppressive activity of hMSCs in clinical trials, the underlying molecular level
mechanisms are still unclear (Le Blanc et al. 2004, Le Blanc et al. 2008).
Several different soluble factors have been identified that may work alone or
together to mediate the immunosuppressive effect in a T-cell proliferation model, i.e.
indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), transforming growth
factor beta (TGF-�) etc. (Lepelletier et al. 2010, Meisel et al. 2004, Ryan et al. 2007).
However, the immunosuppressive function of hMSCs remains controversial with
reference to the actual mechanisms, as data from different laboratories has been
difficult to reproduce. In addition, some studies have shown that cell-cell contacts are
needed for proper immunosuppression mediated by hMSCs. Several cell surface
molecules have been shown to participate in immunosuppression directly, such as Thy-
1 (CD90), or indirectly, such as intercellular adhesion molecule-1 (ICAM-1) and
vascular cell adhesion molecule-1 (VCAM-1) which bind immune cells to the close
proximity of hMSCs and under the paracrine effect of hMSCs (Campioni et al. 2009,
Ren et al. 2010). In addition, it was recently discovered that hMSC also express OX2
(CD200), an anti-inflammatorial protein, that has a central role in regulation of
macrophage activity. The role of CD200 in hMSC mediated immunosuppression has
not been elucidated as yet (Delorme et al. 2008, Hoek et al. 2000).
31
The interactions at a site of injury between hMSCs and immune cells are also
working in both directions, since immune cells and pro-inflammatorial factors
also alter the function and phenotype of hMSCs.
Figure 2. Simplified representation of the intercellular communications between hMSCs and immune
cells at a site of inflammation. hMSCs mediate the immunosuppression by different soluble factors and
cell-cell interactions (green arrows) but also pro-inflammatory conditions modifies the
immunosuppressive activity of hMSCs (red Arrows).
Pro-inflammatory conditions have been shown to modulate immunosuppressive
activity of hMSCs. IFN-� and TNF-�, cytokines that are secreted by different immune
cells at a site of inflammation, have been demonstrated to induce IDO activity,
enhancing proliferation of hMSCs and altering the expression of several cell surface
antigens which are linked to the immunosuppressive activity of hMSCs (Croitoru-
Lamoury et al. 2011, Miettinen et al. 2011, Ryan et al. 2007). In addition, Toll like
receptor (TLR) activation, such as bacterial lipopolysaccharide (LPS) binding to TLR-4,
32
has been shown to possess a similar effect on the immunomodulative function of
hMSCs as TNF-� and IFN-� (Opitz et al. 2009). Most recently, it has been shown that
macrophages alter the cytokine secretion profile of hMSCs and enhances the migration
of hMSCs towards macrophage secreted soluble factors when hMSCs were grown in
macrophage conditioned medium (Anton et al. 2012).
Based on the fact that systemically delivered hMSCs tend to accumulate into lungs
and only small proportion of transplanted hMSCs only transiently targets the site of
injury it is difficult to understand how implanted hMSCs still show healing effect in
animal models and clinical trials (Schrepfer et al. 2007). Recent research has sought to
unravel this paradox as it was shown that hMSCs improve the recovery from
myocardial infarction in a mouse model even though most of the hMSCs were
entrapped in the lungs by secreting anti-inflammatory TNF-� induced protein-6 (TSG-
6) (Lee et al. 2009). It was shown that the hMSCs induced the secretion of TSG-6 from
the lungs which led to the immunosuppressive effects at the site of the myocardial
injury and decreased the damages to cardiac tissue. This was confirmed by silencing
TSG-6 gene by siRNA knockdown in hMSCs which showed no positive effect on the
condition of myocardial infarction. Moreover, by delivering recombinant human TSG-6
(rhTSG-6) in the myocardial infarction mouse model, an almost similar effect was
achieved as with hMSCs. Similar results have also been obtained in a rat model of
corneal injury (Oh et al. 2010, Roddy et al. 2011). Systemic administration of hMSCs
effectively reduced corneal opacity and inflammation even without engraftment of
hMSCs into the corneal site. Also these studies demonstrate the role of TSG-6 as a
mediator of positive effect of hMSCs in corneal healing and the findings could be
confirmed by silencing the TSG-6 gene by siRNA knockdown which caused no anti-
inflammatorial effect and also by delivering hrTSG-6 which had a similar effect as did
hMSCs.
2.3.1.3. Anti-apoptotic effect of hMSCs
In addition to the immunosuppressive properties of hMSCs, recent research has also
shown that hMSCs mediate anti-apoptotic effects at the site of injury. The exact
mechanisms are unknown but hMSC appear to provide anti-apoptotic molecules and
even mitochondria to such a process. hMSCs are capable of rescuing cells that have
non-functional mitochondria by delivering functional mitochondria or mitochondrial
DNA to such cells (Spees et al. 2006). Moreover, mitochondrial transfer occurs also in
33
vivo and it may play a role in recovery of myocardial infarction by reprogramming
myocardial cells into myocardial resident stem cells (Acquistapace et al. 2011).
However, even though the exact mechanisms and the role of mitochondrial transfers
remain open this may present an interesting model of restoring aerobic respiration of
damaged cells at a site of injury.
Mouse model of acute kidney injury has been widely used to study the anti-
apoptotic effect of MSC administration for restoring damaged tissues or cells (Bi et al.
2007, Morigi et al. 2008). Several studies have shown that delivery of in vitro
expanded MSCs induces the recovery of kidney injury but the mechanisms are
unknown and controversial. However, it has recently been demonstrated that
microvesicular secretion by hMSCs may be responsible for the positive outcomes in the
mouse model of acute kidney injury induced by cisplastin (Bruno et al. 2012). The
administration of microvesicles derived from hMSCs rescued the SCID mouse from
acute kidney injury. The administration of microvesicles inhibited apoptosis at the site
of injury, which was seen by the upregulation of anti-apoptotic genes, Bcl-2, Bcl-XL
and downregulation of pro-apoptotic genes, Casp 1, Casp 8 and LTA.
Another example of the anti-apoptotic function of MSCs is the
hypoxia/reoxygenation induced injury of islets in rats (Lu et al. 2010). The co-culture
of islets with MSCs maintained the stimulation index of glucose-stimulated insulin
secretion during hypoxia/reoxygenation and the insulin expression also differed in
transplanted islets that were co-cultured with MSC when compared to islets grown
alone after 7 days of transplantation. The protective effect of MSC was shown to be
carried out by inhibition of apoptosis and it has been postulated that MSC delivered
microvesicles may contain microRNAs, mRNA, proteins and cytokines that have anti-
apoptotic and healing function. Recent co-culture studies of MSCs with different cell
types have also shown that cell-cell interactions are more intense than have been
previously thought (Rappa et al. 2012, Strassburg et al. 2012, Vallabhaneni et al. 2012).
34
2.3.2. Current status of stem cell-based clinical trials
Allogenic stem cell transplants have been used in treatment of many hematological
diseases for decades. However, during the last decade hopes have been raised for use of
stem cells for the treatment of a variety of clinical conditions. Numerous clinical trials
have been conducted with adult stem cells and several products have already been
launched on the market. As can been seen from Table 2, the number of new registered
clinical trials with mesenchymal stem cells have increased dramatically during the last
12 years (clinicaltrials.gov).
Table 2. hMSC-based registered clinical trials during 2000-2012.
Number of new clinical trials reigstered in clinicaltrials.gov
webpage
Condition
2000-2004 2005-2008 2009-2012
Graft versus host
disease
1 10 9
Multiple sclerosis 0 3 5
Myocardial infarction 0 8 11
Osteogenesis
Imperfecta
0 3 0
Diabetes Mellitus 0 4 15
Osteoarthritis 0 1 19
Crohn’s disease 0 3 8
Cartilage diseases 0 0 3
Total 2 61 182
The database clinicaltrials.gov was used and results are based on the search made in June 2012
Even thought the increase in clinical trials is partly explained by the more active use of
clinicaltrials.gov webpage by clinics and research groups, this clearly demonstrates the
hype and hope of stem cell therapies. The most striking increase is evident in the
number of new clinical trials for treatment of different autoimmune diseases, namely
diabetes mellitus, osteoarthritis and Crohn’s diseases. During 2009-2012 the most
active countries or regions for registration of clinical trials with hMSC have been China
(64), Europe (42) and USA (28) (clinicaltrials.gov, search made on June 2012). The
outcomes of the trials have been variable. hMSCs have been shown to be safe but
results from the use of hMSC to treat myocardial infarction, the most popular
35
intervention so far, have been somewhat controversial. Results have varied from
signifcant improvement to mild or no improvement of cardiac function when compared
to placebo (Dill et al. 2009, Tendera et al. 2009, Wollert et al. 2004, Yerebakan et al.
2011). This has raised questions about the feasibility of using undifferentiated hMSCs
in the treatment of myocardial infarction but also about the timing of the
transplantation of hMSC after infarction, which may be critical and could explain some
of the inconsistencies. However, the treatment of autoimmune diseases such as Crohn’s
disease and GVHD have shown promising results and several products have already
been launched on the market in the USA. Prochymal, an allogenic mesenchymal stem
cell product from Osiris Therapeutics (http://www.osiris.com/therapeutics.php), has
been approved by the Food and Drug Administration (FDA) in the USA and the
product has undergone phase III trials for treatment of GVHD and Crohn’s disease (Le
Blanc et al. 2004, Le Blanc et al. 2008).
Contrary to hMSC, hESCs have only recently progressed to the first clinical trials.
Currently, three clinical trials have been registered in clinicaltrials.gov (search made in
June 2012) and all were for treatment of different eye diseases. The first of the studies
has already shown promising short term results for the safety of hESC-derived retinal
pigment epithelium to treat patients with Stargardt’s macular dystrophy and dry age-
related macular degenration (Schwartz et al. 2012).
36
2.4. Current status of European legislation for cellular products
2.4.1. Overview of the regulations
Advances in cell biology, molecular biology and medicine have introduced novel
medicinal products that include living cells. Since the industry is young and constantly
changing, the nomenclature for these products is somewhat mixed and several names
and abbreviations can be found in the literature: i.e. Cell Containing Medicinal
Products (CCMP), Advanced Therapy Medicinal Products (ATMP), Stem Cell Based
Interventions (SCBI), Cell-Based Medicinal Products (CBMP) etc. Such names are used
to describe cellular products in general but according to the EU legislation some of the
names refer to products that are regulated differently and do not represent the same
product category. Therefore when referring to cellular products, it is important to
recognize the basis of their regulatory classification.
An overview of the current EU legislation is presented in Figure 3. Based on the
content, level of manipulation and the final use of the cellular products, CBMP can
roughly be categorized into three groups; medicinal devices, tissues and cells or ATMPs.
As a general rule, the more the product has undergone in vitro manipulation and the
more complex product it is, the more complex is also the regularly burden. Products
that contain exclusively non-viable animal cells are regulated as medical devices
(Directive 93/42/EEC, Directive 90/385/EEC and Directive 2003/32/EC). The most
problematic are ATMPs which may include one of the following products: a gene
therapy medicinal product (as defined in Annex I to Directive 2001/83/EC), a somatic
cell therapy medicinal product (as defined in Annex I to Directive 2001/83/EC) and a
tissue engineered product (as defined in Regulation N:O 1394/2007). ATMPs are
regulated as medicinal products (Directive 2001/83/EC) as further amended by
Regulation on Advanced Therapy Medicinal Products (Regulation N:O 1394/2007)
which sets a strict regulation for controlling the quality of the product. Moreover, the
Directives 2006/17/EC and 2006/86/EC include more specific operational regulations
for the donation, procurement and testing of cells from human origin which all ATMP
suppliers should comply with.
In order to reach the market a CBMP must have Marketing Authorization granted
by regulatory authorities in accordance with Regulations (No 726/2004 and No
1394/2007). The development, manufacture and quality controls for CBMP that are
entering a Marketing Authorization procedure should be performed according to the
Guideline on Human Cell-Based Medicinal Products amended by the Committee for
37
Medicinal Product for Human Use (CHMP) from the European Medicines Agency
(EMEA). The marketing authorization must be prepared in accordance with the
directives and regulations mentioned above (EMEA CHMP 2006). Compliance with
the requirements of the above guideline is also required when planning to enter into
clinical trials with a CBMP.
The third group of products is tissues or cells (Figure 3), which includes only non-
engineered human cells and which are used as an autologous graft within the same
surgical operation. On the other hand, the definition of the engineering of cells has
posed significant challenges and currently cell engineering in legislation is described as
follows: 1.) The cells or tissues have been subjected to substantial manipulation, so
that biological characteristics, physiological functions or structural properties relevant
for intended regeneration, repair or replacement is achieved. 2.) The cells or tissues
are not intended to be used for the same essential function or functions in the recipient
as in the donor (Regulation No 1394/2007). The first definition is clear and it includes
products such as in vitro differentiated stem cells but the second definition is difficult
to interpret since the detailed function of the cells after implantation is not easy to
predict. Because of the continuous development of cellular products and their new
applications there are many cases which fall somewhere between the categories
specified by the current legislation and their regulatory status is interpreted by a case-
by-case approach. Moreover, some exclusions exist at the moment such as ATMPs that
are isolated, manufactured and used within the same Member State hospital under the
exclusive professional responsibility of a medical practitioner in order to comply with
an individual medical prescription for a custom-made product for an individual patient
(Regulation No 1394/2007). In addition, blood and blood products and cells thereof
have a long history of therapeutic use, but are covered by separate regulations and are
not discussed further in the present thesis.
38
Figure 3. The overview of the current regulation of the cell containing medicinal products in Europe reprinted with the permission from original illustrator (Närhi & Nordström 2008).
2.4.2. Quality controls and risk analysis of CBMPs
EMEA guideline (CHMP 2006) describes certain quality controls, characterizations
and in-process controls that the manufacture of an ATMP should fulfill for Marketing
Authorization. These are also recommended for CBMP intended for use in clinical trials.
The producer of CBMP must perform comprehensive risk analysis in order to evaluate
risk versus benefit of the product. Risk analysis is done as a case-by-case basis and
according to the established European legislations and guidelines (Annex I to Directive
2001/83/EC, Guideline EMEACHMP/96268/2005). The risk posed by administration
of CBMPs is highly dependent on the origin of the cells, the level of manipulation and
39
engineering of cells as well as the inteded usage of the cell product. The Guideline on
Human Cell-Based Medicinal Products (EMEA CHMP 2006) recommends the
following risk criteria to be used in the estimation of the overall risk of the CBMP: 1.)
Autologous or allogenic cell origin, 2.) ability of the cell to proliferate or differentiate,
3.) ability of the cell to initiate immune response, 4.) level of cell manipulation, 5.)
mode of administration, 6.) duration of exposure or culture or life span of cell, 7.)
combination product and 8.) availability of clinical data on a similar product.
The manufacture of the CBMP should be carried out with extreme care according to
good manufacture practices (GMP) as stated in Annex 2 to Directive 2003/94/EC.
During manufacture, several in-process controls should be performed at the level of
critical steps, such as after isolating the cell material, after the thawing of cells from
master bank and before release, to monitor the quality of the CBMP throughout the
manufacturing process. In-process controls may include the same validated methods
which are used in characterization of CBMPs. The manufacture of stem cell based
medicinal products dictate certain requirements for their quality, safety and efficacy
which are indicated in Directive 2001/83/EC and certain characteristics that are
suggested to be monitored are stated in the EMEA guideline (CHMP 2006) such as
identity, purity, potency, viability and suitability for the intended use. Especially the
potency and viability are important parameters to monitor but also the most
challenging ones. In addition to the characterization of CBMP, all biologically active
components in the culture media should also be characterized with reference to purity,
sterility, identity, biological activity and absence of adventitious agents. On the other
hand, even though there are a number of established methods for the control of
conventional biological medicinal products, most of them are poorly applicable for the
control of the manufacturing processes of CBMPs, as CBMP are living products with
short shelf-life. Living products are metabolically active and will not be the same at the
beginning of production and prior to release for clinical use. As the shelf-life is short,
there is often limited time to perform extensive testing prior to implantation, and batch
size may also limit the number of controls that can be performed.
40
2.4.3. Microbial safety
Regulatory demands for the manufacture of stem cell based medicinal products dictate
certain requirements also for microbial (bacterial and fungal) and viral safety
(European Parliament and European Council (2001) Directive 2001/83/EC). However,
the regulation builds on quality control of traditional synthetic medicines and therefore
is not directly applicable to CBMPs. One of the major challenges in manufacture of
CBMPs is to ensure microbial safety of CBMP, as living cells can not undergo terminal
sterility in a similar manner as synthetic medicines. Moreover, the short shelf-life of
CBMPs renders detection of microbial contamination detection challenging as
traditional time consuming colony forming unit (cfu)-protocols for e.g. bacteria will
often provide data only after the product has already been implanted and are not ideal
for controlling the microbial quality of CBMPs. Moreover, the possible transmission of
latent and unknown endogeneous viruses is very poorly understood and lacks reliable
tests. The incidents of microbial contamination of cell products seem to be rare, but
still the risk of contamination is a critical issue, as the consequences to patient health
may be unpredictable and even devastating (Kamble et al. 2005, Klein et al. 2006,
Smith et al. 1996, Vanneaux et al. 2007). The most common point of e.g. bacterial
contamination during manufacturing is the in vitro expansion and manipulation of
cells (Smith et al. 1996, Vanneaux et al. 2007). Presumambly viral contamination is
most likely derived from the original donor. Accordingly, the development of relevant
process controls for use in manufacturing to monitor the quality of CBMPs is clearly
pivotal to the successful translation of research into clinical practice.
41
3.AIMS OF THE PRESENT STUDY
The focus of the present thesis is on the mitochondrial characterization of hMSC with
the overall aim of shedding insights on hMSCs as a potential cell type for cellular
therapies. To study the functionality of the hMSC in more detail, the aim was to study
the immunomodulative capacity of hMSC in a macrophage model. The specific aims
were as follows:
1. To increase the knowledge of mitochondrial and bioenergetics characteristics of
hMSCs from different origin and find alternative measurable properties that could
be utilized in definition of hMSCs. New characterization criteria are needed for
more standardized and pure hMSCs products.
2. To establish a new sensitive viability measurement tool based on measurement of
the inner mitochondrial membrane potential of hMSCs for monitoring of the effects
of apoptotic changes on the cells. This will most likely improve the quality of
hMSC-based cell products.
3. To test the suitability of the established viability measurement tool for detecting
apoptotic changes that may be caused by microbial contamination. Such data is
critical to the establishment of validated methods for microbial quality control for
safe and regulatory approved manufacture of cell based products in the future.
4. To study the immunosuppressive properties of hMSCs and especially the role in
controlling innate immunity. The more specific aim was to elucidate the role of the
cell surface marker, CD200, in regulation of macrophage activity by hMSCs. This
specific aspect of hMSC functionality was chosen as the role of hMSCs in the
treatment of autoimmune diseases is currently one of the most promising area for
future cell therapies.
42
4.MATERIALS AND METHODS
The summary of the main methods used in the present studies is shown in Table I.
More detailed information on the methods can be found in the original publications (I-
IV) indicated in brackets after each method.
Table I. Summary of the methods used in this study
Method Publication
Cell culture of BM-MSC I, II, III and IV
Cell culture of UCB-MSC I,IV
Cell culture of THP-1 cell line IV
Flow cytometry I and II
Osteogenic differentiation of hMSCs I and II
Transmission electron microscopy I
Western blot I
Immunofluorescent staining IV
Macrophage differentiation of THP-1 IV
Bacterial cultivation III
Enzyme linked immuno sorbent assay (ELISA) III
Phase contrast microscopy I, III, IV
4.1. Cell Cultures
4.1.1. Human bone marrow-derived mesenchymal stem cells (I,II,III,IV)
In this thesis, BMMSCs were isolated from an unaffected bone site of patients who
were operated for osteoarthritis and from some younger patients operated for
idiopathic scoliosis. Basic proliferation medium for BMMSCs contained alpha
minimum essential medium (�MEM, Sigma Aldrich) buffered with 20 mM HEPES and
containing 10 % heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 0.1
mg/ml streptomycin and 2 mM L-glutamine. Samples from bone marrow were
suspended in basic proliferation medium and then the suspension was transferred into
cell culture flask. BMMSCs were allowed to attach for 48 hours at 37oC under 5 % CO2
43
and 20 % O2. Nonattached cells were removed by changing fresh medium and attached
cells were cultured at the bottom of the flask until they reached 70-80 % confluence.
The medium was changed twice a week and cells in passages 3-6 were used in
experiments included in this thesis.
4.1.2. Umbilical cord blood-derived mesenchymal stem cells (I,IV)
UCBMSCs were provided by the Finnish Red Cross Blood Service. Cord blood
collections were performed at the Helsinki University Central Hospital, Department of
Obstetrics and Gynaecology, and Helsinki Maternity Hospital after written consent and
the study protocol was approved by the ethical review board of Helsinki University
Central Hospital and the Finnish Red Cross Blood Service. The culture conditions for
UCB-MSC differed from BM-MSCs. Mononuclear cells were isolated using Ficoll-
Hypaque (Amersham Biosciences, Piscaway, NJ, USA) gradient centrifugation. Tissue
culture plates (Nunc) were coated with fibronectin (Sigma) and mononuclear cells were
plated at a density of 1 � 106/cm2. The initial umbilical cord blood mesenchymal stem
cell (UCB-MSC) line establishment was performed under hypoxic conditions (5 % CO2,
3 % O2 at 37oC) in medium containing �MEM with Glutamax (Gibco, Grand Island, NY,
USA) and 10% fetal calf serum (Gibco) supplemented with 10 ng/ml epidermal growth
factor (EGF, Sigma), 10 ng/ml recombinant human platelet-derived growth factor
(rhPDGF-BB; R&D Systems, Minneapolis, MN, USA), 50 nM Dexamethasone (Sigma),
100 U/ml penicillin together with 100 �g/ml streptomycin (Invitrogen). Cells were
allowed to adhere overnight and non-adherent cells were washed out with medium
changes. Hence, UCBMSCs were cultured under normoxic conditions (5 % CO2 and
20 % O2 at 37oC) and proliferation medium was renewed twice a week.
4.1.3. Monocyte culture, differentiation and co-culture with hMSCs (IV)
The THP-1 human monocyte cell line from an acute monocytic leukemia patient was
obtained from ATCC (TIB-202). THP-1 cells were expanded in RPMI 1640 medium
(Lonza) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 0.1 mg/ml
streptomycin and 2 mM L-glutamine (expansion medium). The macrophage
differentiation of THP-1 cells was performed as follows: THP-1 cells were suspended in
the expansion medium supplemented with 100 ng/ml phorbol 12-myristate 13-acetate
(PMA, Sigma Aldrich) and then 100 000 cells per well were added to 24-well plates
44
and were incubated for 48 h at 37oC under 5% CO2 and 20% O2. The co-culture assay
medium contained �MEM buffered with 20 mM HEPES supplemented with 100 U/ml
penicillin, 0.1 mg/ml streptomycin and 2 mM L-glutamine. First, 100 000 hMSCs per
well were added (the final ratio of hMSCs and THP-1 macrophage cells were 1:1) in a
24-well plate where PMA-treated THP-1 macrophage-like cells had already been were
plated, following with 100 ng/ ml LPS (purified from Escherichia coli 055:B5; Sigma
Aldrich) or 100 ng/ml IFN-c (Sigma Aldrich) were added. Coculture was performed for
24 hours at 37oC under 5% CO2 and 20% O2 followed by the collection of supernatants
for cytokine assays. The change in TNF-� or IL-10 secretion was determined by
comparing cytokine levels in co-culture with hMSCs in the presence of LPS or IFN-� to
the cytokine levels in THP-1 macrophage-like cells alone in the presence of LPS or IFN-
�. Mouse anti-human PE-conjugated CD200 antibody (BD Biosciences) was used in
some experiments to block CD200 in hMSCs. For blocking, the hMSCs were incubated
with antihuman CD200 antibody for 20 minutes in PBS with 0.5% bovine serum
albumin (BSA) and then washed twice with PBS and suspended in co-culture assay
medium whereas the control cells of the same hMSC pool remained untreated.
4.2. Microbial cultivations and bacterial infection of hMSCs (III)
Five different bacterial strains were used: Pseudomonas aeruginosa PA01 (VTT E-
84219), Staphylococcus aureus Cowan I (DSM 20372) and Staphylococcus
epidermidis ATCC 14990, S. aureus KK1089 and S. epidermidis KK1087. The first
three strains were obtained from commercial culture collections and S. aureus KK1089
and S. epidermidis KK1087 were isolated from discarded, contaminated batches of
thrombocyte preparations. The bacterial strains were stored in skim milk suspension at
-80 oC and before each co-culture experiment new bacterial mass was thawed and
plated onto trypticase soy agar (TSA; Tammer Tutkan Maljat Ltd, Finland) plates and
cultivated at 37 oC over night. Following incubation, a bacterial suspension was
prepared by inoculating single colony into 5 ml of soy casein-broth (Tammer Tutkan
Maljat Ltd., Finland) and the suspension was cultivated at 37 oC over night. Next,
bacteria were pelleted, washed twice with 10 ml of phosphate buffered saline (PBS), pH
7.2, and finally suspended in 1 ml of PBS. The bacterial suspensions were adjusted to
an optical density of 1.0 at A600nm by Shimadzu UV-1700 Pharma Spec
spectrophotometer. This was estimated to correspond to approximately 108
bacteria/ml. Finally, series of 10-fold dilutions of each bacterial suspension was
45
prepared in PBS, and three dilutions with the smallest number of bacteria were
selected in experiments.
The bacterial infection of BM-MSCs: BM-MSCs were washed with PBS, trypsinized
from culture flasks and suspended in expansion medium without antibiotics. Infection
was performed in 96-well plates. BM-MSCs were plated as 10 000 cells per well and
cultured over night at 37oC under 5 % CO2 and 20 % O2. Bacterial dilutions
corresponding approximately to 101,102 and 103 bacteria/well were inoculated, each
individual strain separately, into the 96-well plates where BMMSCs had been grown
over night.
4.3. Induction of osteogenic differentiation (I,II)
hMSCs were cultured for 14, 21 or 28 days in osteogenic induction medium that
contained basic cell culture medium supplemented with 100 nM dexamethasone, 10
mM b-glycerolphosphate, and 0.05 mM ascorbic acid-2-phosphate at 37oC under 5%
CO2 and 20% O2. Basic cell culture medium was used as a control.
4.4. Flow cytometry analysis
4.4.1. Measurement of mitochondrial inner membrane potential (II)
hMSCs were detached from culture flasks and resuspended in phosphate buffer saline
(PBS) (100,000 cells/mL). Samples were stained by incubating with 1 �M JC-1 for 30
min at 37oC. As a positive control, the protonophore carbonyl cyanide m-
chlorophenylhydrazone (CCCP) was used to depolarize the ��m. Samples were
analyzed using FACSCalibur (Becton Dickinson), equipped with laser emitting at 488
nm. JC-1 monomers were detected in FL1 using a 530/30nm bandpass filter. The
emission of JC-1 aggregates was detected in FL2 using a 585/42nm bandpass filter.
Compensation for signal spillover was performed off line with the FlowJo software
(TreeStar Inc. Ashland, OR). Flow cytometry data were also analyzed with FlowJo. Cell
debris was gated out from all samples and the rest of the cell population was analyzed.
46
4.4.2. Measurement of Annexin V and Propidium iodide (II)
After a wash with PBS, cells were incubated with AnnexinV-Alexafluor 647 (Invitrogen,
Carlsbad, CA) for 10 min at room temperature in AnnexinV working buffer containing
10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, and 0.5 % bovine serum albumin (BSA)
pH 7.4. To determine the plasma membrane integrity, cells were incubated with
propidium iodide (PI) (0.25 mg/mL; Becton Dickinson, Heidelberg, Germany) for 15
min at room temperature. Samples were analyzed using FACSCalibur (Becton
Dickinson), equipped with laser emitting at 488 nm. Flow cytometry data were
analyzed with FlowJo. Cell debris was gated out from all samples and the rest of the
cell population was analyzed.
4.4.3. The analysis of cell surface antigens (I,II)
hMSCs were detached from culture flasks, suspended in PBS + 0.5% BSA and
incubated in the presence of the following conjugated antibodies for 20 min at room
temperature: CD90 (FITC; Stem Cell Technologies, Grenoble, France), CD73 (PE; BD
Biosciences), HLA-ABC (APC; BD Biosciences), and CD105 (FITC; Abcam, Cambridge,
United Kingdom). Negative surface antigens for hMSCs were incubated simultaneously
as a group with the same sample: HLA-DR (PE; BD Biosciences), CD34 (PE; BD
Biosciences), CD45 (PE; BD Biosciences), CD14 (PE; BD Biosciences), and CD19 (PE;
BD Biosciences). After incubation, cells were washed with PBS + 0.5 % BSA and
analyzed by FACSAria or FACSCalibur. Flow cytometric data were analyzed with
FlowJo. Cell debris was gated out from all samples. Based on unlabeled cells, the gate
for positivity was defined.
4.4.4. Measurement of mitochondrial superoxide levels (I)
Mito- SOX (Molecular Probes, Invitrogen) was used to measure mitochondrial
superoxide levels. Detached cells were suspended in PBS, incubated with 5 �M
MitoSOX for 10 min at room temperature, and washed with PBS. Flow cytometry
analysis was performed with FACSCalibur and equipped with lasers emitting at 488
and 633 nm. MitoSOX fluorescence was excited at 488 nm, emission was measured at
585/42 nm, and data were analyzed with FlowJo. MitoSOX positive cells were
determined by a negative control. Cell debris was gated out from data analysis.
47
4.5. Transmission electron microscopy (I)
hMSCs grown in cell culture flasks were fixed for 10 min in 1 % glutaraldehyde 4 %
formaldehyde mixture in 0.1 M phosphate buffer, then scraped off, and pelleted,
followed by further fixation for 1 h. Then, the cell pellets were immersed in 2 % agarose
in distilled water, postfixed in 1 % osmiumtetroxide, dehydrated in acetone, and
embedded in Epon LX 112 (Ladd Research Industries). Thin sections were cut with
Leica Ultracut UCT ultramicrotome, stained in uranyl acetate and lead citrate, and
examined in a Philips CM100 transmission electron microscope. Image analysis was
performed with MCID Core 7.0 software (InterFocus Imaging LTD).
4.6. Micro plate reader
4.6.1. Measurement of specific alkaline phosphatase activity (I,II)
Alkaline phosphatase activity (ALP) was used to determine the level of osteogenic
differentiation. 4-p-nitrophenylphosphatenitrophenylphosphate as a substrate (0.1
mM) was used and absorbance was read at 405nm in a plate reader (Victor 2; Wallac
Oy). Four parallel samples were measured in duplicate. The ALP acitivty was
normalized against the protein levels. The protein contents of the wells were
determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The
specific ALP activity was expressed as absorbance at 405 nm/mg/mL protein.
4.6.2. Measurement of calcium content (I,II)
The calcium content was measured as described below. After osteogenic induction, 0.6
M HCL was added to cells and incubated overnight at room temperature. Calcium
content was determined based on the reaction of calcium with o-cresolphthalein-
complexone according to the manufacturer’s instruction (Roche Diagnostics
Corporation, Indianapolis, IN). The colorimetric reaction was measured at 550nm in a
plate reader (Victor 2; Wallac Oy). Four parallel samples were measured in duplicate.
48
4.6.3. Measurement of mitochondrial inner membrane potential (III)
After washing with PBS, mitochondrial inner membrane potential sensitive dye
enhanced 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-
10; Enzo Life Sciences) was used. The JC-10 staining solution (5 �M) was prepared.
Cells were incubated for 45 min at 37oC under 5 % CO2 and 20 % O2. Following
incubation, the staining solution was removed and PBS was added. The JC-10 signal
was detected by a fluorescence plate reader (Victor2 1420 Multilabel counter, Wallac).
Fluorescence was excited at 488 nm and the emission was measured at 595 ± 42 nm
and 535 ± 30 nm. The background fluorescences of 595 nm and 535 nm from wells
without cells were subtracted from the analyzed samples. The 595 nm/535 nm
emission - ratio was determined from four different replicates and the ratio of infected
or CCCP treated cells were compared to the control cells. CCCP was used to depolarize
��m, which was seen as a decrease in the 595 nm/530 nm emission-ratio.
4.6.4. MTT proliferation assay (II)
Five hundred cells per well were plated in 96-well plate, and cell proliferation was
measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay after 1, 7, and 14 days culture as described below. MTT was added to the medium
at a final concentration of 0.5mg/mL, and cells were incubated for 2 h at 37oC under
5 % CO2 and 20 % O2. After incubation, the staining solution was removed and 100 �L
per well of dimethyl sulphoxide was added. The absorbance of the reduced form of
MTT was measured at 550 and 650nm (background) in a plate reader (Victor 2; Wallac
Oy, Turku, Finland).
49
4.6.5. Enzyme linked immuno sorbent assays (ELISA) (IV)
TNF-� and IL-10 levels were determined from supernatants as described below. TNF-�
and IL-10 capture antibodies (TNF-�, BD Pharmingen; IL-10, BD Pharmingen) were
diluted to 1 �g/ml and incubated in maxi-sorp 96-well plates (Nunc) over night at 4oC.
Next, unbound capture antibodies were removed and wells were blocked with PBS with
1.0% BSA (Sigma) for 1 hour. Washes were performed with PBS supplemented with
Tween-20 (Sigma). IL-10 and TNF-� standards (TNF-�, BD Pharmingen; IL-10, BD
Pharmingen) were incubated together with the collected supernatants followed by
thawing and incubation in 96-well maxisorp plates for two hours at room temperature.
After two hours of incubation, plates were washed five times with PBS containing
Tween-20. Biotin-labeled IL-10 and TNF-� antibodies (IL-10, BD Pharmingen; TNF-�,
BD Pharmingen) were diluted to 0.25 �g/ml in PBS with 1.0% BSA and Tween-20 and
incubated for one hour at room temperature. Wells were washed five times with PBS
containing Tween-20. Avidinhorseradish peroxidase (HRP) conjugate (BD
Pharmingen) was diluted in PBS with 1.0% BSA and Tween-20 (1:1000) and was
incubated for 30 minutes. Wells were washed five times with PBS with Tween-20.
Substrates of 3.39, 5.59 tetramethylbenzidine (TMB, PD Pharmingen) reagent set were
mixed in 1:1 and added to the wells and color development was followed. The reaction
was stopped with 1 M phosphoric acid and absorbance was measured at 450 nm with
ELISA plate-reader (MultiScan). Cytokine concentrations were determined by Multi-
Calc software. Results from co-cultures are indicated as %-change in cytokine secretion
when compared to activated THP-1 macrophage-like cells alone.
4.7. Western blot (I)
Samples for western blot were suspended into lysis buffer consisting of 50 mM Tris,
0.9 % NaCl, 0.2 % NaN3, 0.1 % Triton X-100, 0.1 % deoxycholic acid, and protease
inhibitor. Protein concentrations were determined by Bio-Rad Protein Assay (Bio-Rad
Laboratories). Protein samples (10 �g of protein) were separated on 12% SDS-PAGE
and blotted on nitrocellulose membrane. Blocking was performed by using 5% milk
powder in PBS + 0.1% Tween20 (PBST; Sigma-Aldrich). The membrane was incubated
with MnSOD (anti-rabbit, 1:100,000; Abcam) and b-actin (anti-mouse, 1:7,500;
Sigma-Aldrich) primary antibodies in PBST over night at 4oC in a shaker following
50
secondary antibody (anti-rabbit IgG IR800 conjugated 1:1,000 and anti-mouse
IgGIR700 conjugated 1:1,000) incubation for 1 h, the MnSOD and �-actin signals were
measured by Odyssey Infrared Imager System (Li-Cor Biosciences). To exclude
variations in amounts of loaded protein, MnSOD signals were normalized against �-
actin.
4.8. Trypan blue staining (III)
Trypan blue (Stem Cell Technologies) was added onto the cells. Cells were studied by
inverted microscope Leica DM IL LED and photographed by Leica DFC 420 digital
microscope camera.
4.9. Statistics (I, II, III, IV)
Statistical analysis and all diagrams were performed by using the OriginPro version 8.
All data are presented as mean±standard deviation (SD) of the results from different
number of independent replicates indicated in each section. The significance level was
determined by two-sample t-test and one way analysis of variance (ANOVA) with
following means comparison with Tukey’s test when CD200 high, medium and low
BMMSCs were compared together in original publication of IV. The p-value p<0.05
was considered as statistically significant.
51
5.RESULTS
5.1. hMSCs from different origin show identical immunophenotype characteristics but differ by their ultrastructure (I,II)
BM-MSC derived from donors over 50 years old (BM-MSC>50) and BM-MSC from
donors under 18 years old (BM-MSC<18) both showed characteristics of hMSCs by
fulfilling the minimum criteria panel of cell surface antigens established by ISCT and
also the three lineage differentiation potency (II, Figure 1). Also UCB-MSC used in
this study have shown the characteristics of hMSCs, excluding one UCB-MSC line
whose CD90 positivity was under 95 % (data not shown). In this comparison study
three BM-MSC>50, three BM-MSC<18 and three UCB-MSC lines were selected. UCB-
MSCs were expanded in presence of rhPDGF, EGF and dexamethasone whereas BM-
MSCs were grown without supplemented growth factors.
Morphologically UCB-MSC, BM-MSC<18 and BM-MSC>50 showed spindle-shape
but the cell size differed dramatically (I, Figure 1A,B). UCB-MSC possess over three
times smaller cell surface area after one day of plating when compared to BM-MSC>50.
Also cell size in suspension measured by flow cytometry differed between UCB-MSC
and BM-MSC>50 (I, Figure 1C). Interestingly BM-MSC<18 showed more similarity
with UCB-MSC than BM-MSC>50 in their cell size on the bottom of the culture flask
and also in suspension. Ultrastructural analysis of hMSCs from different origin by TEM
showed also remarkable differences between hMSC groups (I, Figure 2). UCB-MSC
showed more abundant rough endoplasmic reticulum (rER) which were organized in a
parallel stack of lines whereas BM-MSC<18 and BM-MSC>50 showed more dilated
rER structures. In addition, BM-MSC>50 showed the presence of vacuoles which were
not evident in UCB-MSC and BM-MSC<18. Mitochondrial morphology in
undifferentiated hMSCs showed native undeveloped form where clear christae were
undetectable. These results clearly indicate that even though these cells are identical in
their immunophenotype characteristics they have remarkable differences in cell size
and ultrastructure, which may indicate different function of the cells. The differences in
ultrastructure and in morphology are most likely explained by the differences in
culture conditions between UCB-MSC and BM-MSC.
52
5.2. Differences in mitochondrial properties between UCB-MSC and BM-MSC before and after osteoinduction (I)
Ultrastructural analysis of BM-MSC<18, BM-MSC>50 and UCB-MSC after two weeks
of osteogenic differentiation showed differences in mitochondrial properties when
compared to undifferentiated ones (I, Figure 3A-L). The mitochondrial area in UCB-
MSC was highest whereas BM-MSC>50 showed the lowest mitochondrial-to-cytoplasm
area ratio (I, Figure 3M). Again, BM-MSC<18 were between the two other groups.
Mitochondrial morphology was changed in UCB-MSC and BM-MSC<18 from native
unenergized mitochondria to the more mature and energized ones. Clear chirstae were
evident after 14–day osteogenic differentiation and mitochondria showed also higher
density when compared to undifferentiated ones. In BM-MSC>50 the changes in
mitochondrial structure was not as dramatic as in the two other hMSC types. However,
the mitochondrial area increased most in BM-MSC>50 after 14-day osteoinduction
showing almost three times higher mitochondrial-to-cytoplasm ratio after
osteoinduction whereas UCB-MSC and BM-MSC<18 showed milder increase in
mitochondrial area (I, Figure 3M).
UCB-MSC and BM-MSC<18 showed clear cristae of mitochondria after 14-day of
osteoinduction and also higher mitochondrial area before differentiation when
compared to BM-MSC>50. On the other hand, BM-MSC>50 showed lower
mitochondrial area before osteoinduction and the highest increase in mitochondrial
area without any changes in mitochondrial ultrastructure. Therefore, it could be
suggested that the increase in mitochondrial area takes place first, after which changes
in mitochondrial ultrastructure, e.g. christae formation are evident. The level of
osteogenic differentiation was also measured after a four weeks differentiation period
and the results were in line with observation on mitochondrial ultrastructures and area
(I, Figure 3N). Three hMSC lines from each group were selected for osteogenic
differentiation assay. After 14-day osteoinduction, UCB-MSC showed highest Ca2+
deposition whereas BM-MSC>50 showed the lowest Ca2+ deposition when compared to
undifferentiated hMSCs. BM-MSC<18 was between UBM-MSC and BM-MSC>50.
Therefore it seems that the higher mitochondrial-to-cytoplasm ratio may also be
related to the osteogenic differentiation efficiency.
A more detailed examination was carried out on mitochondrial properties after 14-
day osteoinduction. Mitochondrial superoxide levels were measured by using MitoSOX
53
reagent and analyzed by flow cytometry (I, Figure 4A). Three different hMSC lines
were selected for the analysis from each group. MitoSOX levels were then compared to
14-day osteoinduced hMSCs. BM-MSC<18 and BM-MSC>50 were mostly MitoSOX
negative but showed significant over 2-fold increase in number of MitoSOX positive
cells after 14-day osteoinduction. Interestingly UCB-MSC lines were mostly positive for
MitoSOX and the positivity did not change dramatically during 14-day osteoinduction.
The results from the present study show that UCB-MSC posses already elevated
mitochondrial ROS levels, which may reflect their more active mitochondrial function.
Since the shift from anaerobic to aerobic metabolism has been shown to occur during
the osteogenic differentiation, this result could indicate that UCB-MSC may represent
early osteoprogenitors when compared to BM-MSC>50 which showed neglient
MitoSOX positivity before 14-day osteoinduction.
MnSOD is an enzyme that neutralizes superoxide in mitochondria to form
hydrogen peroxide. The levels of MnSOD were measured by western blot from
undifferentiated UCB-MSC, BM-MSC<18 and BM-MSC>50 (I, Figure 4I). The level
of MnSOD in UCB-MSC was already elevated when compared to BM-MSC<18 and BM-
MSC>50. In addition, the MnSOD amount increased after 14-day of osteoinduction in
UCB-MSC and BM-MSC<18 lines whereas in BM-MSC>50 there was no increase in the
level of MnSOD. Again, the ROS scavenging enzyme MnSOD was already elevated in
UCB-MSC suggesting more mature mitochondrial function whereas BM-MSC>18
showed increase in MnSOD levels only after 14-day osteoinduction and BM-MSC>50
have negligent MnSOD levels even after 14-day osteoinduction. These results clearly
indicate that mitochondrial changes occur during early osteogenic differentiation, most
likely due to increased energy demand, and by measuring mitochondrial number,
mitochondrial ROS levels and amount of MnSOD it could be possible to distinguish
different subpopulations of hMSCs with different osteogenic differentiation
potency.
54
5.3. Monitoring of ��m: a sensitive quality control tool for hMSCs (II)
The sensitivity of monitoring mitochondrial function as an early apoptosis indicator
was investigated by measuring ��m. First two commercially available and widely used
dyes, Rhodamine123 and JC-1 were tested and compared. However, preliminary
studies with CCCP showed a higher sensitivity of JC-1 to detect changes in ��m and
therefore JC-1 was selected for further experiments. Based on ��m, cellular
morphology and cell membrane integrity, JC-1 loaded hMSCs could be distinguished
into four different groups (II, Figure 2). The first group, R1, contained cells with
normal ��m, cells in R2 showed partly depolarized mitochondria with normal
morphology, R3 group contained cells with severly depolarized mitochondria where
part of the cells showed abnormal membrane integrity (measured by PI) and cell
shrinkage, and R4 cells had completely depolarized mitochondria, a disrupted cell
membrane and showed shrinkage of the cell size.
The sensitivity of JC-1 to detect early apoptosis was tested with mild menadione
exposure for 7 hours to induce apoptosis. Results showed gradual shift first from group
R1 into groups R2 and R3 and finally after 7 hours of exposure into group R4 (II,
Figure 3). Cells at different time points of menadione treatment were also plated for
proliferation and osteogenic differentiation assays to test the functionality of the cells.
Results from ALP and MTT assays showed that the proliferation and differentiation
capacity of menadione exposed hMSCs decreased gradually (II, Figure 4BC).
Following these experiments JC-1 was compared with another early apoptosis marker
AnnexinV. hMSCs loaded with AnnexinV and JC-1 were analyzed by flow cytometry.
Results showed that the changes in ��m precede changes in the organization of
phosphatidyl serines in the cytoplasmic membrane (II, Table 1). The correlation
between ��m and functionality of hMSCs in menadione exposure experiments led also
to analysis of untreated hMSC lines. Analysis of several hMSCs lines showed variability
in JC-1 staining pattern (II, Figure 5.). Tests were performed to study if it is possible
to predict the osteogenic differentiation potency of hMSCs by measuring ��m before
osteogenic differentiation. Results showed that the JC-1 aggregates / JC-1 monomers –
ratio correlated strongly with Ca2+ deposition and ALP activity (II, Figure 7.). The
results from the JC-1 studies clearly indicate the potential of mitochondrial analysis as
a quality control tool for hMSC intended for cellular therapies, since the viability and
also potency of hMSC can be measured more rapidly and accurately when compard to
traditional tools.
55
5.4. Monitoring of ��m: detection of microbial infection caused cellular stress of hMSCs (III)
The potential of ��m sensitive probes to detect cellular stress in hMSCs caused by
microbial infection was studied. The enhanced version of JC-1 (JC-10) was tested and
selected for further experiments, because of its’ better suitability for the plate reader
format. Five different bacteria strains, namely P. aeruginosa PA01, S. aureus Cowan
I, S. aureus KK1089, S. epidermidis ATCC 14990 and S. epidermidis KK1087, were
inoculated as 101, 102 and 103 bacteria/ well in the presence of three different BM-MSC
lines and monitored for 24 hours for signs of infection on the viability of hMSCs.
Preliminary results showed that even the lowest bacterial inocula (101 bacteria /
well) caused similar level of dissipation of hMSCs after 24 hours than many times
higher inoculated bacteria loads (III, Figure 1). On the other hand, the effect of the
different bacterial strains on hMSCs after 24 hours varied. S. aureus strains formed
large aggregates and caused the increase in number of dead cells indicated by trypan
blue staining whereas the other bacterial strains caused total dissipation of
hMSCs. Based on these preliminary results the highest bacterial load was selected
for further experiments. The early effect of the inoculation of 103 bacteria / well was
studied. It was apparent that all the bacteria needed 3-4 hours to adapt hMSCs
culture conditions but after this lag phase a rapid proliferation was evident as
indicated by the increased number of dead hMSCs shown by trypan blue staining
(III, Figure 2). Again there were some differences between the bacteria in
their adaptation time and behavior where P. aeruginosa showed the most
aggressive expansion and destruction of hMSCs during the first 5-6 hours whereas S.
aureus and S.epidermidis strains appeared to need a longer adaptation time.
To test the sensitivity of JC-10 to detect microbial infection caused mitochondrial
damage in hMSCs the 4h time point after infection was selected for measuring ��m. At
the 4h time point no dramatic changes in cellular morphology or in the
number of dead hMSC as measured by trypan blue were evident. The effect
of bacterial infection of ��m was tested with three different BM-MSC lines. Results
showed that most of the bacterial strains and inoculated bacterial loads caused
significant decrease in ��m before cell death was evident as measured by trypan blue
(III, Figure 3). Only S. aureus Cowan I strain had no effect on the ��m even at the
highest inocula whereas some of the strains, such as P. aeruginosa, S. epidermidis
ATCC14990 and S. epidermidis KK1087 caused significant depolarization of � �m
already at the lowest
56
inoculated bacterial load. The sensitivity of ��m analysis to detect cellular stress
induced by microbial infection was clearly superior when compared to traditional
detection of dead cells by trypan blue.
5.5. Mesenchymal stem cells showed different expression pattern ofCD200 (IV)
Study of the cell surface antigens of BM-MSC and UCB-MSC lines revealed differences
in expression of CD200, which has been shown to play a role in immunomodulation of
macrophages. Flow cytometric analysis of CD200 from 20 different hMSC lines (17
BM-MSC + 3 UCB-MSC) showed the variation in CD200 expression from highly
positive (CD200Hi, 50-70 % positivity) to medium (CD200Me, 10-30 % positivity) and
low positive (CD200Lo, 5-8 % positivity) in BM-MSCs, whereas UCB-MSC were
completely negative for CD200 (IV, Table 1.). Most of the BM-MSC lines belonged to
the CD200Me group whereas only few lines showed high positivity to CD200. The
CD200 expression was also confirmed by immunofuorescent microscopy (IV, Figure
1). In addition, the effect of in vitro passaging on the CD200 expression of hMSCs was
tested with the CD200Hi cell line. The positivity did not change dramatically during in
vitro expansion (IV, Table 2). To clarify the role of CD200 in immunosuppression,
the following hMSC lines were chosen for further experiments: CD200Hi 1, CD200Hi 2,
CD200Me 1, CD200Me 2, CD200Lo 1, CD200Lo 2, UCB-MSC 1 and UCB-MSC 2.
5.6. The secretion of TNF-� from THP-1 macrophages is inhibited by BM-MSCs in the presence of IFN-� (IV)
THP-1 monocyte-derived macrophage-like cells were used as a macrophage model to
study the role of CD200-CD200r axis in hMSC mediated immunosuppression of
macrophages. Preliminary studies showed that when THP-1 cells were incubated for 48
hours in PMA (100 ng/ml) the cells started to adhere to the bottom of the wells (IV,
Figure 2). Moreover, a further 24h treatment with LPS caused significant increase in
TNF-� secretion. In addition, IFN-� was used as an activator of macrophages, but the
response to the IFN-� was not as great as with LPS. The preliminary experiments from
the co-cultures between hMSCs and THP-1 macrophages showed that the co-culture
inhibited the secretion of TNF-� from THP-1 macrophages in the presence of IFN-�
57
whereas no significant impact of hMSCs on TNF-� secretion of LPS activated THP-1
macrophages was evident. In addition to TNF-�, the secretion of IL-10 was also
measured (IV, Figure 2B). The activation of THP-1 macrophages with LPS or IFN-�
did not induce IL-10 secretion but the co-culture with hMSCs caused significant
increase in IL-10 levels under all experimental conditions.
Preliminary results indicated that hMSCs may inhibit TNF-� secretion from THP-1
macrophages more efficiently in the presence of IFN-� than LPS. To confirm this, the
co-culture between THP-1 macrophages and eight hMSC lines in the presence of LPS
and IFN-� was repeated and TNF-� levels were measured after 24h of co-culture (IV,
Figure 3). TNF-� concentrations from co-cultures were then compared to the TNF-�
levels of THP-1 macrophages cultured alone. The presence of hMSCs decreased TNF-�
secretion from THP-1 macrophages in the presence of IFN-� more efficiently whereas
in the presence of LPS there were practically no differences in TNF-� levels between
co-cultures and THP-1 macrophages cultured alone.
5.7. The role of CD200-CD200r axis in hMSC mediated immunosuppression of macrophages
The CD200r expression was confirmed from THP-1 macrophages. Immunofluorescent
staining showed that the CD200r expression was evident only in IFN-� activated THP-1
macrophages (IV, Figure 4A-F). In the presence of IFN-� CD200Hi 1 and CD200Hi
2 showed the most striking decrease in the TNF-� secretion of THP-1 macrophages
while CD200Me 1 and CD200Me 2 showed slightly weaker inhibition of TNF-�
secretion from THP-1 macrophages and CD200Lo 1 and CD200Lo 2 showed the lowest
inhibition of TNF-� secretion from THP-1 macrophages when compared between BM-
MSCs (IV, Figure 4G). Moreover, both UCB-MSC lines had only mild impact on the
secretion of TNF-� from THP-1 macrophages in the presence of IFN-�. These results
clearly demonstaretd the correlation between CD200 expression in hMSC and the
inhibition of TNF-� secretion. However, the results from co-cultures in the presence of
LPS were compleately different (IV, Figure 4H). Only few hMSC lines were able to
inhibit TNF-� secretion significantly, namely CD200Me 1, CD200Lo 2 and UCBMSC 2
showing no correlation with the expression of CD200 in hMSC.
58
The role of the CD200-CD200r axis in hMSCs mediated immunomodulation of THP-1
macrophages was also confirmed by antibody blocking experiments (IV, Figure 5).
CD200Hi 1, CD200Hi2 and CD200Me 1 lines were pre-treated with anti-human CD200
antibody and the the ability to inhibit TNF-� secretion from THP-1 macrophages was
compared to the untreated CD200Hi 1, CD200Hi 2 and CD200Me 1 BM-MSCs.
Untreated CD200Hi/Me BM-MSC lines decreased the TNF-α secretion over 50 %
when compared to THP- macrophages alone whereas anti-human CD200 antibody
pretreated CD200Hi/Me (CD200ab block) BM-MSCs decreased TNF-� secretion
from THP-1 macrophages in the presence of IFN-� only about 30 %. These results
clearly indicate that the CD200-CD200r axis has an important role in hMSC
mediated immunosuppression of macrophages.
On the contrary, in the presence of LPS, the anti-human CD200 antibody pre-
treatment of CD200Hi/Me BM-MSC did not have any impact on their inhibiting
capacity of TNF-� secretion from THP-1 macrophages when compared to
CD200Hi/Me untreated BM-MSCs. In addition, the pre-treatment of UCBMCS 1 with
anti-human CD200 antibody did not have any impact on the ability to inhibit TNF-�
secrfetion from THP-1 macrophages in the presence of IFN-�.
59
6.DISCUSSION
6.1. Characterization of hMSCs in accordance with ISCT guideline showed no dramatic differences between UCB-MSC and BM-MSC
The lack of detailed characteristics of hMSCs is one of the major problems and
hindrances in the transfer of hMSC-based cellular products from laboratories to the
clinics. The minimum panel for cell surface antigens established by ISCT is considered
as the first criterion for characterization of hMSCs, althought it has been critized by the
lack of specifity for the markers for hMSCs (Dominici et al. 2006, Haniffa et al. 2009).
In accordance with previous reports, the present study noted no dramatic differences
in the minimum panel of cell surface antigens established by ISCT (Kern et al. 2006,
Rebelatto et al. 2008).
The present study has focused on ultrastructural, mitochondrial and morphological
properties of hMSCs from different source and age; namely umbilical cord blood (UCB-
MSC), bone marrow of under 18 years old donors (BM-MSC<18) and bone marrow of
over 50 years old donors (BM-MSC>50). The limitation of this comparison study is the
different culture conditions for UCB-MSC and BM-MSC. Therefore we can not
conclude that the mitochondrial and cellular differences between these hMSC types
reflect the situation in vivo. However, the focus of this work is to compare two hMSC
types that are potential cell sources for hMSC-based cellular therapies and they are
grown in different culture conditions also in clinical settings. Therefore the comparison
is conducted in their own culture conditions. Only one UCB-MSC line showed
abnormal CD90 expression, but other hMSC lines were identical regardless that UCB-
MSC and BM-MSC were grown in different culture conditions and possess different
cellular properties. In addition to immunophenotype characterization, the tri-lineage
differentiation potency of hMSCs represents the second criterion of hMSC
characterization. Accordingly, also the tri-lineage differentiation potency of UCB-MSC,
BM-MSC<18 and BM-MSC>50 was confirmed. On the other hand, as also reported by
others, difference in efficiency of osteogenic differentiation was noted (Guillot et al.
2008).
60
Interestingly, higher osteogenic differentiation potency was evident for UCB-MSC
than for BM-MSC and BM-MSC<18 also showed higher level of calcium deposition
than BM-MSC>50. In the previous comparison studies the results have varied
markedly, but age dependent decrease of osteogenic differentiation have been shown
when fetal, young and old BM-MSC have been compared (Zaim et al. 2012, Zhou et al.
2008). However, also discrepant results on the effects of aging have been
demonstrated when BM-MSC from adult and elderly cell donors have been compared
(Fickert et al. 2011, Leskela et al. 2003). Therefore it seems that the osteogenic
differentiation of BM-MSC is higher during childhood and in early adulthood when
compared to the elderly. However, the osteogenic differention potency seems not to
decline after adulthood.
The third criterion for hMSCs established by the ISCT is the fibroblast-like spindle
shape. In the present studies, the morphological analysis of UCB-MSC, BM-MSC<18
and BM-MSC>50 showed dramatic differences in the cell surface area of hMSCs.
Morphologically the cells presented as fibroblast-like spindle-shaped cells but the cell
surface area of attached BM-MSC>50 together with cell size in suspension were
dramatically higher when compared to the two other hMSC types. In addition,
ultrastructural analysis of BM-MSC<18, UCB-MSC and BM-MSC>50 showed several
interesting differences. UCB-MSC showed more abundant rER when compared to BM-
MSC<18 and BM-MSC>50. BM-MSC>50 showed also the presence of vacuoles.
However, the identity and specific function of these vacuoles remained unknown in
the present study. Similar kinds of vacuoles have been shown in previous studies of
BM-MSC (Karaoz et al. 2011). These differences in ultrastructure and cell size clearly
indicate that studied hMSCs types may represent different hMSC populations
even though they are immunophenotypically identical.
6.2. Mitochondrial properties as indicators of the osteoprogenitors of hMSCs
Mitochondrial bioenergetics of stem cells has been shown to differ from terminally
differentiated cells (Chen et al. 2008, Lonergan et al. 2006). Therefore it is possible
that mitochondrial and metabolical properties could be used as an alternative
characterization criteria for hMSCs. In the present studies, analysis and comparison of
several mitochondrial properties from UCB-MSC, BM-MSC<18, BM-MSC>50 and 14-
61
day osteoinduced cells were performed. Based on the results, it is suggested that
determination of mitochondrial number, mitochondrial superoxide levels and
antioxidant defense system could be utilized as useful indicators to distinguish
different subpopulations of hMSCs. Comparisons were carried out on the
mitochondrial area between BM-MSC<18, BM-MSC>50, UCB-MSC and 14-day
osteoinduced cells. UCB-MSC showed the highest mitochondrial area whereas BM-
MSC>50 showed the lowest mitochondrial area, while BM-MSC<18 were in-between
UCB-MSC and BM-MSC<50. In addition, during 14-day osteoinduction, the
mitochondrial area increased significantly in all hMSC types. However, the increase
was greatest in BM-MSC>50 and lowest in UCB-MSC, which indicates that in UCB-
MSC the mitochondrial number has already increased, which was also evident
in comparison between undifferentiated UCB-MSCs and BM-MSCs.
The increase in mitochondrial area measured from TEM slices may also reflect
increased mitochondrial volume due to oxidative stress and swelling of the
mitochondria. However, in the present studies, the area of individual mitochondria
seemed to stay normal, but the number of individual mitochondria increased.
Therefore it may be postulated that the increased mitochondrial area indicates an
increase in the number of mitochondria. In addition, other studies have used similar
methods to evaluate mitochondrial number during osteoinduction of murine MSCs and
demonstrated the increase in mitochondrial number during early differentiation (An et
al. 2010).
There is some inconsistency in observations of mitochondrial biogenesis during
early osteogenic differentiation. The study by Chen et al. (2008) did not note any
change in mitochondrial mass even though the copy number of mtDNA increased and
expression of genes related to mitochondrial biogenesis increase. However, it is
important to notice that in that study BM-MSC were cultured under mix of growth
factors (Chen et al. 2008). Regardless, the present results suggest that the increase in
mitochondrial number does occur at the early phase of differentiation. Moreover, it is
possible that the differences in mitochondrial number and properties could indicate
hMSC population that is already committed to the osteogenic lineage when compared
to more multipotent hMSCs with low mitochondrial number and activity and could
therefore be utilized in characterizations of hMSCs. As a support for that, the
osteogenic differentiation efficiency was also in line with differences in mitochondrial
properties where UCB-MSC showed highest Ca2+ deposition and BM-MSC>50 lowest.
TEM analysis also showed that after 14-day osteoinduction, mitochondrial christae
were evident in UCB-MSC, which indicates more energized and mature mitochondria.
62
BM-MSC>50 showed only an increase in mitochondrial number but not yet clear
mitochondrial christae, which may reflect a slower osteogenic differentiation process.
It is obvious that the differences seen in mitochondrial properties and in osteogenic
differentiation potency is due to differences in culture conditions. UCB-MSCs are
cultivated under the influence of growth factors whereas BM-MSCs only in presence of
serum. However, this still shows that by mitochondrial analysis it could be possible to
distinguish more osteoprogenitor type cells from multipotent hMSCs.
The aging of BM-MSCs may explain the differences between BM-MSC<18 and BM-
MSC>50. That is supported by some recent work where hMSCs from rats of different
ages were compared and similar differences in mitochondrial number between neo-
natal, young and adult MSCs were seen as in the data of the presently discussed thesis
(Mantovani et al. 2012). Interestingly, in the studies of this thesis also the BM-MSC<18
lines showed similarities with UCB-MSC with regards to several properties supporting
the role of aging in function of hMSCs.
Mitochondria are the main source for intracellular ROS. There is no agreement
about the exact location and mechanisms for the production of mitochondrial
superoxide but the sites in complex I and complex III have been introduced (Hirst et al.
2008). In the present study, the mitochondrial superoxide levels were measured and
compared by MitoSOX as was also the amount of superoxide scavenging enzyme
MnSOD. Most of the BM-MSC<18 and BM-MSC>50 hMSCs were negative for
MitoSOX whereas UCBMSC were mostly positive. However, during 14-day
osteoinduction, in UCBMSC there was no significant change in MitoSOX positivity
whereas both BM-MSC groups showed dramatic increase in positivity. In addition,
levels of MnSOD from undifferentiated hMSCs and after 14-day osteoinduction were
measured. Interestingly, UCBMSC and BM-MSC<18 lines showed clear increase in
MnSOD levels after 14-day osteoinduction whereas in BM-MSC>50 lines there was no
change. The initial levels of MnSOD were already elevated in UCBMSC whereas both
BM-MSC groups showed negligible MnSOD levels. An increase in MnSOD levels was
also evident in UCBMSC and BM-MSC<18 which is in line with previous work, even
though the changes in mitochondrial superoxide levels differed (Chen et al. 2008).
The present studies showed clear increase in number of MitoSOX positive BM-
MSCs whereas previous work has shown decrease in superoxide levels during early
osteogenic differentiation (Chen et al. 2008). Again this could be explained by the
differences in culture conditions between BM-MSC, since in previous work, BM-
MSCs were grown in the presence of growth factors. Therefore, the initial levels
of ROS before osteogenic differentiation may differ between these studies. This is
also supported by the fact that
63
UCB-MSC which also were grown with a specific cocktail of growth factors did not
show increase in MitoSOX levels after 14-day osteoinduction. Owusu-Ansah & Banerjee
(2009) have shown that in hematopoietic stem cells the intracellular ROS levels are
tightly regulated during development of stem cells. Transient increase in ROS levels
occurs during the progenitor phase of stem cells whereas more native stem cells and
terminally differentiated blood cells show low levels of ROS. Moreover, ROS has been
shown to regulate the differentiation of hESC into cardiomyocytes (Crespo et al. 2010).
These data also support the regulative role of ROS in the fate of stem cells which
demonstrates that the analysis of ROS is a useful measure of stem cell potency. The
increase in ROS levels as was evident in BM-MSCs may have a key role in initiation of
differentiation but after long term of differentiation the activation of antidoxidant
defense system detoxifies the excess of ROS and thus prevents their toxic effect as has
been demonstrated in the hematopoietic system (Owusu & Banerjee 2009).
The present hypothesis that UCB-MSC may represent different subpopulation of
hMSCs with more active mitochondrial function when compared to BM-MSCs is also
supported by the differences in ROS and MnSOD levels. BM-MSC>50 showed increase
in mitochondrial superoxide levels but not in MnSOD after 14-day osteoinduction
whereas UCB-MSC showed already elevated ROS levels and slight decrease in ROS
levels after short term osteoinduction possibly due to increased MnSOD levels.
Mitochondrial analysis in parallel with immunophenotype analysis will provide more
detailed characteristics of the lineage commitment of hMSCs which could be utilized in
isolation of multipotent stem cells and thus producing more homogenous hMSCs
cultures (Figure 4.). Higher mitochondrial acitivty may reflect the commitment of
hMSCs towards osteogenic lineage. However, this should be confirmed by indicating
lower adipogenic or chondrogenic differentiation potency of these cells in the future.
Moreover, mitochondrial activity and dormacy may also directly regulate the
differentiation of hMSCs in a manner, which currently remains to be elucidated, as has
been demonstrated with hESCs (Mandal et al. 2011, dashed lines in Figure 4.).
64
Figure 4. Simplified illustration of mitochondrial and metabolical changes during early differentiation.
A) Mitochondrial number increases, mitochondrial ultrastructure changes and shift from anaerobic to
aerobic metabolism occurs shortly after differentiation (1.). ROS levels increases and differentiation
initiates simultaneously with inhibition of expression of stem cell markers (2.). After a long term
differentiation the antioxidant scavenging system activates and detoxifies excess ROS (3.). B)
Mitochondrial and bioenergetic properties could be used in parallel with immunophenotype analysis to
produce homogeneous hMSC cultures and/or products. Abbreviations: OCT4, octamer-binding
transcription factor 4; SOX2, sex determining region Y-box 2; HK, Hexokinase; LDH, lactate
dehydrogenase; PFK, phosphofructokinase; Pol �, DNA polymerase subunit gamma; mtTFA, transcription
factor A, mitochondrial; PGC-1�, peroxisome proliferator-activated receptor gamma coactivator 1-alpha.
65
6.3. Monitoring ��m as a quality control tool for hMSCs
Regulators, developers and cell therapy providers alike emphasize the need to develop
and establish sensitive, rapid and robust quality control tools for hMSCs intended for
cellular therapies. Standardization of the quality and safety of hMSC-based cellular
products and ensuring their functionality is essential. The most challenging
characteristics to show are the potency and viability of the hMSC products which are
also demanded by European legislation.
Current viability tools, such as trypan blue and propidium iodide staining based
methods, lack sufficient sensitivity and only recognize live cells from dead cells. Cells in
early apoptosis, severly stressed or senescent cells can not be detected by such methods.
However, by monitoring ��m it is possible to detect early changes in apoptosis but also
to monitor mitochondrial energy state which reflects the activity and metabolic status
of hMSCs. The depolarization of ��m and MPTP opening are well known and
characterized processes during early apoptosis (Heiskanen et al. 1999, Kroemer & Reed.
2000).
The present study has shown sensitivity of monitoring ��m to detect oxidative
stress and apoptosis when induced by menadione. Menadione has been shown to
induce oxidative stress by increasing ROS levels, increasing intracellular Ca2+
concentration and decreasing cellular ATP levels (Criddle et al. 2006, Gabai et al.
1990). Moreover, the comparison with AnnexinV and PI showed that the changes in
��m precede changes in plasma membrane organization and integrity. AnnexinV
detects the exposure of phosphatidyl serines (PS) on cell surface and PI the integrity of
the plasma membrane (van den Eijnde et al. 1998). Phosphatidyl serines are known to
change their orientation on the cell surface during early apoptosis and the plasma
membrane starts to leak during late apoptosis making cells sensitive for PI and trypan
blue. In addition to PI and tryban blue, currently several different proliferation assays
are also used, such as MTT, Alarm Blue, CFU-method etc. However, the most critical
disadvantage of these proliferation assays is their time consuming nature. CBMP have
short shelf-life and the viability and potency should be determined within hours
whereas determination of proliferation will take several days. The present studies have
shown that monitoring of ��m correlates with the MTT assay, which also monitors
mitochondrial activity, but the JC-1 based assay takes only few hours to perform.
Therefore it is more suitable for CBMP quality control monitoring than traditional
proliferation assays.
66
The most challenging characteristic to demonstrate is the potency of the CBMPs.
The potency assay for the product should be developed and established by a case-by-
case approach according to the essential function of the cell product after
administration. However, there are problems in development of assays in certain
applications since the exact mode of action of the product can not be elucidated.
Therefore, surrogate potency tests may also be useful. The studies of the present thesis
show that by monitoring mitochondrial function it is possible to predict osteogenic
differentiation potency of hMSC. In the future, it would also be beneficial to study
whether the similar kind of correlation is also evident between ��m and adipogenic or
chondrogenic differentiation potency. However, the reason for the correlation between
��m and osteogenic differentiation remains unknown. It is possible that the variability
in the quality of hMSC preparations may differ and hMSCs with lower ��m may
represent more stressed cells and thus lower differentiation potency. In addition,
hMSCs in vitro are sensitive to senescence and during senescene the depolarization of
��m has been shown to occur (Carracedo et al. 2011). Moreover, during differentiation
the ATP demand increases and mitochondrial energy production increases, and thus
mild depolarization of ��m is also induced (Nicholls. 1974). Therefore, hMSCs with
lower ��m may also represent already differentiated cells, such as fibroblasts,
myoblasts etc.
However, controversial results have been shown with pluripotent stem cells.
Previous research has shown that hESC population with low ��m was more prone to
differentiate into mesodermal lineage and showed lower teratoma formation than a cell
population with high ��m (Schieke et al. 2008). However, it is possible that the
mitochondrial energy state and function differs at different developmental stages and
therefore would explain these controversial obervation between pluripotent hESCs and
multipotent BM-MSCs. Regardless, monitoring of ��m could be utilized as a surrogate
potency assay for hMSCs in applications where osteogenic differentiation is needed.
This will be a significant tool for hMSCs therapy providers as currently the osteogenic
differentiation assays take several weeks to perform whereas the measurement of ��m
takes only a couple of hours.
To transfer the JC-1 viability assay to clinical use as a quality control tool for hMSC
based cellular therapies, it is necessary to validate the assay and to test the robustnes
and sensititivy at a larger scale. Moreover, dyes for monitoring ��m are extremely
sensitive to environmental changes and experimental conditions. In addition, several
normal mitochondrial conditions and functions are known to alter ��m, such as
67
increased ATP demand and presence of uncoupling proteins (UCPs) (Nicholls. 1974,
Stuart et al. 1999). It is also important to recognize that ��m sensitive dyes also have
several disadvantages that should be noted when results of the assay are interpreted,
such as mitotoxicity at high concentrations and sensitivity to plasma membrane
potential (Dedkova & Blatter. 2012). Therefore extreme care should be taken in
intepretation of the results and selection of the proper dye for a particular use as well
as paying attention to the experimental conditions. Moreover, previous research has
shown that there are limitations in measuring ��m, Annexin V and PI to detect
apoptosis when used independently (Lugli et al. 2005, Suzuki et al. 2012). Therefore it
is recommended that a broad selection of different changes in cellular function are
monitored in order to obtain a reliable, overall estimate of viability. Currently there are
also some tools already available for monitoring mitochondrial function established
and provided by companies, such as HemoGenix. HemoGenix offers FDA complient
viability and potency assays which measures cellular ATP levels.
(http://www.hemogenix.com/prod_LUMENESC.php?tab=2#TabbedPanels1).
6.4. Early detection of microbial contamination induced cellular stress by monitoring ��m
Many microbial toxins have been shown to be also mitochondrial toxins (Arnoult et al.
2009, Kozjak-Pavlovic et al. 2008). It has been demonstrated that �-toxins and
Panton-Valentine leukocidin secreted by S. aureus directly targets host mitochondria
and induces apoptosis (Bantel et al. 2001, Genestier et al. 2005). In addition, P.
aeruginosa and S. epidermidis have been shown to induce mitochondrial mediated
apoptosis in infected host cells (Jendrossek et al. 2001, Pharmakakis et al. 2009). The
present studies showed that even the smallest bacterial contaminations of all five
bacterial strains used caused equally severe destruction of co-cultured hMSCs as did all
higher loads of bacteria within 24 h. The strains used included highly aggressive strains
such as P. aeruginosa but also less virulent S .epidermidis which also caused severe
consequences to hMSCs viability during 24 hours. This is extremely alarming, since
even low levels of microbial contamination may be devastating in the long term and
cause inefficiency of administrated hMSCs.
Moreover, all pathogens except S. aureus Cowan I seemed to cause also systematic
depolarization of ��m already after 4 h of infection. The four hour time point was
selected since no signs of cellular stress or cell death as measured by trypan blue were
68
evident at that time point. However, P. aeruginosa had induced some visible changes
in cell membrane integrity and viability at higher bacterial loads. The results from
analysis of ��m at the 4 h time point showed that in some cases even the lowest
numbers of bacteria caused a significant decrease in ��m when compared to
uninfected controls. Thus measurement of ��m appears to be a sensitive method for
detection of the decrease in the viability and thus the condition of a cell product caused
by microbial infection. As discussed also earlier, the short shelf-life of cellular products
set certain limitations for viability methods and therefore the use of ��m sensitive dyes
could be beneficial. Moreover, the ��m assay also showed better sensitivity to detect
microbial infection induced decrease in viability of hMSCs than trypan blue. This study
clearly showed that ��m sensitive dyes are suitable for measuring viability of hMSCs
which enhances the safety of hMSC products since the depolarization of ��m may also
reflect microbial contamination of hMSCs. Moreover, to ensure the microbial safety of
hMSC products also rapid diagnostic tests to detect microbes from cellular products
are needed. Traditional CFU tests to detect bacterial contamination are not ideal due to
their time consuming nature. The sterility and viability of the product should be
demonstrated in a manner of hours.
6.5. CD200 positive BM-MSC as effective regulators of macrophages
The innate immunity plays a major role in early phase of injury, such as myocardial
infarction. Macrophages are the main players in innate immunity and they are present
in different phenotypes, pro-inflammatorial M1 and anti-inflammatorial M2. However,
recent studies have shown that the polarity of macrophages is more complicated and
several different phenotypes are present (Bystrom et al. 2008, Mosser & Edwards.
2008). In the regulation of macrophage activity the CD200-CD200r axis has been
shown to be important (Hoek et al. 2000). In addition, recently the expression of
CD200 was demonstrated for hMSCs (Delorme et al. 2008). Moreover, Delorme et al.
(2008) suggested that CD200 could be a marker for native hMSC since its expression
was rapidly decreased after short term of osteoinduction.
In the studies of the present thesis, the cell surface expression of CD200 was
characterized from 20 different hMSCs lines from bone marrow and umbilical cord
blood. Interestingly the expression of CD200 differed between BM-MSC from low
positivity to medium and to high positivity. UCB-MSC lines were negative for CD200
69
and they also showed lowest inhibition ability of TNF-� secretion from THP-1
macrophages. However, due to the different origin of UCB-MSC and BM-MSC it is not
possible to confirm that the lower immunosuppressive acitvity of UCB-MSC towards
macrophages is explained by the lack of CD200. BM-MSCs are shown to reside in a
close proximity with macrophage progenitors in bone marrow and thus they may be
already educated by the environment to control macrophage activity, which could thus
explain their higher immunosuppression potency when compared to UCB-MSC (Chow
et al. 2011).
In the present study, the role of the CD200-CD200r axis in hMSCs mediated
regulation of macrophages was tested by using a THP-1 macrophage model. THP-1
monocytes have been shown to mature into macrophages in the presence of PMA and
their activation with LPS has been demonstrated (Asseffa et al. 1993, Kang et al. 2007).
Results of the present study showed also the maturation of monocytes into
macrophage-like cells which were seen as attachment of cells and increased TNF-
�secretion. In addition, 24 hours LPS activation caused dramatic increase in TNF-
�secretion whereas IFN-� showed milder activation. To clarify the role of CD200-
CD200r axis in hMSC mediated immunosuppression of THP-1 macrophages, a
comparison of CD200Hi, CD200Me and CD200Lo BM-MSC was carried out in THP-1
macrophage co-culture. It was evident that CD200Hi, CD200Me and CD200Lo have a
different ability to inhibit TNF-� secretion from THP-1 macrophages. Moreover, UCB-
MSC showed lowest inhibition capacity. However, the ability of BM-MSC to inhibit
TNF-� secretion from THP-1 macrophages was dependent on activation. Only IFN-�
activated THP-1 macrophages showed sensitivity for BM-MSC inhibition of TNF-
�secretion. The IFN-� did not induce as strong activation of TNF-� secretion as LPS
which was in line with recent data on characterization of LPS and IFN-� activated
macrophages (Brown et al. 2010). The study by Brown et al. (2010) suggested that
IFN-� primes macrophages whereas LPS activates macrophages more strongly. It is
therefore evident that the maturity and the type of activation of macrophages have an
effect on the outcome of hMSCs mediated immunosuppression of macrophages.
The present studies also measured IL-10 levels from THP-1 macrophages and
showed that the co-culture with hMSCs induced IL-10 secretion from THP-1
macrophages. However, the induction of IL-10 secretion did not correlate with CD200
expression. Also previous studies have shown that hMSCs change the polarity of
macrophages from pro-inflammatorial M1 to IL-10 secreting anti-inflammatorial M2
phenotype in human (Kim & Hematti. 2009, Zhang et al. 2010). The studies presented
70
in this thesis also confirmed the role of the CD200-CD200r axis in hMSC mediated
immunosuppression. The blocking of CD200 from the cell surface of hMSCs showed
that the CD200-CD200r signalling mediates only part of the inhibitory effect of hMSCs,
since the CD200 antibody treatment only partially blocked the inhibition of TNF-�
secretion from THP-1 macrophages by hMSC. Several different mainly soluble but also
cell-cell independent mechanisms have been suggested to play a role in hMSC
mediated immunosuppression (Campioni et al. 2009, Meisel et al. 2004, Ren et al.
2010, Ryan et al. 2007). Therefore it is not suprising that CD200 blocking could not
completely interfere with the immunosuppressive effect of THP-1 macrophages by BM-
MSC.
This study demonstrated that CD200 expression on the cell surface of hMSCs could
be utilized to isolate a population of hMSCs that possess higher immunosuppressive
activity towards macrophages. However, these results also showed that CD200 positive
hMSCs were more efficient in immunosuppression of THP-1 macrophages only in the
presence of IFN-�. The role of IFN-� in many autoimmune diseases and inflammatory
conditions has been demonstrated and therefore the use of CD200 positive hMSCs may
be beneficial in such conditions (Shi et al. 2006, Young & Hardy. 1995). This reflects
the complexity of the whole immune system and its’ regulation since multiple parallel
mechamisms are working simultaneously and their combinational effect is needed for
the immunosuppression at a site of injury. Therefore selecting only one population
with ability to inhibit only certain immune cells may not be beneficial. One interesting
solution could be to combine hMSC types or subpopulations from different origins with
alternative immunosuppressive acitivities towards different immune cells. UBM-MSC
have been shown to be effective inhibitors of T-cell proliferation and in such a process
the CD200-CD200r axis seemed to have no direct function (Najar et al. 2012, Wang et
al. 2009)(Simelyte et al. 2010). Therefore, combination of UCB-MSC and BM-MSC
may be beneficial. One of the recent studies also showed that the expression of CD200
in MSCs is enhanced by IFN-�, which may also partly explain the observations of the
present thesis on differences in inhibition of TNF-� from THP-1 macrophages
mediated by hMSCs in presence of LPS and IFN-� (Najar et al. 2012). However, Najar
et al. (2012) were unable to show a direct role of the CD200-CD200r axis in MSC
mediated inhibition of T-cell proliferation, but MSCs were able to regulate CD200 and
CD200r expression of T-cells. This is in line with previous studies where the role of
CD200 mediated immunosuppression of T-cells was questinoned (Simelyte et al. 2010).
Clearly, the interactions between hMSCs and different immune cells need to be studied
71
in more detail to clarify the role of different mechanisms and factors. Moreover, the
role of different hMSCs types and subpopulations in immunosuppression processess
needs to be elucidated, in addition to the effect of pro-inflammatorial conditions on the
function of hMSCs.
6.6. Mitochondrial analyses combined with the cell surface characterizations ensure highly functional hMSC products
To conclude, mitochondrial analyses offer alternative parameters in addition to ISCT
characteristics that could be utilized to define hMSC and thus produce highly
homogeneous and functional hMSC products intended for cellular therapies. The
purity and viability of the hMSCs are essential for the efficient use of hMSC in cellular
therapies. Moreover, extension of ISCT minimum criteria panel for hMSCs with CD200
could produce highly immunosuppressive hMSC preparations. However, the final use
of the hMSC product will define the most appropriate potency tests. Therefore, it is
recommended to test different functional parameters based on the intended use, i.e.
immunosuppression assays for inflammatory (Figure 5.).
72
Figure 5. A suggested process flow with in-process characterizations and controls for hMSC intended for
cellular therapies. The red process indicates the production of hMSC for treatment of inflammatorial
conditions and green process indicates production of hMSC for treatment of large bone defects. In case of
green process the analyses of ��m could be used as a potency assay before in vitro differentiation or if the
hMSC are differentiated in vivo the analyses could be used as a release test (dashed line).
73
7.CONCLUSIONS
This thesis has focused on the mitochondrial characterization of hMSCs as well as on
the establishment of improved and more reliable quality control tools for ensuring the
safety and efficacy of hMSCs in future therapies. As treatment of autoimmune diseases
through immunomodulation is one of the most promising areas for cell therapies in the
future, the study has also aimed at providing a more detailed understanding of the
mechanisms of immunosuppressive activity of hMSCs, which is also inherently tied to
the viability and functionality of these cells. The more detailed conclusions of these
studies are:
1. Mitochondrial characterization of hMSCs offers new methods for identifying the
hMSCs in addition to the traditional immunophenotype characterization. The
mitochondrial and metabolic status of stem cells differs from terminally
differentiated cells. This unique status of stem cells could be utilized to distinguish
osteoprogenitors from more multipotent hMSCs. It is suggested that methods to
measure mitochondrial number, ROS levels and antioxidant defence systems from
hMSCs could be ideal parameters for defining the multipotent hMSCs.
2. By monitoring ��m it is possible to detect early changes in oxidative stress induced
apoptosis which precede changes in plasma membrane organization and integrity.
��m sensitive dyes, such as JC-1, have several advantages when compared to
traditional viability tools, they are: a) more sensitive than PI and trypan blue, b)
rapid and therefore more suitable for hMSC-based cellular products than time
consuming proliferation assays, and c) mitochondrial ��m also correlates to the
osteogenic differentiation capacity of hMSCs.
3. Microbial infection induced cellular stress can be detected rapidly by ��m analysis.
Changes in ��m precede changes in plasma membrane integrity during microbial
infection induced cell death.
4. In this study it has been demonstrated for the first time, that the
immunosuppression of macrophages by hMSCs is mediated partly through the
74
CD200-CD200r axis. This offers possbilities to isolate macrophage inhibiting
hMSCs populations for use in clinical interventions where there is a need to
suppress the activity of innate immunity.
5. The analysis of mitochondrial number and energy state combined with cell surface
characterization of minimum criteria panel established by ISCT and extended by
CD200 characterization could be used as a quality control tool during manufacture
of homogeneous and highly functional hMSC products intended for clinical use to
ameliorate inflammatorial conditions.
75
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ISBN 978-952-60-4889-5 ISBN 978-952-60-4890-1 (pdf) ISSN-L 1799-4934 ISSN 1799-4934 ISSN 1799-4942 (pdf) Aalto University School of Chemical Technology Department of Biotechnology and Chemical Technology, Aalto University
BUSINESS + ECONOMY ART + DESIGN + ARCHITECTURE SCIENCE + TECHNOLOGY CROSSOVER DOCTORAL DISSERTATIONS
Aalto-D
D 161
/2012
Pietilä M
ika F
unctionality of Hum
an Mesenchym
al Stem C
ells Assessed by E
nergy Metabolism
and Imm
unomodulative
Capacity
Aalto
Unive
rsity
Department of Biotechnology and Chemical Technology, Aalto University
Functionality of Human Mesenchymal Stem Cells Assessed by Energy Metabolism and Immunomodulative Capacity
Pietilä Mika
DOCTORAL DISSERTATIONS
