Thesis
Reference
Transgenic enrichment strategies for cardiac cells derived from mouse embryonic stem cells using HSV1-TK-ganciclovir and
CD-5-fluorouracil
HEUKING, Pernilla
Abstract
The aim of this thesis was to enrich for cardiac cells derived from mouse embryonic stem cells (mESC) during differentiation. In order to achieve this aim we tested two different suicide gene-based systems and used lentivectors as means of transgene delivery into the genome of undifferentiated mESC. One transgenic enrichment approach was based on the use of herpes simplex virus thymidine kinase 1 (HSV1-TK). An enrichment of ~20 fold was obtained and demonstrated using flow cytometry after targeting cardiac troponin T. Enriched cells showed retained cardiac phenotypes as shown via RT-PCR and immunocytochemistry.
Spontaneous beating and reactivity to pharmacological stimulation was observed via confocal microscopy after fluo-2 incubation. The second approach based on the suicide gene system composed by cytosine deaminase (CD) and the prodrug 5-fluorocytosine (5-FC). Cardiac enrichment monitored by flow cytometry after staining for cardiac Troponin T reached ~9 fold.
Also in this case, enriched cardiac cells retained their cardiac phenotype. In summary, we report the successful enrichment of cardiac cells derived from mESC [...]
HEUKING, Pernilla. Transgenic enrichment strategies for cardiac cells derived from mouse embryonic stem cells using HSV1-TK-ganciclovir and CD-5-fluorouracil. Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4542
URN : urn:nbn:ch:unige-277304
DOI : 10.13097/archive-ouverte/unige:27730
Available at:
http://archive-ouverte.unige.ch/unige:27730
Docteur Marisa Jaconi
Transgenic Enrichment Strategies For Cardiac Cells Derived From Mouse Embryonic Stem Cells Using HSV1-TK-Ganciclovir And CD-5-Fluorouracil
THÈSE
présentée à la faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur és sciences, mention sciences pharmaceutiques
par
Pernilla HEUKING
de Holzminden (Allemagne)
Thèse no 4542
GENÈVE
Atelier d’impression ReproMail
2013
To
Margareta and Per‐Ove,
Irmgard and Siegfried,
Eva and Rainer,
Inga and Michael,
Simon and Emma.
„Wer nicht mehr liebt und nicht mehr irrt,
der lasse sich begraben.“
Johann Wolfgang von Goethe
stem cells as well as thereof derived differentiated cells are considered potential tools for detection of pharmacological or side effects within screening assays.
Since spontaneous differentiation of mouse embryonic stem cells (mESC) into cardiac cells results in a relatively low percentage of cells within the whole beating embryoid body [1], enrichment strategies for increasing the cardiac cell number are desirable. The total number of cardiac cells needed to replace dead cells in a heart after an infarct have been estimated to be around 1 billion cells [2]. A homogenous population of cardiac cells would be beneficial for the discovery of new cardiac drugs and the detection of cardiotoxicity of drugs in preclinical phases [3].
The aim of this thesis was to enrich for cardiac cells derived from mouse embryonic stem cells (mESC) during differentiation. In order to achieve this aim we tested two different suicide gene‐based systems and used lentivectors as means of transgene delivery into the genome of undifferentiated mESC. Transduced and wild type cells were indistinguishable based on the assays performed.
One transgenic enrichment approach was based on the use of herpes simplex virus thymidine kinase 1 (HSV1‐TK). Upon administration of ganciclovir HSV1‐TK initiates the phosphorylation of the per se nontoxic prodrug. The activated drug leads to death in dividing cells. In order to protect differentiating and proliferating cardiac cells this system was meant to be improved by tetR‐Krab / tetO2 based transcriptional inhibition of the HSV1‐
TK in cardiac cells. The tetR‐Krab expression is driven by a Nkx‐2.5 cardiac promoter. The
concentration of 50 μmol ganciclovir was demonstrated to lead to enrichment of cardiac cells. An enrichment of ~20 fold was obtained and demonstrated using flow cytometry after targeting cardiac troponin T. Enriched cells showed retained cardiac phenotypes as shown via RT‐PCR and immunocytochemistry. Spontaneous beating and reactivity to pharmacological stimulation was observed via confocal microscopy after fluo‐2 incubation.
The second approach based on the suicide gene system composed by cytosine deaminase (CD) and the prodrug 5‐fluorocytosine (5‐FC). Since ganciclovir is routinely used in a clinical setting, having an alternative suicide strategy for transplanted cells at hand would be a benefit. We tested the CD / 5‐FC suicide system in mESC after lentiviral delivery into the genome of mESC. Transduced cells were analyzed in comparison to wild type and mock transduced controls. Transduced and wild type cells were indistinguishable based on the assays performed. The system’s functionality in combination with the prodrug 5‐
fluorocytosine could be shown in mESC and during differentiation. A bystander effect for the CD/5‐FC suicide system was noticed in mESC, however it was less strong than the one observed for the HSV1‐TK / ganciclovir system. No synergistic effect concerning cell killing was observed after combining the CD and HSV1‐TK based suicide genes in transduced mESC.
Cardiac enrichment monitored by flow cytometry after staining for cardiac Troponin T reached ~9 fold. Also in this case, enriched cardiac cells retained their cardiac phenotype.
In summary, we report the successful enrichment of cardiac cells derived from mESC after lentiviral transduction with either the CD / 5‐FC suicide gene system or the HSV1‐TK / ganciclovir suicide gene system in combination with the tetR‐Krab / tetO2.
outils pour des études pharmacologiques, comme la détection d’effet pharmacologique et aussi la recherche d’effet secondaire lors des « screenings ».
Le taux de différentiation spontanée des cellules souches embryonnaires de souris (mESC) en cellules cardiaques est relativement faible au sein des corps embryonnaires battants [1], suggérant de nouvelles stratégies d’enrichissement des cellules cardiaques. Le nombre total de cellules cardiaques nécessaires pour remplacer les cellules mortes dans un cœur après un infarctus a été estimé à 1 milliard de cellules [2]. Une population homogène de cellules cardiaques serait favorable pour la découverte de nouveaux médicaments pour le cœur et aussi pour la détection de la cardiotoxicité des médicaments lors des phases précliniques [3].
L’objectif de cette thèse a été d’augmenter l’efficacité de différentiation des cellules souches embryonnaires de souris (mESC) en cellules cardiaques. Pour atteindre cet objectif, nous avons testé deux différents systèmes de gêne suicide en utilisant des lentivecteurs comme système de transport et d’intégration des transgènes dans le génome des mESC indifférenciées. La distinction entre les cellules transduites et les cellules sauvages étaient impossible à effectuer sur la base des analyses effectuées.
Une approche transgénique d’enrichissement a été basée sur l’utilisation du gène Herpès simplex virus thymidine kinase 1 (HSV1‐TK). Lors de l’administration de ganciclovir sur les cellules, cette dernière est phosphorylée par HSV1‐TK et devient ainsi toxique, entrainant la mort des cellules en division. Afin de protéger les cellules cardiaques prolifératives et en
de gêne suicide est fonctionnel dans les mESC pendant la différentiation cardiaque. La distinction entre les cellules transduites et les cellules sauvages étaient impossible à effectuer sur la base des analyses effectuées. Une utilisation de 50 µmol de ganciclovir
pendant 6 jours entre le 8ème jour et le 14ème jour de différentiation permet d’obtenir un enrichissement des cellules cardiaques. Un enrichissement par un facteur 20 a été obtenu et quantifié par cytométrie en flux en vérifiant l’expression du facteur cardiaque Troponine T.
Les cellules enrichies conservent un phénotype cardiaque vérifié par RT‐PCR et immunomarquage. Après incubation avec du fluo‐2, la réactivité à des stimulations pharmacologiques des battements spontanés des cellules cardiaques a été enregistrée par microscopie confocale.
La seconde approche a consisté en un système de gêne suicide composé par la cytosine déaminase (CD) et le pro‐médicament 5‐fluorocytosine (5‐FC). Comme le ganciclovir est couramment utilisé en milieu clinique, une stratégie alternative pour la transplantation de cellules cardiaques semble intéressante. Nous avons testés le système CD / 5‐FC dans les mESC après transduction dans le génome de ces dernières. Les cellules transduites ont été analysées en comparaison des cellules sauvages et des cellules contrôles négatives mock. La distinction entre les cellules transduite et les cellules sauvages étaient impossibles à effectuer sur la base des analyses effectuées. La fonctionnalité du système en combinaison avec le pro‐médicament 5‐FC a pu être démontrée dans les mESC en différentiation. Un effet « bystander » a été observé dans ce système ce dernier restant cependant moins important que dans le système HSV1‐TK / ganciclovir. Aucun effet synergique concernant la destruction des cellules a été observé en combinant les deux systèmes dans les mESC
cardiaques enrichies ont conservées leur phénotype.
En résumé, nous présentons la réussite de l’enrichissement de cellules cardiaques dérivées de mESC après transduction lentiviral soit avec le système de gêne suicide CD / 5‐FC, soit avec le système de gêne suicide HSV1‐TK / Ganciclovir en combinaison avec la construction TetR‐Krab / tetO2.
ABBREVIATIONS ... XVII THESIS OBJECTIVE... XX
INTRODUCTION
1. BRIEF HISTORY OF STEM CELLS ... 1
2. EMBRYONIC STEM CELLS ... 6
2.1. THE DEVELOPING EMBRYO ... 6
2.2. DERIVATION OF PLURIPOTENT STEM CELLS FROM THE MOUSE EMBRYO ... 7
2.3. PLURIPOTENCY ... 9
2.4. PATHWAYS IN PLURIPOTENCY ... 10
2.4.1. LIF ... 10
2.4.2. BMP4 ... 12
2.4.3. Wnt/β‐catenin ... 12
2.5. TRANSCRIPTION FACTORS INVOLVED IN PLURIPOTENCY ... 13
2.5.1. Nanog ... 13
2.5.2. Oct4 ... 14
2.5.3 Sox2 ... 15
2.6. THE CHROMATIN STATE IN ESC ... 16
2.7. THE METABOLISM IN ESC ... 16
3. CARDIAC DIFFERENTIATION ... 18
3.1. THE HEART ... 18
3.2. CARDIOGENESIS ... 18
3.2.1. Markers throughout cardiogenesis ... 20
3.3. THE HEART FIELDS ... 25
3.3.1. The first heart field and its markers ... 27
3.3.2. The second heart field and its markers ... 27
4. ENRICHMENT STRATEGIES FOR CARDIAC CELLS ... 29
4.1. NON TRANSGENIC CARDIAC ENRICHMENT ... 31
4.1.1. Isolation of cardiac progenitors ... 32
4.1.2. Membrane proteins used for isolation and enrichment ... 34
4.1.3. Stimulation to cardiac commitment via supplements ... 35
4.1.4. Physical means ... 41
4.1.5. Cardiomyocyte specific dyes ... 44
4.2. TRANSGENIC CARDIAC ENRICHMENT ... 46
4.2.1. Drug resistance genes ... 47
4.2.2. Fluorescent reporter genes ... 50
4.2.3. HSV1‐TK ... 51
5.2. CYTOSINE DEAMINASE ... 64
6. TRANSGENIC PROMOTER REPRESSION ... 68
6.1. TRANSCRIPTION REGULATING SYSTEM BASED ON LIGANDS ... 68
6.1.1. Tetracycline (Tc)‐controlled trans‐activator (tTa) tet‐off system ... 69
6.1.2. Reverse tc‐controlled trans‐activator (rtTa) tet‐on system ... 70
6.1.3. Trans‐silencer tTs system ... 70
6.1.4. TetRKrab‐tet0 system ... 71
7. REFERENCES FOR INTRODUCTION ... 77
MATERIALS AND METHODS 8. CELL CULTURE ... 107
8.1. MESC CULTURE ... 107
8.2. DIFFERENTIATION OF MESC TO MEBS ... 108
8.3. % BEATING DURING DIFFERENTIATION ... 109
8.4. HL‐1 CELLS ... 109
8.5. 293T HEK CELLS ... 110
8.6. LIF PRODUCTION ... 110
8.7. GANCICLOVIR AND 5‐FLUOROCYTOSINE USED IN CELL CULTURE ... 110
9. LENTIVIRUSES... 112
9.1. LENTIVIRAL PRODUCTION ... 112
9.2. LENTIVIRAL TRANSDUCTION AND SELECTION OF TRANSDUCED MESC ... 112
9.3. LENTIVIRUS TITRATION ... 113
10. PROLIFERATION AND APOPTOSIS ANALYSIS ... 114
10.1. GROWTH CURVES ... 114
10.2. DNA LADDERING ... 114
10.3. XTT PROLIFERATION ANALYSIS ... 115
11. IMMUNOCYTOCHEMISTRY ... 117
12. CELL CYCLE ANALYSIS ... 119
13. CARDIAC TROPONIN T STAINING FOR FACS ANALYSIS ... 120
14. FLUO‐2 INCUBATION ... 122
15. WESTERN BLOT ... 123
16.4. PURIFICATION OF VECTORS FROM BACTERIA... 127
16.5. DIGESTION AND LIGATION OF VECTORS ... 127
16.6. PCR GELS ... 128
16.7. GENOMIC DNA EXTRACTION AND PCR FOR TRANSGENE INTEGRATION ... 128
16.8. RNA ISOLATION AND RT‐PCR ... 130
16.9. QUANTITATIVE RT‐PCR ... 132
17. CLONING ... 133
17.1 EF1Α‐HSV1‐TK ... 133
17.2. EF1Α‐CD LENTIVECTOR ... 133
17.3. SFFV‐CD LENTIVECTOR... 138
17.4. SFFV‐TK LENTIVECTOR ... 139
17.5. EF1Α‐GFP LENTIVECTOR ... 140
17.6. HSV1‐TK MOCK ... 140
18. STATISTICAL ANALYSIS ... 141
19. REFERENCES FOR MATERIALS AND METHODS ... 142
RESULTS 20. ENRICHMENT MESC USING HSV1‐TK AND TETR‐KRAB ... 143
20.1. TRANSDUCTION, SELECTION AND ANALYSIS OF TRANSGENIC MESC ... 143
20.2. CHARACTERIZATION OF TRANSGENIC CELLS DURING DIFFERENTIATION ... 153
20.3. CHARACTERIZATION OF THE EF1Α PROMOTER ... 159
20.4. CHARACTERIZATION OF NKX‐2.5/E PROMOTER ... 166
20.5. TIME FRAME FOR GANCICLOVIR EXPOSURE DURING DIFFERENTIATION FOR CARDIAC ENRICHMENT ... 172
20.6. ANALYSIS OF CARDIAC ENRICHMENT ... 180
20.7. ANALYSIS OF ENRICHED CARDIAC CELLS ... 187
21. HL‐1 CELLS AS A MODEL OF PROLIFERATING CARDIAC CELLS ... 193
21.1. TRANSDUCTION AND SELECTION OF TRANSGENIC HL‐1 CELLS ... 194
21.2. CARDIAC STAINING VIA ICC ON HL‐1 CELLS ... 195
21.3. CELL PROLIFERATION ASSAY DURING GANCICLOVIR EXPOSURE ... 196
21.4 REALTIME PCR TARGETING HSV1‐TK EXPRESSION LEVELS IN HL‐1 CELLS ... 198
21.5 WESTERN BLOT ... 200
22. RESULTS CYTOSINE DEAMINASE ... 201
22.1 TRANSDUCTION, SELECTION AND CHARACTERIZATION OF TRANSGENIC MESC/MEBS ... 201
22.2 PROLIFERATION ANALYSIS FOR CD TRANSDUCED MESC IN THE PRESENCE OF 5‐FC ... 212
DISCUSSION
24. TRANSDUCTION OF TRANSGENIC CELLS ... 237
25. CHARACTERIZATION OF TRANSGENEIC MESC ... 240
26. DIFFERENTIATION ... 246
27. TIME FRAME FOR CARDIAC ENRICHMENT AND CARDIAC ENRICHMENT ... 252
28. TRANSCRIPTIONAL INHIBITION OF HSV1‐TK EXPRESSION ... 256
29. CD BASED SUICIDE SYSTEM ... 261
30. REFERENCES FOR DISCUSSION ... 266
ANNEXES 31. VIABILITY ASSAY OF A MOCK HSV1‐TK CONSTRUCT ... 277
32. KOZAK SEQUENCE UPSTREAM THE START CODON OF THE CD TRANSGENE ... 280
33. HANGING DROPS ... 280
34. CELL CYCLE IN MESC IN COMPARISON TO MEBS ON DAY EIGHT OF DIFFERENTIATION ... 281
35. EXPRESSION OF CONNEXINS IN MESC AND MEBS ... 282
36. RESULTS CLONING ... 283
36.1. RESTRICTION DIGESTION OF NKX‐2.5‐TETR‐KRAB AND EF1Α‐TETO2‐HSV1‐TK ... 283
36.2. RESTRICTION DIGESTION OF NKX‐2.5‐GFP ... 283
36.3. RESTRICTION DIGESTION OF EF1Α‐PENTR ... 284
36.4. RESTRICTION DIGESTION OF CD‐PENTR ... 284
36.5. RESTRICTION DIGESTION OF LENTIVECTOR P2K7 ... 285
36.6. RESTRICTION DIGESTION OF EF1Α‐CD ... 285
36.7. RESTRICTION DIGESTION OF LENTIVECTOR LEGO‐IG2‐PUROR ... 286
36.8. RESTRICTION DIGESTION OF CD‐PT7BLUE3 ... 286
36.9. RESTRICTION DIGESTION OF SFFV‐CD ... 287
36.10. RESTRICTION DIGESTION OF SFFV‐HSV1‐TK ... 287
37.4. TETRKRAB IN NKX‐2.5P/E‐TETRKRAB ... 298
37.5. EF1Α IN EF1ΑTETO2‐GFP ... 300
37.6. GFP IN EF1ΑTETO2‐GFP ... 302
37.7. NKX‐2.5P/E IN NKX‐2.5P/E‐GFP ... 304
37.8. GFP IN NKX‐2.5‐GFP ... 307
37.9. CD IN ENTRY CLONE ... 309
37.10. EF1Α IN EF1Α‐CD ... 313
37.11. CD IN EF1Α‐CD ... 317
37.12 CD IN SFFV‐CD ... 319
37.13. TK IN SSFV‐TK ... 322
38. REFERENCES FOR ANNEXES ... 324
39. CURRICULUM VITAE ... 325
40. ACKNOWLEDGEMENTS ... 327
BMP bone morphogenetic protein BSA bovine serum albumin CBT cell based therapy CD cytosine deaminase CGR8 mouse embryonic stem cells
CMV promoter element derived from the cytomegalovirus CO2 carbon dioxide
cPPT central polypurine tract cTNT cardiac troponin T
d refers to the day of in vitro mESC differentiation DMSO dimethylsulfoxide
DNA deoxyribonucleic acid E enhancer element
Ex x days of embryonic development in vitro after fertilization EBs embryoid bodies
EC embryonal carcinoma EF1α elongation factor 1 α EpiSC epiblast stem cell ESC embryonic stem cells
FACS fluorescence activated cell sorting 5‐FC 5‐Fluorocytosine
FDA food and drug administration FGF basic fibroblast growth factor FHF first heart field
5‐FU 5‐Fluorouracil GCV ganciclovir
GDEPT Gene directed enzyme prodrug therapy GFP green fluorescent protein
GJIC gap junctional intercellular communication hESC human embryonic stem cells
HF head fold
hiPSC human induced pluripotent stem cells HSV1‐TK herpes simplex virus 1 thymidine kinase ICM inner cell mass
IRES internal ribosome entry site Isl1 Insulin gene enhancer protein
ISSCR international society for stem cell research ihESC induced human embryonic stem cells imESC induced mouse embryonic stem cells iPSC induced pluripotent stem cells Jak janus kinase signal
LIV leukemia inhibitory factor LTR long terminal repeat
MAPK mitogen‐activated protein kinase MEF mouse embryonic fibroblasts mESC mouse embryonic stem cells mRNA messenger Ribonucleic acid MHC myosin heavy chain
MICP multipotent Isl1+ cardiovascular progenitor cells mRNA messenger Ribonucleic acid
MS mass spectrometry
NIH national institute of health
PFA parafinformaldehyd PGI2 prostaglandin I2 PrEct primitive ectoderm PrEn primitive endoderm PS primitive streak
qRT‐PCR quantitative reverse transcription polymerase chain reaction RCR replication competent retrovirus
RT‐PCR reverse transcription polymerase chain reaction rESC EpiSC reprogrammed into mESC like cells
SHF second heart field
smMHC smooth muscle myosin heavy chain SSEA‐1 stage‐specific embryonic antigen 1
SV40 promoter derived from the Simian virus 40 TetR tet repressor
TK thymidine kinase
TMRM tetramethylrhodamine methyl ester perchlorate VE visceral endoderm
WPRE woodchuck hepatitis virus posttranscriptional regulatory element
The aim of the thesis was to enrich and characterize mESC derived cardiomyocytes. An enriched population of mESC derived cardiac cells could be beneficial for drug testing assays or animal experiments with the aim of cell replacement therapies.
To achieve this aim, we transduced mESC with the HSV1‐TK and exposed cells to the prodrug ganciclovir during differentiation in order to enrich for cardiac cells via a negative selection. The HSV1‐TK suicide gene has been used on hESC for cardiac enrichment already by Anderson et al. [4]. We wanted to improve the system to select cardiac cells not only after their exit of the cell cycle, but already earlier during the first commitment of cells to the cardiac lineage. Therefore we used a system that should repress expression of the suicide protein HSV1‐TK in early cardiac cells. By these means the susceptibility of early, still dividing cardiac cells to the activated ganciclovir should be inhibited.
The transcriptional inhibition of HSV1‐TK in early cardiac cells was based on TetR‐Krab expression [2]. As Nkx‐2.5 is one of the earliest cardiac markers [3], we made use of a Nkx‐
2.5 promoter to drive expression of the TetR‐Krab in cardiac cells exclusively. This promoter should trigger expression of a TetR‐Krab protein in early cardiac cells. Furthermore, the EF1α promoter, driving expression of the HSV1‐TK, was modified. Two repeats of the tetO
sequence, named tetO2 after fusion, were cloned into the EF1α promoter. In early cardiac cells, the Nkx‐2.5 promoter should trigger expression of the TetR‐Krab transcriptional
repressor that would bind to the tetO2 sequence of the EF1α promoter. By these means the expression of HSV1‐TK is meant to be silenced in early cardiac cells.
To achieve the aim mentioned above, we tested a second suicide system for enrichment of cardiac cells, that involves the use of cytosine deaminase (CD) in combination with the
diffusion. Since cardiac cells are within the earliest cells to exit the cell cycle, non cardiac and still cycling cells are sensible to the activated 5‐FC and die. By a negative selection non cardiac cells should get killed while cardiac cells survive 5‐FC treatment.
The thesis novelty consists in the evaluation of the TetR‐Krab / tetO2 as well as the CD system in mESC during differentiation for cardiac enrichment, the characterization of transduced and enriched cardiac cells. To our best knowledge, the TetR‐Krab modified HSV1‐TK / ganciclovir system has not been studied before for cardiac enrichment. While HSV1‐TK / ganciclovir based strategies have been used for the enrichment of hESC derived cardiac cells, this strategy has not yet been combined with the TetR‐Krab / tetO2 system. As far as we know, neither the suicide system CD / 5‐FC has been characterized in mESC nor used with the aim of cardiac enrichment on mESC.
INTRODUCTION
1. Brief history of stem cells
Research on stem cells could be considered quite a young filed of research taking into account the fact that first mouse embryonic stem cell lines were obtained in the 1980s [5, 6]. It took almost two decades to see the successful derivation of the first human embryonic stem cell line [7]. However, the beginnings of stem cell research date back as long as 50 years [8].
Research on pluripotent stem cells emerged with the derivation of a murine inbred 129 strain in 1954, showing a 10% higher incidence [9] of testicular teratoma in comparison to other mouse strains. Stevens et al. demonstrated that a certain degree of the observed cancer cells remained in an undifferentiated state while, at the same time, some cells within the teratoma had the ability to differentiate into different somatic cell types of the adult mouse organism [9].
An important achievement was gained by Kleinsmith and Pierce. They strengthened Stevens’ findings by demonstrating the multipotent potential of single embryonal carcinoma cells. They transplanted one embryonal carcinoma cell per animal before analyzing and characterizing so derived multipotential cell lines [10].
The discovery of the multipotentiality of described carcinoma cells as well the similarity of structures in the derived tumors to early embryos inspired researchers to directly implant d1 to d6 embryo cells into testes of the 129 mouse strain [8]. Resulting cells had the same characteristics as the testicular teratoma cell lines of the 129 strain mice. By these means
demonstrated. Both resulting carcinoma lines could be transplanted for prolonged periods and were demonstrated to originate from pluripotent stem cells [11].
Over years, conditions for the culture of pluripotent teratocarcinoma cell lines have considerably improved [8], together with the knowledge on culture conditions as well as tools to examine cell characteristics like for e.g. pluripotency markers. The enzyme alkaline phosphatase was the very first target to be found to be expressed in embryonal carcinoma cells [12]. Later on, pluripotency‐related antibodies, like stage‐specific embryonic antigen 1 (SSEA‐1) was found to be specific for mouse embryonal carcinoma cells, while stage‐specific embryonic antigens 3 (SSEA‐3) and 4 (SSEA‐4) were found to be specific for human embryonal carcinoma cells [8]. Alkaline phosphatase assays and the described antibodies to define pluripotent cells are now widely used for the in vitro characterization of bona fide pluripotent stem cells.
As mentioned above, the two labs of Evans and Kaufman as well as Martin were the first to report the successful derivation of mouse ESC from mouse blastocysts that could be cultured continuously [5, 6] before Thomson et al. [7] published the derivation of human ESC about 17 years later. In 2001 the group of Lior Gepstein characterized the first cardiomyocytes derived from hESC [13] demonstrating the occurrence of beating clusters within the 8% of differentiating embryoid bodies (EB), obtained from aggregated ESC clusters allowed to differentiate.
It is estimated that close to 1000 hESC have been derived so far [14, 15]. Quite some effort has been performed to set general standards for characterizing and comparing hESC lines [16]. The National Institute of Health (NIH) currently has 91 hESC lines approved for NIH‐ or publically‐funded research and further 83 cell lines pending for approval. Besides the NIH,
the International Stem Cell Forum has set up standards for comparing different derived lines. The forum was founded in 2003 by experts in the field during a meeting in London.
Until 2005, 75 hESC lines were studied by the participating labs of the International Stem Cell Initiatives (ISCI I and II) [17, 18].
In 2006 stem cell science was “revolutionized” with findings of a Japanese lab. Yamanaka and Takahashi were the first researchers to demonstrate the successful reprogramming of somatic cells into embryonic like stem cells, called induced pluripotent stem cells (iPSC) [19].
By forced expression after retroviral transduction of the following transgenes Oct3/4, Sox2, c‐Myc, and Klf4, mouse fibroblasts could adopt a stem cell‐like phenotype. Yamanaka, together with John B. Gurdon, was awarded with the Nobel Prize in Physiologie or Medicine in 2012 for his findings [20]. In 2007 the successful reprogramming of human fibroblasts was achieved [21]. Derivation and culture of induced miPSC and hPSC would probably not have been possible without the knowledge on the marker expression profile of ESC. 2007 was an important year for stem cell research for another reason as well. Besides the successful derivation of hiPSC, Sir Martin J. Evans together with Mario R. Capecchi and Oliver Smithies obtained the Nobel Prize in Physiology/Medicine "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells" [22].
Ever since iPSC have been raising the hope of circumventing the ethically critical destruction of human embryos for deriving hESC lines. Improving the efficiency of deriving iPSC as well as defining, characterizing and comparing iPSC with other pluripotent cell lines are highly investigated nowadays, as differences are becoming evident and seem to greatly depend on epigenetic modifications linked to the method used for the reprogramming.
Another feature rendering iPSC very important to the scientific community is the possibility of generating iPSC as models of human diseases, when iPSC are derived from patient cells.
In doing so, researchers are able to investigate the molecular basis of diseases during early embryogenic development. The possibility at hand to investigate on e.g. genetic modifications and to follow up pharmacological effects at a cellular level of the pathologic disease profile renders iPSC highly valuable and interesting.
Besides fundamental research, the pluripotent characteristics of iPSC have raised the hope to circumvent the immune rejection drawback of hESC, making iPSC valuable candidates in clinical settings addressing future cell therapy options [23]. iPSC are assumed to sidestep immunological difficulties in the context of cellular transplantation therapies, the rational for this assumption being that iPSC derived from the patient are autologous. However, only very recently Zhao et al. demonstrated that among ESC and iPSC, both derived from B6 mice and injected back into B6 mice, the iPSC are the ones triggering the higher level of immune response in the B6 mice. The authors explained these observations with the finding that the expression profile and level were different for some immunogenicity related genes in iPSC compared to ESC [24]. These findings indicate that more effort needs to be invested in understanding transcriptional and epigenetic differences between iPSC and ESC before evaluating their possible contribution to regenerative medicine.
Several countries have commissioned their regulatory authorities to guide cell therapies towards clinical applications. How to define safety in patients, quality of cells and how to validate upscaling procedures are hurdles to overcome before proposing various types of stem cells for cell therapy [25]. Furthermore, many nonprofit organizations (see reference [26] for extensive review), including the International Society for Stem Cell Research (ISSCR),
published guidelines that point out some valuable considerations and recommendations for the process of taking stem cell research into clinics [23].
Nowadays, clinical stem cell trials approved by American or British authorities account for myocardial infarctions, diabetes and injuries of the central nervous system, for instance [27].
2. Embryonic stem cells
2.1. The developing embryo
After fertilization, the mammalian embryo starts dividing independently of extrinsic signals [28]. Blastomeres at the 4‐cell stage can be divided into two groups based on their Oct4 kinetics [29]. At the 8‐cell stage each blastomere undergoes apical basal polarization [30]. In this developmental phase different forms of cellular divisions occur [31]. There are some cells that perform cell division in a symmetric way. Both resulting daughter cells will be part of the outside region of the embryo. Cells that, on the other hand, perform asymmetric cell division will donate one daughter cell to the outer and the other daughter cell to the inner region of the developing embryo [29]. Also intermediate forms between symmetric and asymmetric cell divisions exist [31]. The inside region will eventually develop into the inner cell mass and subsequently the epiblast [29].
After the very first divisions in murine embryos, the morula is formed at day 2.5 after fertilization [32] and will give rise to the inner cell mass (ICM) in the early blastocyst [33], as can be seen in Figure 1. At this time point, the trophoectoderm (TE) surrounds the ICM and will develop into the extraembryonic cells that, at later stages, support the embryo as placenta tissue [32]. In vivo, the ICM gives rise to the epiblast and the primitive endoderm (PrEn) during the late blastocyst stage (Figure 1). The epiblast will form the primitive ectoderm (PrEct) and subsequently the embryo [34]. PrEct is still pluripotent but will lose this characteristic upon the subsequent formation of the three germ layers [32].
Figure 1. Representative illustration of the early mouse embryonic development of the morula, over the early and late blastocyst‐ until the egg cylinder stage. The morula divides and develops into the cells of the inner cell mass (ICM) and the trophoectoderm (TE). Later in development, the ICM will give rise to the epiblast and the primitive endoderm (PrEn) during the late blastocyst stage. At the egg cylinder stage, the early embryo implants into the uterus. The epiblast will develop into the primitive ectoderm (PrEct), while the primitive endoderm will result in visceral endoderm (VE) and parietal (PE). (Taken from [32]).
The egg cylinder stage (Figure 1) is reached after implantation into the uterus. From this time point on, the embryo is receptive to extrinsic signaling [28]. During further development, the PrEn gives rise to the visceral (VE) and parietal (PE) endoderm.
2.2. Derivation of pluripotent stem cells from the mouse embryo
So far, mESC have been derived from preimplantation embryos [35]. To keep mESC pluripotent they need to be cultured either on growth‐inactivated fibroblast feeder cells, in conditioned media or in the presence of the leukemia inhibitory factor (LIF).
While adult stem cells divide asymmetrically and by these means retain their number as well as sustain the tissue they differentiate into, ESC differentiate by symmetric divisions. In
main characteristics of ESC are that they can be kept in culture for extended periods of time while they keep the feature of pluripotency [37]. ESC can therefore give rise to the three germ layers and during further differentiation develop into every somatic cell type in the adult organism. However, ESC cannot organize themselves to form an individual [34].
Regardless if mESC have been derived from epiblast or earlier pre‐epiblast embryonic stages, mESC lines are reported to share the same molecular and phenotypic characteristics [28]. mESC lines derived from later stages than the preimplantation embryo have been successfully obtained as well. These cells are no longer called ESC, but post implantation epiblast stem cells (EpiSCs). Morphologically they seem to resemble human ESC (hESC) rather than mESC, since they grow as thinner layers. Similarly, they need FGF2 and activin to keep their pluripotent state and, by this dependency, even more resemble hESC rather than mESC [38]. The epigenetic state as well as the signals regulating pluripotency and differentiation in mEpiSCs are different from the ones in mESC [39]. These findings put new emphasis on the question whether mouse and human ESC are derived from the same embryonic developmental stage. Nevertheless, by culturing EpiSCs in the presence of LIF for 2‐5 weeks, Bao et al. demonstrated that EpiSCs can be reprogrammed in mESC like cells, that authors named reprogrammed ESC (rESC) [40]. Differentiation of mESC into EpiSCs is in turn possible as well by culturing mESC in the presence of FGF2 and activin [41].
The morphology of mouse mESC is marked by a high nucleus to cytoplasma volume ratio and they grow as tightly packed three dimensional colonies. hESC lines rahter grow as monolayers [32].
2.3. Pluripotency
Pluripotency defines the capacity of ESC to develop into every cell type that exists in an adult organism. When injected into a blastocyst, ESC are able to form an entire organism.
However, since they cannot contribute to the formation of trophoectoderm, for example, ESC are considered not totipotent, like a fertilized egg is, but pluripotent. To ascertain pluripotency in vitro, cells are injected into SCID mice and resulting teratoma are analyzed for the expression of all three germ layers [32].
Markers for pluripotency analysis include both transcription factors and surface markers.
Best studied transcription factors are Oct3/4, Sox2 and Nanog. These three factors have target genes in common [42] and are important key regulators of pluripotency. They are characteristic for both human and mouse ESC. Surface markers, on the other hand, differ between species [32]. SSEA‐1 for example is a pluripotency marker for mouse ESC, while SSEA‐3, SSEA‐4 and Tra‐1‐81 are rather surface markers that are expressed on human ESC.
Another marker of pluripotency for both human and mouse ESC is the alkaline phosphatase [43].
Both EpiSCs and mESC can form teratomas upon injection into immunocompromized animals, the hallmark of pluripotency. Furthermore, ESC are able to functionally integrate and contribute to the formation of chimeric organisms, upon transfer of ESC into the ICM of a E3.5 blastocyst [43]. This ability is not shared by EpiSCs and only mESC can contribute to chimerism, and by these means reveal what is called “naïve pluripotency” by Nichols and Smith [28]. EpiSCs on the other hand are considered to possess a “primed pluripotency” as they can form teratomas upon injection into mice but were so far unable to integrated into
2.4. Pathways in pluripotency
2.4.1. LIF
As can be seen in Figure 2, leukemia inhibitory factor (LIF) is a cytokine that belongs to the IL6 family. It is one important factor required for the maintenance of the pluripotent state in mESC in the presence of serum. It is produced by feeder cells, is present in conditioned media from feeders and can also be added extrinsically.
Upon binding of LIF to its receptor, the formation of a heterodimer consisting of the LIF receptor β and gp130 on the cell membrane is initiated. Subsequently the transcription factor Stat3 is activated over a Janus Kinase (Jak) signal [32]. After phosphorylation and dimerization, Stat3 moves into the nucleus and acts as a transcription factor activating genes necessary for pluripotency. See Figure 2 for illustration.
The group of Takashi created a fusion protein consisting of Stat3 and the binding domain of an estrogen receptor. A synthetic tamoxifen derivative could maintain the pluripotent state of mESC being transfected with the fusion protein upon withdrawal of LIF but still in the presence of serum [44].
Figure 2. Overview of the different signaling pathways mediating pluripotency in mESC. The Wnt/βcatenin, BMP4 and LIF pathway represent important signaling cascades regulating pluripotency in the nucleus. These pathways result in the activation of important pluripotency related transcription factors like Sox2, Oct4, Nanog and Stat3, which ultimately preserve pluripotency in mESC. (Taken from [43]).
One target of the LIF‐Stat3 signaling is c‐myc [32] that was shown to be important also within mESC [45]. Upon withdrawal of LIF, Myc mRNA levels decrease and the Myc protein gets phosphorylated and subsequently degraded [45].
LIF, however, has been found dispensable for the embryonic development in mice in vivo as some LIF‐mutant mice were shown to survive until adulthood [46]. Moreover, serum seems to be required in addition to LIF to maintain the pluripotency of mESC [43].
2.4.2. BMP4
There is another pathway being important to sustain self‐renewal, which is a characteristic of pluripotency. This pathway is signaling over bone morphogenetic protein‐4 (BMP4) as illustrated in Figure 2. In contrast to LIF, BMP4 is present in fetal calf serum. In BMP4‐free media, LIF alone cannot sustain pluripotency and differentiation into neurons occurs. In other words, LIF and BMP4 together are able to maintain pluripotency in the absence of feeder cells and serum [47].
BMP4 is acting via the Smad pathway and triggers the expression of “inhibitor of differentiation” (Id) genes [47]. In the presence of LIF, Id genes activated by BMP4 prevent the differentiation into the neural lineage. In the absence of LIF; BMP4 triggers differentiation of cells into mesoderm and endoderm [47].
2.4.3. WNT/Β‐CATENIN
Besides LIF and BMP4 signaling, the canonical Wnt/β‐catenin pathway is also involved in maintaining the pluripotent state of both mouse and human ESC [48]. As can be seen in Figures 2 and 3, upon binding of the Wnt protein on the surface receptor Frizzled (Fzd), the glycogen‐synthase kinase‐3 (GSK3) gets inhibited, leading to a reduced degradation and, thus, increased levels of β‐catenin [43]. After translocation to the nucleus, β‐catenin acts together with members of the Tcf transcription factors to form complexes that mediate gene expression. As shown in Figure 3, Tcf3 represses the pluripotency genes Oct4, Nanog and Sox2. β‐catenin blocks the suppressive function of Tcf3 [49, 50]. Subsequently, the target genes Oct4, Nanog and Sox2 are activated [48]. Berge et al. demonstrated the
dependency of mESC for Wnt signals, in contrast to EpiSCs [51]. β‐catenin does not only operate with Tcf3, but promotes pluripotency together with Tcf1 [50].
Figure 3. Wnt/βcatenin, E‐cadherin and LIF pathways in mESC are involved in the maintenance of pluripotency in mESC. The correlation between the Wnt pathway and the Tcf3 and Tcf1 proteins are illustrated. After mobilization of βcatenin into the nucleus, it complexes with Tcf3. By these means Tcf3’s suppressive function on pluripotency genes like Oct4 is abolished and pluripotency is maintained. In a complex with Tcf1, βcatenin activates pluripotency associated genes. (Taken from [49]).
2.5. Transcription factors involved in pluripotency
Three transcription factors have been shown to play a major role in maintaining the pluripotent state of both the inner cell mass and embryonic stem cells: Nanog, Oct3/4 and Sox2.
2.5.1. NANOG is a transcription factor that is found in both human and mouse pluripotent stem cells [52]. While Nanog is necessary for the formation of germ cells [52], it does not
reported a trend towards differentiation of cells upon transient downregulation of Nanog, but not commitment to differentiation. Furthermore, Nanog seems indispensable for the
formation of germ cells since Nanog‐/‐ cells cannot reach the genital ridge during development [53]. Nanog can preserve pluripotency in mESC cells in the absence of LIF, and by these means LIF/Stat3 signaling is circumvented [52]. The sequence that Nanog binds to has been proposed but it is still a controversial matter [33].
Loh et al. point out the importance of Nanog as a key regulator of pluripotency by controlling Oct4 and Sox2 expression levels. Indeed, physiological levels of Oct4 and Sox2 prevent differentiation, while overexpression of Oct4 triggers differentiation [54]. Both Nanog and Oct4 have Esrrb and Rif1 as common targets. These have been shown to be important for conserving pluripotency in mESC [55].
2.5.2. OCT4 is member of the octamer transcription factor class. As the class name indicates, Oct4 binds to a DNA site composted of the eight base pairs ATGCAAAT [56, 57].
Oct4 is member of the POU (Pit, Oct and Unc) transcription factor class that interacts with DNA through two DNA binding domains [33], one low and one high affinity binding domain [58].
To uncover the role of Oct3/4 in ESC, Niwa et al. regulated the expression levels of Oct3/4 via a tetracycline‐regulated transactivator and a transactivator‐responsive Oct3/4 transgene in Oct3/4 null mESC [54]. They show that ESC do not keep their pluripotent state but differentiate into trophoectoderm upon downregulation of Oct3/4, regardless of the presence or absence of LIF. Since an overexpression of Oct3/4 triggers differentiation as well, Niwa et al. concluded that a controlled Oct3/4 level within ES cells is necessary to keep
the stem cell phenotype. They defined the differentiation threshold at 50% of the endogenous Oct3/4 expression levels.
Loh et al. determined the targets of Oct4 and Nanog in the whole mESC genome using, among other methods, paired‐end ditag technology in combination with chromatin immunoprecipitation (ChIP) [55]. Between the human and the mouse genome, Oct4 and Nanog targets did not result to overlap much, as this accounted for 9% for Oct4 and 13% for Nanog, with ~1000 binding sites for Oct4 and ~3000 for Nanog. Nevertheless, Loh et al.
demonstrated that Oct4 and Nanog shared some important targets. Downstream genes of Nanog and Oct binding targets were shown to be involved in maintaining pluripotency.
Nanog is not the only transcription factor binding at the same genomic location as Oct4. In fact, Oct4 was proposed as “anchor point” for the further binding of other transcription factors [38]. Furthermore, Oct4 and other factors seem to autoregulate their own expression levels [38]. Enhancer regions of Oct4 and Nanog are bound by additional transcriptions factors.
2.5.3 SOX2 belongs to a family of proteins that contains a “high mobility group (HMG) box DNA binding domain box”. Sox2 was shown to bind in minor grooves of DNA at defined binding sites [33]. Loh et al. demonstrated that Sox2 binding sites overlap to a great extends with Oct4 sites, indicating that Sox2 and Oct4 act together to mediate their target genes.
2.6. The chromatin state in ESC
Transcription factors are not the only key actors for conserving the pluripotent state of ESC:
non‐coding‐ and microRNA as well as the chromatin status of ESC [38] also play an important role.
In ESC chromatin is organized to a higher degree as euchromatin in comparison to differentiated cells. While cells commit to different lineages during differentiation, they reorganize their chromatin structure and more compact heterochromatin is found [59, 60].
Chromatin structure is influenced by the methylation status of DNA, as well as the modifications of histones. In addition, ATP‐dependent enzymes remodeling chromatin have been shown to influence the chromatin status in ESC [60].
2.7. The metabolism in ESC
Knowledge has been gained on the genetic and epigenetic status of pluripotent stem cells.
Chromatin status as well as DNA methylation pattern change extensively during the transit from pluripotent to differentiated cells.
Besides the genetic and epigenetic status being important to keep the pluripotent characteristics within a ESC, the group of Banerjee pointed out the importance of metabolism in the control of ESC proliferation and differentiation [61]. They used the chemical component Carbonyl Cyanide m‐Chlorophenylhydrazone to knock down mitochondrial function. While the cell cycle phase was not affected, the proliferation rate of mESC was slowed down in comparison to untreated mESC, indicating the importance of mitochondria on ESC proliferation, also during differentiation. Indeed, the expression
profiling after seven days of treatment revealed significant differences in genes related to development and differentiation. Further investigation needs to be done to understand the role of mitochondria within proliferating and differentiating ESC. However, these results underline the importance of grasping the impact of mESC metabolism regulation in pluripotency and differentiation conditions.
3. Cardiac differentiation
3.1. The heart
In line with the pivotal function of the heart in vertebrates, the heart is among the earliest organs to develop from mesoderm [62‐65]. After proliferation, maturation and organization of cardiac cells, the mammalian heart is build up by four chambers, the left atrium (LA) and right atrium (RA) as well as the left ventricle (LV) and the right ventricle (RV) [63].
Figure 4. Schematic illustration of the heart anatomy and the directions of blood flow (picture modified from http://www.fetal.com/FetalEcho/02%20Anatomy.html).
3.2. Cardiogenesis
Human cardiogenesis takes place over several weeks. In contrast, the murine development is finalized after 48 hours [62]. In mice, heart progenitors derive from a region that is anterior to the primitive streak (PS) [66], see Figure 5 for illustrations. From there, the cells position in two areas left and right to the midline (ML) and posterior to the head fold (HF)
