ISSN 1990-519X, Cell and Tissue Biology, 2009, Vol. 3, No. 3, pp. 199–212. © Pleiades Publishing, Ltd., 2009. Original Russian Text © A.S. Grigoryan, P.V. Kruglyakov, 2009, published in Tsitologiya, Vol. 51, No. 5, 2009, pp. 442–454.

Murine Embryonic Stem Cells as a Model for Human Embryonic Stem-Cell Research A. S. Grigoryan and P. V. Kruglyakov Trans Technologies LTD, St. Petersburg, Russia e-mail: [email protected] Received September 29, 2008

Abstract—Over the last several decades, murine embryonic stem cells (mESCs) have been used as a model for human embryonic stem cell (hESC) research. The relevance of this approach has not yet been proven. There is a great deal of evidence that is indicative of substantial differences between these two cell types. An analysis of the literature shows that the differences concern ESC proliferation, self-renewal, and differentiation. Consequently, mESC may be considered as a model object for hESC studies only for some aspects of their biology. The alternative model objects, such as primate ESC, are also discussed briefly in this review. Key words: murine embryonic stem cells, human embryonic stem cells, ESC application, ESC markers, ESC differentiation. DOI: 10.1134/S1990519X09030018

Abbreviations: ICM, inner cell mass; ESC, embryonic stem cells; mESC, murine embryonic stem cells; hESC, human embryonic stem cells.

to examine an object is a metaphor of a real process and reflects the properties of the object. The choice of model is defined by its adequacy in evaluating the process; the more complicated the process, the higher the requirements for the model. Immortalized cell lines are widely used in cell biology, biochemistry, and genetics (Sinz and Kim, 2006). Most of these models are specific to their application and are used to examine a particular process (metabolic, signaling, etc.). In the case of mESC and hECS, we speculate on a general model that may be effective in many aspects of its application. mESCs are applied to study various aspects of hESC biology. It seems that phenomenological features of proliferation, e.g., the stemness and differentiation in the ESCs of both species, are very similar. However, the conclusion that the regulation of these processes is identical is not so obvious. The aim of this review is to survey the literature data on mESCs and hESCs to compare the external regulation of their behavior in culture and transplantation into the recipient organism, as well as intracellular regulation of ESC-specific attributes (self-renewal and pluripotency). This approach will help us to understand whether the data obtained on mESCs can be extrapolated to hESCs.

About 4000 papers on murine embryonic stem cells (mESC) applied as a model in human embryonic stem cell (hESC) research have been published. The approach is quite explainable, as mice are classic objects of experimental biology. About a million studies in cell biology, genetics, embryology, and medicine have been performed on mice in the last century (US National Library of Medicine Data, http://www.nlm.nih.gov). However, whether and to what degree mESCs may be regarded as adequate objects for studying hESCs remains to be determined. Research on hESCs faces the human moral dilemma; therefore, the search for model objects that are both suitable and well-examined is being undertaken as a compulsory measure. In fact, we must admit that, currently, mESCs are not regarded as the only acceptable model for hESC research. Thus, stem cell lines established from mouse epiblasts of postimplantation embryos were suggested as more adequate models for hESC research than mESCs derived from the inner cell mass (ICM) of blastocysts (Tesar et al., 2007). This is a single publication; therefore, it is difficult to conclude whether or not this is true. However, the relevance of this study is justified simply based on its goal of finding a new model for hESC research. Modern science is characterized by increasing interpretative activity; therefore, one cannot underestimate the importance of proper models. Every model applied

Historical Aspect In 1998, J. Thomson and colleagues published a paper (Thomson et al., 1998) in Science on the first human embryonic stem cell line established from surplus human blastocyst available in IVF clinics. The 199

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cells displayed unlimited self-renewal capacity and the ability to differentiate into multiple cell lines. The use of hESCs is a promising strategy for regenerative medicine due to their ability to differentiate into all embryonic and adult tissues; however, it as prompted a great deal of discussion regarding legislation on the current applications of hESCs and future therapeutic uses. Several countries, including Germany, Austria, and Ireland proclaimed a ban on certain types of hESC research, whereas United States policy restricts the state funding of hESC research. Despite these limitations, 155 hESC lines were obtained up until 2005 (Weiss, 2005). The moral aspects of hESC research concern their derivation, maintenance in culture, characterization, induced differentiation, and transplantation into recipients. Opponents of hESC research based on cell lines derived from ICN suggest the use of cells with properties similar to those of hESCs. For this purpose, the following approaches are proposed: 1. Nuclear reprogramming of differentiated somatic cells by fusing them with ESCs. However, the elimination of all ESC chromosomes from hybrid cells has not yet been successfully achieved (Cowan et al., 2005). 2. The derivation of ESCs from blastocyst-like structures. This method is based on the in vitro transfer of the nucleus from somatic cells into zygotes and subsequent establishment of pluripotent cells from the developing blastocyst-like structure (Hurlbut, 2005). To restrict stem cell potency in order to generate viable embryos in vivo (Meissner and Jaenisch, 2006), the expression of genes responsible for ESC differentiation into trophectoderm, e.g., Cdx2, (Niwa et al., 2005), is suppressed. 3. The derivation of hESCs from single blastomeres of IVF blastocytes (Klimanskaya et al., 2006; Krylova et al., 2003, 2005). 4. The generation of hESC-like cells by the induced differentiation of somatic cells by the reactivated expression of genes responsible for pluripotency. Induced pluripotent cells were successfully produced in humans by transfection with Oct-4, Sox-2, Nanog, and Lin28 (Yu et al., 2007) or Oct-4, Sox-2, c-Myc, and Klf4 genes (Takahashi and Yamanaka, 2007). Induced and true pluripotent cells are very similar. However, at present, the application of induced pluripotent cells for therapy seems hardly possible. The cells are produced by retroviral or lentiviral transfection and may potentially be oncogenic (Hanna et al., 2007). Thus, hESC-like cells are not totally adequate models for hESC research and hESCs generated by alternative methods are limited in their possible clinical applications. Therefore, a large part of hESC research is performed on animal cells, particularly on ESCs derived from mouse blastocysts. The results obtained on animal cells are then extrapolated to processes that occur in hESCs. It should be noted that mESCs were obtained much earlier than hESCs (Evans and Kaufman, 1981).

The authors used a technique previously utilized to isolate rabbit ESCs (Cole et al., 1966). In 2007, Dr. Martin Evans was awarded the Nobel prize in physiology and medicine for his great contribution to modeling human diseases on mESCs with directed genetic modifications (Deb and Sarda, 2008). The biology of mammalian ESCs, mostly mESCs, has been studied more comprehensively than the biology of hESCs. The comparison of mESCs and hESCs mostly concerns transcriptome and proteome profiles. Similar transcription, however, does not indicate identical protein expression in both cell types. The particular gene transcription and mRNA synthesis does not always define the expression or activity of the protein in cells (Wei et al., 2005). Protein profiles of hESCs and mESCs (Van Hoof et al., 2006) does not provide information whether intracellular protein functions are similar or different; knowledge on protein involvement in signal pathways is also frequently missing. The large body of evidence from screening publications is difficult to interpret due to the lack of sufficient information on particular genes and their products. There are no published attempts to compare mESCs and hESCs based on the data available for unambiguous interpretation and to solve the problem whether it is possible to extrapolate results gained from mESC to hESC biology. mESC and hESC Cultures ESCs are blastocyst inner cell masses (ICMs) transferred in culture (figure). It should be emphasized that the behavior of ICMs in vivo and ESCs in vitro is significantly different. The main feature of ESCs is pluripotency, i.e., the ability to differentiate into any embryonic or adult tissues; all cells of a particular line are identical. In ICMs, morphogenes direct separate populations to differentiate into particular cell lines; this process is mostly controlled by bone morphogenetic proteins (BMPs), the wingless and integration site growth factor (Wnt), and the sonic hedgehog growth factor (Shh). The spatial location in blastocysts of cells and their interactions with the surrounding trophectoderm also play an important morphogenetic role (Itskovitz-Eldor et al., 2000; Buehr and Smith, 2003). Stable mESC populations capable of unlimited proliferation in culture have only been established from mouse strains 129 (129/Ola and 129/Sv) and C57 BL/6. Standard protocol for isolating and cultivating these strains was published in 1996–1997 and remains almost unchanged (McWhir et al., 1996; Brook and Gardner, 1997). mESC cell lines derived from other mouse strains are unstable: the number of euploid cells decreases during long-term cultivation (Mitalipov et al., 1994). Why stable mESC can only be established from two mouse strains and a few other animal species is unclear. Cells isolated from murine ICMs are highly clogenic (Brook and Gardner, 1997). It is unknown CELL AND TISSUE BIOLOGY

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Table 1. mESC and hESC markers Markers

Mouse ESC

Human ESC

Source

SSEA-1

+

–

Solter and Knowles, 1978; Krylova, 2003

SSEA-3/4

–

+

Thomson et al., 1998; Reubinoff et al., 2000; Xu et al., 2001; Henderson et al., 2002

TRA-1-60/81

–

+

Thomson et al., 1998; Reubinoff et al., 2000; Xu et al., 2001; Henderson et al., 2002

TRA-2-54

–

+

Henderson et al., 2002

GCTM-2

–

+

Pera et al., 2000; Reubinoff et al., 2000

TG 343

?

+

Henderson et al., 2002

TG 30

?

+

Pera et al., 2000

CD 9

+

+

Pera et al., 2000

CD 133/prominin

+

+

Carpenter et al., 2004; Kania et al., 2005

Alkaline phosphatase

+

+

Wobus et al., 1984; Thomson et al., 1998

Oct-4

+

+

Thomson et al., 1998; Pesce et al., 1999

Nanog

+

+

Chambers et al., 2003; Mitsui et al., 2003

Sox-2

+

+

Avilion et al., 2003; Ginis et al., 2004

FGF4

+

–

Ginis et al., 2004

LIF-R

+

+/–

Richards et al., 2004

Other characteristics Telomerase activity

+

+

Thomson et al., 1998; Armstrong et al., 2000

Regulation of self-renewal

Feeder or LIF in culture medium

Feeder (inactivated mouse or human fibtoblasts), serum and bFGF

Niwa et al., 1998; Thomson et al., 1998; Xu et al., 2001; Chambers et al., 2003; Ying et al., 2003

Cell culture morphology

Round multilayer colonies

Monolayer colonies with uneven edges

Thomson et al., 1998

Generation of embryoid bodies in suspension

Simple or cystic embryoid bodies

Cystic embryoid bodies

Itskovitz-Eldor et al., 2000; Jaenisch, 2000

Teratoma production in vivo

+

+

whether single cells isolated from human blastocysts also have this property because hESCs are derived from whole intact blastocysts (Avery et al., 2006). mESCs are cultured either on a feeder layer (confluent embryonic fibroblasts irradiated or treated with chemicals to inhibit mitotic activity) or media supplemented with LIF (leukemia inhibitory factor). LIF is a soluble glycoprotein from the IL-6 cytokine family that activates signaling through the gp130 receptor and JAK/STAT3-1/3 factors, thus triggering MAP-kinase cascade (Burdon et al., 1999a; 1999b). Cytokines related to LIF, specifically IL-6, IL-11, OSM (oncostatin-M), CNTF (ciliary neurotrophic factor), and CT-1 (cardiotrophin-1), also facilitate mESC maintenance in vitro (Niwa et al., 1998). These cytokines also support mESC self-renewal and the expression of pluripotent markers (mostly Oct4) and inhibit spontaneous differentiation (Buehr et al., 2002). The lack of IL-6 family cytokines and feeder, as well as the blockage of STAT3 CELL AND TISSUE BIOLOGY

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Wobus et al., 1984; Thomson et al., 1998

signaling, induces mESC spontaneous differentiation (Boeuf et al., 1997). The properties of mESCs make them very helpful in fundamental research in development biology, genetics, and biotechnology; they are very useful in studying the inactivation of the X-chromosome, as well as in testing biologically active and potentially toxic substances in vitro (Keller, 1995; Thomson et al., 1998; Heard et al., 1999). mESCs are widely used to obtain transgenic animals to study human genetic diseases (Clarke, 1994). Some authors believe that mESCs may be used to model hESC differentiation and apply mESC culture conditions to induce their specific differentiation (Sukoyan et al., 2002). However, it should be noted that the cells differ in behavior and transcriptomes. Expression of intracellular and surface markers in mESCs and hESCs. mESC and hESCs are different in surface marker expression (Table 1). Thus, stage-spe-

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cific embryonic antigen-1 (SSEA-1) expressed in mESCs is down regulated during differentiation, whereas, in hESCs, SSEA-1 expression is not registered. SSEA-1 is only identified in predifferentiated human cells. mESCs and hESCs have both unique and common markers. Surface markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and GCTM2 are expressed only in hESC (Boiani and Scholer, 2005). Pluripotent markers Oct4 (Niwa, 2000), Nanog (Chambers et al., 2003; Mitsui et al., 2003; Hart et al., 2004), Sox2 (Yuan et al., 1995; Avilion et al., 2003), and Utf-1 (Sato et al., 2003) are expressed in ESCs of both species and are highly conservative. A comparative analysis of surface and intracellular marker expression, as well as proteomic assay, demonstrated that protein sets of mESCs and hESCs are different. Factors that define unique ESC properties. mESC and hESC transcriptome (the set of expressed genes) assay showed that stem cell properties unique to ESCs are maintained differently in these species. About 100 out of 400 genes analyzed in mESCs and hESCs have distinguished expression pattern (Ginis et al., 2004). The expression of genes responsible for LIF, TGF-β, Wnt, and FGF2 signaling, as well as the metabolic, intracellular matrix, and cytoskeleton proteins in these cell types, may differ dramatically (Wei et al., 2005). It was proposed that mESCs and hESCs differ in the mechanisms underlying the regulation of proliferation, population self-renewal, apoptosis, and maintenance of pluripotency. ESC pluripotency markers are Oct4, Sox2, and Nanog. These proteins bind both transcription active and inactive genes in mouse and human ESCs. The active genes are Oct4, Sox2, and Nanog, as well as Rif1, which controls telomerase activity and constant chromosome telomere length in ESCs (Adams and McLaren, 2004; Rodda et al., 2005). The Jarid2 and Smarcad1 genes are regarded to be important regulators in the embryonic development (Schoor et al., 1993; Jung et al., 2005), however, their role in pluripotency maintenance is unclear. It should be noted that most inactive genes for Sox2, Nanog, and Oct4 binding are engaged in ESC specific differentiation (Boyer et al., 2005; Loh et al., 2005). Sox2, Nanog, and Oct4 are presumably direct regulators of transcriptional factors, which are hierarchally activated during ESC differentiation, in not only three germ layers, but also in extraembryonic tissues. Each of these proteins, including the FoxD3 factor, which activates the Nanog gene, interacts with promoter regions of other genes which regulate plutipotency (Pan et al., 2006). Nanog and Oct4 gene targets are different in mESCs and hESCs. Thus, Hand1 and Myst3 genes, which are responsible for ESC differentiation in neuronal and cardiomyocyte directions, are only controlled by Nanog and Oct4 in hESCs, whereas the Esrrb (estrogenrelated receptor b) gene is only identified in mESCs.

Despite that, in mESCs, the Hand1 gene has no binding sites with Oct4 and Nanog, its expression increases if Esrrb and Rif1 genes have been inhibited by the RNA interference (Loh et al., 2005). Out of all identified gene-targets in mESCs and hESCs, only 9.1% for Oct4 and 13% for Nanog are common (Avery et al., 2006; Loh et al., 2005). It is possible that the smaller number of orthologycal gene targets in mESCs than in hESCs is defined by fundamental differences in the regulation of the transcription system in these species. It is highly probable, however, that the difference results from various experimental approaches. The problem of these differences is very important. The solution should answer whether it is possible to extrapolate data on transcriptome analysis obtained on animal ESCs to hESCs. For example, data on gene expression in mESCs and hESCs obtained by various techniques (microarray, serial analysis of gene expression (SAGE), proteomic analysis) differ significantly. Heterogeneous genome expression in various cell lines within a species complicates the interpretation. Nevertheless, most publications show that the expression of key signaling molecules triggered by LIF (STAT3, LIFR, gp130) is higher in mESCs, whereas the expression of pluripotency factors Oct-3/4 and Sox2 is more apparent in hESC (for details, see review by Wobus and Boheler, 2005). Experiments on the selective suppression or hyperactivation of Nanog and Oct4, together with an analysis of genome expression, show which targets for these factors are important for ESC pluripotency and selfrenewal. These experiments confirmed the role of the Esrrb factor and, in mESCs, revealed two new markers of undifferentiated cells, Tcl1 (T-cell lymphoma-1) and Tbx3 (T-box protein 3) (Ivanova et al., 2006). It was found that both Esrrb and Nanog factors are required for the proliferation of germ cells and placenta development (Mitsunaga et al., 2004). Tcl1 stimulated the proliferation of ESCs (Mitsui et al., 2003) and protected them from apoptotic death (Niwa et al., 2005; Meshorer and Misteli, 2006). It is unclear which factors controlled by Oct4, Nanog, and Sox2 are responsible for these processes in hESCs. mESC and hESC requirements for morphogenetic and growth factors. hESC population doubling time (30–35 h) is higher than in mESCs (12–15 h) (Amit et al., 2000). The properties of hESCs are maintained by the feeder and neither LIF nor other cytokines from the IL-6 family support Oct4 or Nanog expression. LIF in combination with the BMP-4 factor ensures the selfrenewal of ESCs, as well as their ability to differentiate into multiple cell lineages, the colonization of embryo tissues after transplantation into the blastocyst, and the generation of germ line cells. BMP-4 induces expression of Id-genes (genes inhibiting differentiation) in hESC via signaling mediated by Smad proteins (Humphrey et al., 2004; Wobus and Boheler, 2005). Without CELL AND TISSUE BIOLOGY

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feeder and serum hESC may be maintained only if three factors are added; LIF, FGF2 and TGFβ. However, the presence of LIF does not seem to be critical (Amit et al., 2004). Unlike mESCs, hESC self-renewal and pluripotency requires activin/Nodal morphogenetic factors (Brons et al., 2007). It would be quite interesting to compare the intracellular signaling triggered in mESCs and hESCs by the same conservative factors. Comparison of signal cascades activated by morphogenetic factors in mESC and hESC. mESC and hESC differently respond to LIF, TGFβ, S1P, BMP-4, and Wnt (Avery et al., 2006). The most examined is the cell response to Wnt. Signaling activated by Wnt is responsible for many processes in mammalian embryogenesis, more specifically embryonic induction, cell polarization, etc. (Boyer et al., 2005). Ligands of the Wnt family bind with the frizzled receptor and activate the disheveled protein, which results in the removal of glycogen-synthetase kinase 3β (GSK-3β) from the APC complex (axin/adenomatous polyposis coli complex) and its inactivation, which prevents the ubiquitin-dependent degradation of β-catenin, which is a transcriptional factor that activates the expression of genes responsible for stem cell proliferation and self-renewal (Austin et al., 1997; Reya et al., 2003). The data on Wnt implication in mESC differentiation are controversial; mESC treatment with retinoic acid induces the neuronal differentiation inhibited by stable Wnt1 expression or the hyperproduction of Sfrp2 protein, which is a Wnt antagonist (Austin et al., 1997). mESC differentiation potential is modulated by the β-catenin dosage (Kielman et al., 2002). On the other hand, mESC treatment with GSK-3β pharmacological inhibitors (Ding et al., 2003) and enhanced Wnt1 expression (Tang et al., 2002a, 2002b) promote mESC neuronal differentiation. The expression of Wnt pathway molecules is highly variable in hESC lines (Sato et al., 2004; Brandenberger et al., 2004; Walsh and Andrews, 2003). There is a lack of transcripts of ligands from the Wnt family in hESCs, despite the presence of their antagonists and inhibitors (albeit with varying dosage in hESC lines) (Brandenberger et al., 2004). It was proposed that Wnt sustained the self-renewal of both mESCs and hESCs (Sato et al., 2004). However, it was later found that the inhibition of GSK-3β with lithium chloride, which triggers Wnt signaling provoked the differentiation of hESCs into multiple lineages (Avery et al., 2006). Similar results were obtained with human embryonic carcinoma cells. Most likely, in hESCs, GSK-3β is engaged in other signal cascades besides in mESCs. Thus, in mESCs, Wnt-signaling is mainly responsible for self-renewal and pluripotency, whereas, in hESCs, it controls proliferation and differentiation. In mESCs, β-catenin is active in the undifferentiated popCELL AND TISSUE BIOLOGY

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ulation, contrary to hESCs, which are characterized by a low level of β-catenin prior to differentiation (Dravid et al., 2005). ESC Differentiation ESCs transplanted into an adult organism produce teratomas (benign tumors) and teratocarcinomas (malignant tumors) comprised of three germ-layer derivates (ecto-, endo-, and mesoderm). This was demonstrated by the ectopic transplantation of both mESCs and hESCs into animals with induced immunodeficiency. hESCs injected subcutaneously and intramuscularly into the testicles of nude mice or into cardiac muscle of nudes or mice with functioning immune systems generate teratomas or teratocarcinomas (ItskovitzEldor et al., 2000; Odorico et al., 2001; Wernig et al., 2004; Nussbaum et al., 2007). Because ESCs are teratogenic, it seems reasonable to use ESCs for transplantation that have differentiated in vitro into precursors of particular tissues. However, the purity of both mESC and hESC differentiated populations is no more than 80% (Deb and Sarda, 2008); therefore, the application of ESCs is not safe for cell therapy. The presence of even a single undifferentiated cell in culture assigned for transplantation into a recipient organism makes these cells potentially risky for the development of teratomas. However, there is a single publication on the pure population of ectodermal cells derived from hESC treated with BMP and β-mercaptoethanol and cultured under rather complicated scheme. These differentiated cells were cultured for 16 passages (about 60 population doubling) and then died due to senescence. During the cultivation period, no karyotypic deviations were registered. The cells transplanted into SCID mice did not produce teratomas (Aberdam et al., 2008). Although this is currently the first and only success in this field, these results are cause for hope. Many protocols for the differentiation of mESCs and hESCs in various directions have been published (Table 2). Similar conditions are usually applied to differentiate mESCs and hESCs into specific lineages. Thus, both types of cells cultivated with the addition of β-mercaptoethanol and ascorbic acid without feeder differentiate into cardiomyocytes (Guo et al., 2006; Sartiani et al., 2007). Erythropoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1α and IL-3 are applied for mESC and hESC differentiation into hematopoietic stem cells (Valtieri et al., 1989; Wiles and Keller, 1991). The reduced expression of the pluripotency genes was already registered 12 h after differentiation was induced (Sene et al., 2007). Gene expression was assayed during the spontaneous differentiation of ESCs in embryoid bodies. Embryoid bodies are formed by

204

GRIGORYAN, KRUGLYAKOV Zygote

TOTIPOTENT

Morula

Return transplantation

Ectoderm Germ cells

Blastocyst

Mesoderm ICM

ESC

Entoderm

PLURIPOTENT In vitro Primordial germ cells

Ectoderm

Mesoderm

Entoderm

Embryonic germ cells

MULTIPOTENT

Progenitors

Organs And other derivatives of ectoderm

In vivo

And other derivatives of mesoderm

And other derivatives of entoderm

Derivation and differentiation of embryonic stem cells (ESC).

ESC maintenance without feeder; furthermore, LIF in the suspension culture prevented the adhesion of cells to the plastic surface (Wobus and Boheler, 2005). It was found that the expression of 24 genes was reduced during the spontaneous differentiation of mESCs, in particular Oct4/Pou5f1 (Loh et al., 2005), Cripto/Tdgf1 (Strizzi et al., 2005), Rex1 (Thomson and Gudas, 2002) and the expression of 12 genes with unknown functions (but that are homologous to human genes) was enhanced. It is of interest to elucidate how this process occurs in hESCs, as ESC differentiation is very important for their therapeutic applications, because the above-mentioned transplantation of undifferentiated cells into recipient organism may produce tumor. On the other hand, the transplantation of terminally differentiated cells with limited life spans may be senseless.

CONCLUSIONS Taking into consideration the data accumulated, one can assert that mESCs are the universal model for hESC research. The main directions of mESC research are (1) pluripotency, (2) proliferation, and (3) differentiation into precursors of various tissues. mESCs and hESCs are phenomenologically similar. This is also true for ESCs of other species taking into account the common function of these cells. However, signal pathways that determine cell functioning in mESC and hESCs are different. The prognosis of hESC behavior in culture and in organisms after transplantation based on mESC research may lead to mistakes. However, mESCs and hESCs are differentiated in specific lineages under similar culture conditions. CELL AND TISSUE BIOLOGY

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Table 2. hESC and mESC differention Lineage* Trophoblast

MouseESC

Human ESC

Only after the deletion of Oct-3/4

Source

+

Xu et al., 2002; Rao and Orkin, 2006

Adipocytes

+

+

Dani et al., 1997; Kang et al., 2007

Osteoblasts and osteocytes

+

+

Schuldiner et al., 2000; Buttery et al., 2001

Chonroblasts abd chondrocytes

+

+

Thomson et al., 1998; Kramer et al., 2000

Smooth muscle cells

+

+

Drab et al., 1997; Yamashita et al., 2000; Xie et al., 2007

Striated muscle cells

+

+

Rohwedel et al., 1994; Schuldiner et al., 2000

Cardiomyocytes

+

+

Doetschman et al., 1985; Maltsev et al., 1993; Maltsev et al., 1994; Metzger et al., 1996; Itskovitz-Eldor et al., 2000; Kehat et al., 2001

Hematopoietic cells

+

+

Doetschman et al., 1985; Wiles and Keller, 1991; Nakano et al., 1996; Nishikawa et al., 1998; Itskovitz-Eldor et al., 2000

Lymphoid, myeloid, erythroid and granulocyte-macrophage cell precursors

+

+

Potochnik et al., 1994; Odorico et al., 2001; Kaufman, Thomson, 2002

Endothelium cells

+

+

Risau et al., 1988; Yamashita et al., 2000; Kappas and Bautch, 2007

Dendrites

+

+

Fairchild et al., 2000; Sluvkin et al., 2006

Mast cells

+

Not shown

Dopaminergic neurons

+

+

Serotoninergic neurons

+

Not shown

Gabaergic neurons

+

+

Cholinergic neurons

+

Not shown

Glutamatergic neurons

+

+

Strubing et al., 1995; Finley et al., 1996; Carpenter et al., 2001

Tsai et al., 2000 Kawasaki et al., 2000; Lee et al., 2000; Carpenter et al., 2001; Rolletschek et al., 2001 Lee et al., 2000 Bain et al., 1995; Strubing et al., 2005; Carpenter et al., 2001 Fraichard et al., 1995

Not shown

+

Carpenter et al., 2001

Motor neurons

+

+

Wichterle et al., 2002; Lee et al., 2007

Oligodendrites

+

+

Angelov et al., 1998; Brustle et al., 1999; Liu et al., 2000; Tropepe et al., 2001; Billon et al., 2002; Shin et al., 2005

Astrocytes

+

+

Fraichard et al., 1995; Andressen et al., 2001; Carpenter et al., 2001; Rolletschek et al., 2001; Tang et al., 2002

Hepatocytes

+

+

Hamazaki et al., 2001; Jones et al., 2002; Rambhatla et al., 2003; Kania et al., 2004

Insulin-producing cells

+

+

Assady et al., 2001; Lumelsky et al., 2001; Hori et al., 2002; Blyszczuk et al., 2003; Segev et al., 2001

Keratinocytes

+

+

Bagutti et al., 1996; Ji et al., 2006; Haase et al., 2007

Germ cells

+

+

Aflatoonian, Moore, 2006

Glycinergic neurons

Note: A comparison of 17 hESC cell lines showed marked differences in their propensity to differentiate spontaneously in embryoid bodies (Osafune et al., 2007). CELL AND TISSUE BIOLOGY

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ESC research started, at least, two decades ago. During this period, ESC lines were isolated from blastocytes of domestic animals (Prelle et al., 1999), chickens (Pain et al., 1996; Chang et al., 1997), hamsters (Doetschman et al., 1988), rabbits (Graves and Moreadith, 1993; Schoonjans et al., 1996), and rats (Iannaccone et al., 1994; Brenin et al., 1997; Vassileva et al., 2000; Buehr et al., 2002). However, only mouse and chicken ESCs transplanted into animal blastocysts were able to yield germ cells. For hESC research, the most important result was the establishment of primate ESCs, including rhesus macaque (Thomson et al., 1995; Pau and Wolf, 2004), common marmoset (Thomson et al., 1996), and cynomolgus monkey (Suemori et al., 2001). Primate ESCs are characterized by high telomerase and alkaline phosphatase activity, as well as by the expression of common hESC markers (Oct4, SSEA-4, TRA-1-60, TRA-1-81); additionally, they retain normal karyotype and pluripotency during long-term cultivation (Thomson et al., 1995; Kawasaki et al., 2002). It seems probable that primate ESCs are a more adequate model for hESC research than mESCs. ACKNOWLEDGMENTS

Angelov, D.N., Arnhold, S., Andressen, C., Grabsch, H., Puschmann, M., Hescheler, J., and Addicks, K., Temporospatial Relationships between Macroglia and Microglia During in Vitro Differentiation of Murine Stem Cells, Dev. Neurosci., 1998, vol. 20, pp. 42–51. Armstrong, L., Lako, M., Lincoln, J., Cairns, P.M., and Hole, N., MTert Expression Correlates with Telomerase Activity during the Differentiation of Murine Embryonic Stem Cells, Mech. Dev., 2000, vol. 97, pp. 109–116. Assady, S., Maor, G., Amit, M., Itskovitz-Eldor, J., Skorecki, K.L., and Tzukerman, M., Insulin Production by Human Embryonic Stem Cells, Diabetes, 2001, vol. 50, pp. 1691–1697. Austin, T.W., Solar, G.P., Ziegler, F.C., Liem, L., and Matthews, W., A Role for the Wnt Gene Family in Hematopoiesis: Expansion of Multilineage Progenitor Cells, Blood, 1997, vol. 89, pp. 3624–3635. Avery, S., Inniss, K., and Moore, H., The Regulation of SelfRenewal in Human Embryonic Stem Cells, Stem Cells and Development, 2006, vol. 15, pp. 729–740. Avilion, A.A., Nicolis, S.K., Pevny, L.H., Perez, L., Vivian, N., and Lovell-Badge, R., Multipotent Cell Lineages in Early Mouse Development Depend on SOX2 Function, Genes Dev., 2003, vol. 17, pp. 126–140.

The authors are grateful to V.S. Sergeev for his help in preparaing the illustrations for this review.

Bagutti, C., Wobus, A.M., Fassler, R., and Watt, F.M., Differentiation of Embryonic Stem Cells into Keratinocytes: Comparison of Wildtype and Beta 1 Integrin-Deficient Cells, Dev. Biol., 1996, vol. 179, pp. 184–196.

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