In vitro differentiation of embryonic stem cells

In vitro differentiation of embryonic stem cells

In vitro differentiation of embryonic stem cells Gordon M Keller National Jewish Center for Immunology and Respiratory Sciences Under appropriat...

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In vitro differentiation

of embryonic stem cells

Gordon M Keller National

Jewish Center for Immunology

and Respiratory


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Introduction The establishment of embryonic stem (ES) cell lines has opened many new experimental approaches in the field of mammalian developmental biology. ES cells are totipotent lines derived from the inner cell mass of developing blastocysts [l-3]. When maintained on embryonic fibroblasts in culture, ES cells retain their totipotential capacity and are able to generate cells of all lineages, including the germ line, after being introduced into host blastocysts [3,4]. Mutations introduced into murine ES cells by homologous recombination are easily carried into the germ line with this approach, resulting in the generation of mice with specific genetic deletions (reviewed in [5]). In addition to displaying these unique properties in viva, ES cells are able to spontaneously differentiate and to generate various lineages under appropriate conditions in culture [2,3,6-9,10*,11*]. Differentiation of ES cells in vitro provides a powerful model system for addressing questions related to lineage commitment, and offers several advantages over comparable approaches in the whole embryo. First, the generation of mature lineages from ES cells in culture provides access to populations of early precursors that are difficult, if not impossible, to access in oivo. Second, the developmental potential of ES cells carrying targeted mutations of genes essential for embryonic development can be determined in culture. Analyses of these mutations in viva are often complicated by early death of the embryo in utero. For in vitro studies, ES cells can be maintained either on embryonic flbroblasts, or in the presence of leukemia



inhibitory factor (LIF) in the absence of fibroblast feeder cells [12,13]. Differentiation of ES cells grown under either set of conditions is remarkably straightforward, and can be achieved by a number of different methods (see Fig.1). The technique used most frequently is to simply remove the ES cells from contact with the feeder cells, or ti-om the presence of LIF, and culture them in liquid or methyl cellulose containing media in bacterial grade petri dishes (see Fig. la). Under these conditions, the ES cells are unable to adhere to the surface of the culture dish, and generate a colony of differentiated cells known as an embryoid body (EB). A modification of this method, used primarily for studies on hematopoietic development, involves the differentiation of ES cells cultured directly on stromal cells (see Fig. lb). The rationale for using this approach is that the stromal cells can provide a supportive environment for the hematopoietic cells as they develop within the EBs. A third method of initiating the development of EBs is to culture the ES cells in ‘hanging drops’ for several days (see Fig. lc). The close association of the ES cells in these cultures promotes the efficient generation of EBs. Once formed, these EBs can be transferred to standard liquid cultures to complete their development. This method may be advantageous when differentiating ES lines that form EBs inefficiently when placed directly into methyl cellulose or liquid differentiation cultures. EBs generated by any of these culture systems can be assayed at various stages of development for the presence of specific populations. Some of the assay systems used to identify and characterize the various precursor populations that develop in EBs generated by the above methods are shown in Figure 1.

Abbreviations BMP-&bone morphogenetic protein 4; EBGmbryoid body; ES-embryonic stem; FCS-fetal calf serum; LIF-leukemia inhibitory factor; LTRSC-longterm repopulating stem cell; RAG-recombinase activating gene; SCID-severe combined immunodeficient.


0 Current


Ltd ISSN 0955-0674

In vitro differentiation of embryonic stem cells Keller

Assays for differentiated progeny

Differentiation protocol

Intact EBs analyzed for presence of differentiated populations

lineages detected

Hematopoietic: erythroid, myeloid, lymphoid Endothelial Muscle Muscle, neuronal

Intact EBs plated on cell development

Differentiation: ES cells cultured in MeC or liquid medium in petri dishes

Hematopoietic: myeloid


Hematopoietic: lymphoid


Dissociated EB-derived cells assayed in MeC for hematopoietic precursors (b)

Differentiation: ES cells cultured directly on stromal cells

Developing EBs with surrounding differentiated hematopoietic cells

Hematopoietic: lymphoid, erythroid, myeloid, hematopoietic stem cells

EB-derived cells transplanted into recipient mice

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Intact EBs p!ated on tissue culture plastrc for adhesive cell development as in (2), above


Q 1995 Current Opinion in Cell Biology

Fig. 1. Culture systems used for differentiating ES cells in vitro. Three different culture systems (a,b and c) used to induce the differentiation of ES cells in vitro are represented. (a) ES cells are induced to form EBs in liquid or methyl cellulose (MeC) containing media in bacterial grade petri dishes. (b) ES cells in direct contact with stromal cells generate EBs. (c) ES cells are induced to form EBs in ‘hanging drop’ cultures. EB development is completed in standard liquid differentiation cultures. The assays used with each of the differentiation protocols are also shown. (1) Differentiated populations can be identified in intact EBs by direct morphological analysis (e.g. presence of erythroid cells or of beating cardiac muscle), by histological analyses and by RNA analyses (using the polymerase chain reaction or Northern blotting). (2) Intact EBs can be plated directly on tissue culture plastic to enhance the growth of differentiated adhesive cells (e.g. muscle cells). (3) EBs can be dissociated with trypsin and/or collagenase, and the cells can then be assayed in methyl cellulose cultures with appropriate growth factors for the presence of hematopoietic precursors. (4) Dissociated EB cells can be stained with various antibodies for the presence of lineage-specific, cell-surface antigens (e.g. immunoglobulin can be used to stain B lymphocytes); staining can be detected by FACS analysis. (5) Dissociated EB cells can be used to repopulate the hematopoietic system of recipient animals. Appropriate references for the different parts of the figure are indicated here: (a) Hematopoietic: erythroid, myeloid, lymphoid [6,7,14-l 7,22*,24,32]; endothelial [6,27]; muscle [6,9,28-301; muscle, neuronal [11,40]. (b) Hematopoietic: myeloid, lymphoid [18,20,22-241; hematopoietic: lymphoid, erythroid, myeloid, hematopoietic stem cells [20,21,23,26**]. fc) Muscle [lo*].

Within the first few days of differentiation, EBs generate populations of cells that express genes indicative of primitive endoderm and mesoderm [2,3,14], suggesting that some of the earliest differentiated cell types found during normal embryogenesis also develop in vitro. After extended periods in culture, EBs can generate cells of the hematopoietic [6,7,14-21,22*,23-25,26**], endothelial [8,27], muscle [9,10*,28,29,30] and neuronal lineages [ll*]. One of the most intriguing aspects of these studies is the observation that the sequence of events leading to lineage commitment in viva is often also followed within EBs, suggesting that the in vitro model faithfully obeys the rules established in the whole animal. In this

review, I will discuss recent studies that highlight the potential of the ES cell differentiation system to widen our knowledge of gene function in early development.

Lineages generated Hematopoietic

from ES cells in culture


Within EBs, the establishment of the hematopoietic system is the most thoroughly analyzed developmental program [6,7,14-21,22’,23-251. Since the initial de+ cription of the formation of volk sac like blood islands


Cell differentiation

the earliest hematopoietic population. This lineage is present as early as day 4 of differentiation (see Fig. 2) and disappears by day 10-12 of differentiation [14]. Shortly after the emergence of the primitive erythroid lineage, precursors of the definitive (adult) erythroid and myeloid lineages (see Fig. 2) develop in both the yolk sac [36] and the EBs [14]. The early establishment and transient behavior of the primitive erythroid population, followed by the development of the definitive erythroid and myeloid lineages, strongly suggest that the molecular mechanisms involved in the establishment of the hematopoietic system in vioo also function within EBs in vitro. Further support for this notion is provided by studies demonstrating the expression of genes that are involved in early hematopoietic development before and during the onset of hematopoiesis both in the embryo and in EBs [14,17,32].

in cystic embryoid bodies [6], a large number of studies have documented the development of various erythroid, myeloid and, to a lesser extent, lymphoid lineages within EBs [6,7,14-21,22*,23-251. Several interesting and important findings have emerged from these studies. First, under optimal conditions hematopoietic development within EBs is efficient and highly reproducible. Second, commitment to hematopoiesis takes place in the absence of added growth factors with the exception of those present in fetal calf serum (FCS). Although not clearly defined, growth factors present in FCS appear to be important, as EBs generated in serum-free cultures show poor hematopoietic development [31*]. The fact that bone morphogenetic protein 4 (BMP-4) induces both mesoderm formation and hematopoiesis within the EBs in these defined culture conditions [31*] suggests that it could be one of the active components found in serum. In addition to factors present in FCS, the EBs themselves probably provide an environment that supports hematopoiesis, as genes encoding various growth factors and growth factor receptors are expressed early in EB development [14,17,32].

In addition to precursors of the erythroid and myeloid lineages, precursors with lymphoid potential also develop within EBs in differentiation cultures. Cells with lymphoid characteristics have been identified either following extended growth of EB-derived cells on stromal lines [20,22,23], or directly within EBs that have been generated in a environment containing a low oxygen concentration [24], or following the immortalization of EB-derived cells with transforming retroviruses [18]. The lymphoid nature of the cells which develop from these precursors has been defined by the expression of specific surface molecules (i.e. B220 and Thy1 [22,24]), by the expression of the genes RAG (recombinase activating gene)-1 and RAG-2 which are involved in immunoglobulin (Ig) and T-cell receptor (TCR) rearrangement [23,24], and by the presence of rearranged IgH and TCR 6 genes [22,24]. Cells displaying these characteristics were not detected before 15-20 days of differentiation, indicating that the lymphoid developmental program is established following the development of the erythroid and myeloid lineages (see Fig. 2). Although these in vitro studies demonstrate that cells with lymphoid characteristics are generated within the ES differentiation cultures, analysis of individual EBs indicates that the process is inefficient as only 10% of those that develop myeloid precursors also

Third and perhaps most important are the findings that the earliest stages of hematopoietic development within EBs (as defined by either the onset of expression of specific genes or the appearance of specific precursor populations) follow an ordered sequence of events similar to those observed in the developing embryo [14,16,17,32]. These similarities are best illustrated by studies that have analyzed the kinetics of development of the earliest hematopoietic lineages. The first sign of hematopoiesis in the mouse embryo is the appearance of large, nucleated, erythroid cells within the blood islands of the yolk sac at approximately 7.5 days of gestation [33]. These embryonic or primitive erythroid cells are characterized by the expression of the embryonic forms of globin, and constitute the predominant hematopoietic population found during early development [33,34]. As hematopoietic activity shifts from the yolk sac to the fetal liver between days 10 and 11 of gestation, this primitive erythroid population disappears [33-351. Similarly, within EBs the primitive erythroid lineage appears as

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In vitro differentiation of embrvonic stem cells Keller

contain lymphoid precursors [22]. These findings suggest that the in vitro differentiation conditions are not optimal for the generation of lymphoid cells. Further documentation of the development of lymphoid precursors in EBs has been provided by repopulation experiments using immunodeficient SCID (severe combined immunodeficient) or RAG-2-lmice as recipients [20,21,23]. As SCID mice can produce some endogenous lymphoid cells, studies with RAG-,?‘recipients, which are unable to generate any B or T cells, are easier to interpret. When transplanted into RAG-Z-‘recipients, ES-derived cells have been shown to generate both B lymphocytes expressing surface immunoglobulin (sIg) and CD3+ T lymphocytes [23]. In addition, serum immunoglobulin was also detected in animals, indicating that these ES-derived B cells were able to function in a normal fashion. Although this study demonstrated that ES cells can generate both T and B cell progeny, the extent to which they are able to do so is unclear as the total numbers of lymphoid cells were not indicated. Whether or not EB-derived lymphoid precursors can be identified reproducibly by this approach remains to be determined. One of the most troublesome aspects of hematopoietic development within the ES/EB system has been the inability to detect primitive stem cells capable of longterm multilineage repopulation of recipient mice, despite numerous attempts by a number of different investigators. A recent report by Palacios et al. [26**], which demonstrates multilineage repopulation of SCID recipients transplanted with ES-derived cells, suggests that this barrier has been broken. In this study, ES cells were allowed to differentiate directly on stromal cells that were presumably selected for their capacity to support the growth of hematopoietic cells (see In addition, the cultures were supplemented with interleukin-3, interleukin-6 and a conditioned medium from another stromal cell line. Differentiated cells from these cultures were injected into SCID recipients, which were then analyzed at various times after transplantation for the presence of ES-derived hematopoietic cells. Hematopoietic cells of ES origin were identified on the basis of the expression of MHC class I antigens, which differ from those of the recipient, and the presence of the SRY male specific gene in female mice repopulated with male ES cells. Using these markers, ES-derived lymphoid and myeloid cells were detected in primary recipients 12-18 weeks after transplantation, and in secondary recipients 16-20 weeks after transfer of bone marrow cells from the primary repopulated animals. The longterm multilineage nature of the repopulation in both primary and secondary recipients strongly suggests that primitive longterm repopulating stem cells (LTRSCs), generated fi-om ES cells in these differentiation cultures, were transplanted into these recipients. As indicated in this study, the generation of LTRSCs from ES cells is dependent both upon the appropriate supportive stromal layer, as not all lines are able to function in

this capacity, and upon the addition of stromal cell conditioned medium to the differentiation cultures. If future studies demonstrate that these conditions support the development of LTRSCs from other ES cell lines, and if these conditions (i.e. stromal cells and conditioned media) are easily adaptable to other laboratories, these findings could be considered a significant step forward in our approach to understanding the molecular events that regulate hematopoietic stem cell development, growth and differentiation.

Endothelial cells

Within the blood islands of the yolk sac endothelial cells develop in close association with the earliest hematopoietic cells. This finding has led to the hypothesis that these lineages arise from a common precursor, the hemangioblast (reviewed in [37]). Given the close developmental association of these lineages in the embryo, it is not surprising that endothelial cells develop within EBs in culture. Cells of the endothelial lineage were first identified surrounding pockets of primitive erythroid cells that develop in the blood island like structure of cystic embryoid bodies [6]. More recent studies have demonstrated extensive vasculogenesis (i.e. the development of new vessels) in EBs generated in the peritoneal cavity of mice, as well as in those that develop over extended periods of time in culture [8,27]. Under both sets of conditions, the endothelial cells form vascular channels that often contain hematopoietic cells. In addition to supporting vasculogenesis, EBs can also induce an angiogenic response (i.e. the sprouting of existing vessels) when cultured on the chorioallantoic membrane of quail embryos [8]. Thus, as observed with early hematopoiesis, many aspects of normal endothelial cell development, growth and differentiation take place within EBs.

Muscle and neuronal development

A regular occurrence in ES differentiation cultures is the development of foci of cells within EBs that begin rhythmic contractions, which are an indication of cardiac muscle development [6]. Molecular analyses of developing EBs have documented the expression of myosin heavy and light chain genes, including myosin light chain-2 (MLC-2V), a gene that is expressed exclusively in cells destined to become ventricular muscle [9]. These findings indicate that not only is the cardiac myogenesis program active within the EBs but that signals leading to cardiac muscle specification are also functioning. Following the onset of cardiac myogenesis, genes involved in skeletal muscle commitment (my&5, myogenin, A4yoD) are expressed within EBs in a pattern that duplicates their temporal activation in normal embryogenesis [9,10’]. When EBs are plated onto tissue culture plastic for one to two weeks, myocytes develop that subsequently fuse to form recognizable myotubes that are characteristic of developing skeletal muscle [lo*] _


Cell differentiation

These myocytes express appropriate well as functional nicotinic receptors, characteristics of maturing cells.

Ca2+ channels as both of which are

Unlike the lineages described thus far which develop in cultures supplemented with FCS only, efficient generation of neuron-like outgrowths horn EBs requires an additional inductive stimulus provided by retinoic acid [ll’]. The neuronal nature of the cells that develop in these cultures is defined by the presence of specific proteins (neurofilament M and class III b-tubulin) and by the presence of electrophysiological properties characteristic of neuron-like cells. Together, the studies reviewed in this section indicate that under appropriate conditions ES cells can generate multiple lineages in culture. As culture conditions that promote the growth of other lineages become better defined, it is likely that precursor populations of these lineages will be identified within EBs.


of genetically

altered ES cells

As indicated earlier, the differentiation of ES cells carrying targeted mutations provides an approach that is complementary to the analysis of knockout mice when defining the role of a gene in early lineage development [38**,39**,40,41,42’,43*]. Studies on the in vi&o differentiation of GATA-l(X-linked) [38**], GATA-2-i[39”], and vad[42*,43’] ES cells demonstrate several advantages of the in vitro system in analyzing the effects of specific mutations. GATA-1 and GATA-2 are members of a family of transcription factors that bind to GATA motifs found in enhancers and promoters of numerous genes that are expressed in hematopoietic cells [44]. Both factors are expressed in cells of the erythroid, mast cell and megakaryocyte lineages. In addition, GATA-2 is expressed in populations that represent early stages of hematopoietic development as well as in endothelial cells and cells of the embryonic brain. When GATA-lES cells were injected into blastocysts [45], they failed to generate mature erythroid cells but did generate white blood cells, plus cells of most other tissues, in the resulting chimeric mice. These findings demonstrated that a functional GATA-1 gene is required for erythropoiesis, but they did not identify the specific stage at which the lineage is blocked. Analysis of GATA-lEBs in vitro [38**] revealed an absence of primitive erythroid precursors but near normal numbers of definitive erythroid, macrophage, and mast cell precursors. These definitive erythroid precursors did, however, show a developmental arrest at the pro-erythroblast stage, indicating that an intact GATA-1 gene is not required for establishment of the erythroid lineage but rather provides a function that is essential for late stage maturation. Analysis of the GATA-lpro-erythroblasts before their death demonstrated the

expression of genes thought to be regulated by GATA-1. A 50-fold increase in the levels of GATA-2 in these cells suggests that this factor compensates for the loss of GATA-1 in the early stages of erythroid lineage development but is unable to compensate for late stage functions. In contrast to the erythroid-specific defect observed in GATA-lES cells, ES cells lacking functional GATA-2 alleles have greatly reduced hematopoietic potential compared with wild-type cells [39**]. The number of primitive erythroid and macrophage precursors is reduced approximately lO-20-fold, whereas the c-Kit ligand responsive definitive erythroid and mast cell precursors are virtually absent in GATA-2-iEBs. Homozygous GATA-2-lmutant mice die between days 9.5 and 10 of gestation. These animals generate some primitive erythroid cells, but appear to be unable to generate definitive erythroid cells. This finding correlates well with the limited potential of the GATA-2-iES cells in culture. The

hematopoietic-specific expression pattern of the was considered to provide strong evidence that this gene plays a pivotal role in some aspect of hematopoietic development [46]. Consequently, the early death of vadembryos during or shortly aher implantation was unexpected. It also precluded any hematopoietic analysis of these embryos [43*]. In vitro analysis indicated that va&ES cells are able to generate cells of the erythroid and myeloid lineages, a finding which demonstrates that a functional vav gene is not required for the early stages of hematopoietic development [42.,43*]. Studies with chimeric animals that were generated by injecting vav-I- ES cells into immunodeficient RAG-.?/blastocysts have provided solid evidence that vav plays an important role in signal transduction mediated by T and B cell antigen receptors [47-491. vav proto-oncogene

These three examples demonstrate that the ES/EB system provides an approach for defining the function of genes in early lineage development that is complementary to analysis of the whole animal. Two aspects of these studies are worth highlighting. First, when compared directly, the defects observed in EBs are similar to those found in vivo. Second, easy access to the early developing populations in vitro allows a more precise characterization of the resulting defect (e.g. a GATA-lphenotype) than is possible in vim. The above studies represent the first analyses of genetically altered ES cells in vitro. They also establish an approach that will become increasingly popular as the number of available ES cell lines carrying targeted mutations rapidly increases.

Conclusions The development of differentiated lineages from ES cells in culture provides a unique model system for defining

In vitro differentiation of embrvonic stem cells

the earliest steps of commitment from the respective The majority of studies with precursor populations. the ES/EB system to date have been descriptive and essentially document the development of a particular lineage. Analyses of ES cells containing targeted mutations represent the newest line of experimentation and clearly demonstrate certain advantages of in vitro differentiation over in viva development for defining the role of a specific gene. One of the challenges for the future will be to utilize the true potential of this model developmental system, through the isolation of the earliest precursors that are restricted to a given lineage. Access to such populations will enable a complete characterization of their developmental potential, the identification of the f&tors involved in their growth and differentiation and the elucidation of the molecular events involved in their establishment. The questions to be addressed are similar in all lineages. Below is a summary of some of the outstanding issues in developmental hematopoiesis that can be addressed through access to early precursor populations in EBs. Our current understanding of the structure of the hematopoietic system, including the identification and characterization of multipotential stem cells, derives largely from studies on the adult mouse. Consequently little is known about the potential of the earliest hematopoietic cell to develop within the embryo, or about the relationships between the earliest lineages. As indicated earlier, a long-standing hypothesis suggests that the hematopoietic and endothelial lineages arise h-om a common precursor, the hemangioblast. Recent studies demonstrating the absence of both endothelial and hematopoietic cells in mice lacking a functionalJk-1 gene (which encodes a receptor tyrosine kinase) support this hypothesis [50**]. Access to early populations within the EBs, prior to the establishment of the endothelial and hematopoietic lineages, provides a unique opportunity to isolate this common precursor, if it exists. A closely related question pertains to the origin of the primitive erythroid lineage. The appearance of mature erythroid cells in the yolk sac before the development of all other hematopoietic populations has led to the suggestion that these early cells arise from mesoderm as an independent hneage. Characterization of precursor populations isolated from EBs before the onset of primitive erythropoiesis will ultimately define their origin. As with studies characterizing lineage relationships and precursor developmental potentials, most of our knowledge of growth regulation within the hematopoietic system comes from experiments with adult bone marrow. Isolation of early embryonic precursors could provide a means of identieing novel regulators that act at early stages of hematopoietic development. Of particular interest will be the identification of those molecules that promote the development of hematopoietic cells horn pre-hematopoietic mesoderm. A recent report [32] indicates that BMP-4 may play an important role in this


early transition step within EBs. Future studies should identifjr additional molecules that are involved in these early stages of hematopoietic commitment. Finally, access to the earliest precursors of the hematopoietic system, cells that presumably represent the immediate progeny of mesodermal precursors, will provide a unique population for the isolation of genes involved in hematopoietic commitment. Functional characterization of such genes will greatly enhance our understanding of the molecular events involved in lineage commitment in general, and in the establishment of the early hematopoietic system in particular.

Acknowledgements I wish to thank John Shannon, Leif Carlsson, Mitch Weiss, George Lacaud and Marion Kennedy for critically reading the manuscript, and Allyson Nash and Kathy Ryan for help in its preparation.

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Zhang R, Ah R, Davidson L, Orkin S, Swat W: Defective signalling through the T-and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene. Nature 1995, 374z470-473.

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GM Keller, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, 5GB Denver, Colorado 80206, USA.