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REVIEW
Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA
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Abstract |
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[Keywords: ES cells; differentiation; mesoderm; endoderm; ectoderm; embryonic development]
; Martin 1981). Their importance to modern biology and medicine derives from two unique characteristics that distinguish them from all other organ-specific stem cells identified to date. First, they can be maintained and expanded as pure populations of undifferentiated cells for extended periods of time, possibly indefinitely, in culture. Unlike transformed tumor cell lines, ES cells can retain normal karyotypes following extensive passaging in culture. Second, they are pluripotent, possessing the capacity to generate every cell type in the body. The pluripotent nature of mouse ES cells was formally demonstrated by their ability to contribute to all tissues of adult mice, including the germline, following their injection into host blastocysts (Bradley et al. 1984). In addition to their developmental potential in vivo, ES cells display a remarkable capacity to form differentiated cell types in culture (Keller 1995; Smith 2001). Studies during the past 20 years have led to the development of appropriate culture conditions and protocols for the generation of a broad spectrum of lineages. The ability to derive multiple lineages from ES cells opens exciting new opportunities to model embryonic development in vitro for studying the events regulating the earliest stages of lineage induction and specification. Comparable studies are difficult in the mouse embryo and impossible in the human embryo. In addition to providing a model of early development, the ES cell differentiation system is viewed by many as a novel and unlimited source of cells and tissues for transplantation for the treatment of a broad spectrum of diseases. The isolation of human ES cells (hES) in 1998 dramatically elevated the interest in the cell therapy aspect of ES cells and moved this concept one step closer to reality (Thomson et al. 1998). This review details the current status of mouse and human ES cell differentiation from both the developmental biology and cell replacement perspectives. The first sections of the review highlight successes to date in the generation and characterization of mature populations, while the final section outlines the challenges for the future with a focus on the identification of progenitor cells representing the earliest stages of embryonic lineage development. The reader is referred to other recent reviews that provide additional details for many of the subjects covered here (Kyba and Daley 2003; Nir et al. 2003; Hornstein and Benvenisty 2004; Lang et al. 2004; Pera and Trounson 2004; Rippon and Bishop 2004; West and Daley 2004). For the purpose of this review, the term ES will be used in reference to mouse cells and hES for human cells. |
Maintaining undifferentiated ES cells |
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; Martin 1981). Subsequent studies identified leukemia inhibitory factor (LIF) as one of the feeder-cell-derived molecules that plays a pivotal role in the maintenance of these cells (Smith et al. 1988; Williams et al. 1988; Stewart et al. 1992). In the presence of appropriate batches of fetal calf serum (FCS), recombinant LIF can replace the feeder cell function and support the growth of undifferentiated ES cells (Smith et al. 1988; Williams et al. 1988). Recently, Ying et al. (2003a) have uncovered a role for BMP4 in ES cell growth and demonstrated that in the presence of LIF, it can replace the requirement for serum. With these new developments, it is now possible to grow ES cells with defined factors in the absence of serum or feeder cells. Molecular analyses have revealed that LIF functions through the gp130 activation of STAT3 (Niwa et al. 1998; Matsuda et al. 1999), whereas the effect of BMP4 on undifferentiated ES cells is mediated by Smad activation and the subsequent induction of the helix–loop–helix Id factors. In addition to STAT3 and Id, two other transcription factors, Oct3/4 (Niwa et al. 2000) and nanog (Chambers et al. 2003; Mitsui et al. 2003), have been shown to play pivotal roles in maintaining the undifferentiated state of ES cells. The role of these transcription factors in ES cell renewal has been recently reviewed (Chambers and Smith 2004) and will not be discussed further here.
The regulation of hES cell growth is less well understood and differs from that of the mouse in that LIF and STAT3 appear to play no role in their self-renewal (Thomson et al. 1998; Reubinoff et al. 2000; Daheron et al. 2004). With current protocols, hES cells can be maintained on feeder cells in serum-free medium supplemented with bFGF (Amit et al. 2000). hES cells can also be grown in the absence of feeder cells, if cultured on matrigel- or laminin-coated plates in medium supplemented with mouse embryonic fibroblast conditioned medium (MEF CM) (Xu et al. 2001). While not as well defined as the conditions for the growth of mouse cells, this protocol does provide for relatively easy maintenance of hES cell populations. Cells grown in these conditions for >100 population doublings retained normal karyotypes and stem cell characteristics, including their in vitro and in vivo pluripotent differentiation potential. Recently, Sato et al. (2004) demonstrated that activation of the canonical Wnt pathway could replace the requirement of MEF CM in the maintenance of undifferentiated hES cells for short periods of time (5–7 d). Whether or not Wnt signaling has an effect on hES cell self-renewal over longer periods through multiple passages remains to be determined. hES cells do express both Oct4 (Ginis et al. 2004) and nanog (Daheron et al. 2004; Richards et al. 2004; Sato et al. 2004), suggesting that this aspect of their regulation may be similar to that observed in mouse ES cells. Future studies will no doubt define specific molecules for the maintenance of hES cells and uncover the molecular mechanisms that regulate their self-renewal.
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Differentiation of ES cells in culture |
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; Smith 2001). Wild-type ES cells do not differentiate to trophectoderm in culture and, in this respect, reflect the potential of their founder embryonic population, the inner cell mass (Fig. 1). hES cells differ from mouse cells in this respect, as when induced with BMP4, they will give rise to cells that display characteristics of the trophoblast lineage (Xu et al. 2002). The reason for this difference is not clear, but may indicate that at least some of the hES cell lines represent earlier stages of development than the comparable mouse populations.
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; Keller 1995). In the second method, ES cells are cultured directly on stromal cells, and differentiation takes place in contact with these cells (Nakano et al. 1994). The most commonly used stromal cell line for such differentiation studies is OP9 (Nakano et al. 1994), originally isolated from CSF-1-deficient op/op mice (Yoshida et al. 1990). The third protocol involves differentiating ES cells in a monolayer on extracellular matrix proteins (Nishikawa et al. 1998).
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Three criteria should be considered when using the ES cell model for lineage-specific differentiation. First, protocols need to be established that promote the efficient and reproducible development of the cell type of interest. If possible, selection strategies should be combined with optimal differentiation schemes to enable the isolation of highly enriched cell populations. Second, lineage development from ES cells should recapitulate the developmental program that establishes the lineage in the early embryo. Third, the mature cell populations that develop in these cultures must display appropriate functional properties both in culture and when transplanted into appropriate animal models.
The three differentiation methods described above have been used to generate a broad spectrum of cell types from ES cells (Keller 1995; Smith 2001). For lineages that have been studied in detail, the first two criteria outlined above have been met as efficient protocols for their differentiation have been established and the sequence of events leading to their development in culture was found to faithfully recapitulate those in the early embryo. The third aim has yet to be fulfilled for most populations and represents one of the major challenges in the field today. Since their derivation, progress has been made with the differentiation of hES cells. Although not nearly as advanced as the studies with the mouse system, the findings to date indicate that it will be possible to generate a broad spectrum of cell types from them in culture (Schuldiner et al. 2000; Odorico et al. 2001; Pera and Trounson 2004).
Many differentiation protocols have been optimized using FCS as a growth supplement and/or as a source of inducing factors. Although these approaches have been successful for the development of certain lineages, the use of FCS has several serious drawbacks that include batch-to-batch variability and the lack of identity of the inducing factors contained in it. As discussed in a later section of this review, several recent studies have eliminated serum and have begun to identify factors required for lineage-specific differentiation. As more protocols incorporate these approaches, both mouse and human ES cell multilineage differentiation will become a routine technology in many labs.
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Differentiated cell types from ES cells |
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Mesoderm-derived lineages, including the hematopoietic, vascular, and cardiac, are among the easiest to generate from ES cells and have been studied in considerable detail. Of these, hematopoietic development is the best characterized. Findings from several different studies have demonstrated striking parallels between the ES cell model and the early embryo, providing important insights into the embryonic origins of the hematopoietic system. Given these findings, hematopoietic development is covered in the greatest detail in this review.
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Hematopoietic development |
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), there was significant interest in modeling various aspects of hematopoietic commitment using the ES cell differentiation model (Burkert et al. 1991; Schmitt et al. 1991; Keller et al. 1993; Nakano et al. 1994). One of the initial goals of many investigators in the field was to generate hematopoietic stem cells (HSCs) from ES cells with the aim of developing a readily accessible supply of transplantable stem cells. HSCs are typically assayed by their ability to provide long-term multilineage hematopoietic repopulation following intravenous (IV) transplantation into hematopoietically deficient animals (Kondo et al. 2003). For a stem cell to function in this assay, it must be multipotent and possess the capacity to home to the bone marrow following transplantation. Despite extensive efforts by many groups during the past 10 years, the development of HSCs from ES cells remains a challenge as most attempts to identify these cells in the differentiation cultures have failed (Muller and Dzierzak 1993; G. Lacaud, V. Kouskoff, M. Kennedy, and G. Keller, unpubl.). These failures prompted a re-evaluation of hematopoietic commitment in the ES cell differentiation cultures from the perspective of hematopoiesis in the early embryo. Patterning hematopoietic development of ES cells in culture on hematopoietic commitment in the early embryo was important as the hematopoietic system undergoes dramatic changes throughout embryonic life (Metcalf and Moore 1971; Russel 1979; Keller et al. 1999). These changes need to be reproduced in the ES cell system if it is to be a valid model of early development and ultimately a source of HSCs. |
Hematopoietic development in the embryo |
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; Russel 1979; Godin et al. 1995; Medvinsky and Dzierzak 1996; Palis et al. 1999). Detailed analysis of hematopoietic development in the early embryo strongly suggests that the programs generated in these two regions are different. Hematopoietic commitment is detected first in the yolk sac, where distinct blood islands appear, shortly following gastrulation (Moore and Metcalf 1970; Haar and Ackerman 1971; Palis et al. 1995, 1999). These blood islands consist of an inner cluster of maturing erythrocytes surrounded by a layer of developing endothelial cells (Haar and Ackerman 1971). The erythroid cells within these blood islands, known as primitive erythrocytes, are distinct from fetal and adult erythrocytes in that they are large, circulate in the bloodstream as nucleated cells for much of their life span, and contain an embryonic form of hemoglobin (Barker 1968; Brotherton et al. 1979; Russel 1979; Kingsley et al. 2004). Production of primitive erythrocytes is known as primitive erythropoiesis and is restricted to the yolk sac during a narrow window of development in the mouse embryo (Palis et al. 1999). Development of all other blood cell lineages including myeloid, fetal, and adult erythroid and lymphoid is referred to as definitive hematopoiesis. Definitive erythroid cells enucleate prior to entering the bloodstream, are smaller than those of the primitive lineage, and produce adult forms of hemoglobin.
In addition to primitive erythrocytes, the yolk sac generates a subset of lineages from the definitive hematopoietic program including the macrophage, definitive erythroid, and mast cell (Palis et al. 1999). Kinetic analysis of the developing yolk sac revealed that these lineages are produced in a defined temporal pattern with primitive erythroid and macrophage appearing first, followed by definitive erythroid, which, in turn, is followed by mast cells. While the yolk sac does have potential beyond that of primitive erythropoiesis, it does not appear to be capable of generating lymphocytes or HSCs, when analyzed prior to the onset of circulation (Cumano et al. 2001). Parallel studies have demonstrated that the hematopoietic program initiated in the P-Sp includes the generation of the myeloid, lymphoid, and definitive erythroid lineages as well as the HSCs (Muller et al. 1994; Godin et al. 1995; Cumano et al. 2001). The P-Sp does not, however, generate primitive erythrocytes. Thus, the distinguishing features of the early yolk sac are the generation of the primitive erythroid lineage and a lack of lymphoid and HSC potential, while the P-Sp program can be defined by the development of the lymphoid lineages and HSCs. Collectively, these observations indicate that the hematopoietic system initiates with the production of a limited number of specialized lineages in the yolk sac and matures with time into a full multilineage system with the switch to the P-Sp. While somewhat unusual, this pattern is logical, as the system is responding to the requirements of the embryo at different developmental stages. These dramatic changes in the hematopoietic system, in particular the early and transient appearance of the primitive erythroid lineage, provide a developmental map for monitoring hematopoietic commitment in the ES cell differentiation cultures.
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ES-cell-derived primitive and definitive hematopoiesis |
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). Hematopoietic commitment within these cultures can be easily monitored by gene expression patterns (Schmitt et al. 1991; Keller et al. 1993; Robertson et al. 2000), the appearance of specific cell surface markers (Kabrun et al. 1997; Nishikawa et al. 1998), and the development of clonable progenitor cells (Schmitt et al. 1991; Keller et al. 1993). With these assays, it has been possible to demonstrate that development of the hematopoietic lineages is highly reproducible and efficient. Under appropriate culture conditions, >50% of the cells in the differentiation cultures will express the hematopoietic/vascular receptor tyrosine kinase Flk-1 (VEGF receptor 2) (Kabrun et al. 1997) and up to 5% can represent a clonable hematopoietic progenitor (Keller et al. 1993). Detailed analyses of the early stages of hematopoietic commitment have shown that both gene expression patterns and the kinetics of lineage development within EBs accurately reflect that found in the yolk sac (Keller et al. 1993; Palis et al. 1999; Robertson et al. 2000). Most notable was the finding that the primitive erythroid lineage develops earliest and represents a transient population that persists in the EBs for 
4 d (Keller et al. 1993). The macrophage, definitive erythroid, and mast cell lineages appear following the onset of primitive erythropoiesis and develop in the temporal order found in the yolk sac (Keller et al. 1993). Lymphoid progenitors and HSCs have not been identified among the progeny of early stage EBs, suggesting that the initial stages of EB hematopoiesis represent the equivalent of yolk sac hematopoiesis.
The faithful recapitulation of this yolk sac developmental program provides strong evidence that regulation of hematopoietic commitment in the ES/EB model is similar, if not identical, to that of the early embryo. Support for this interpretation has been provided by gene targeting studies that helped define the role of specific transcription factors including Scl/tal-1 (Begley et al. 1989), Runx1 (Wang and Speck 1992; Ogawa et al. 1993), and GATA-1 (Orkin 1992) in the establishment of the hematopoietic system. Each of these factors functions at specific stages of blood cell differentiation as demonstrated by the observations that Scl/tal-1 is required for the development of all hematopoietic (primitive erythroid and definitive) lineages (Robb et al. 1995; Shivdasani et al. 1995), Runx1 for the definitive lineages but not primitive erythropoiesis (Okuda et al. 1996; Wang et al. 1996), and GATA-1 for late-stage primitive and definitive erythroid maturation (Pevny et al. 1991; Weiss et al. 1994). All of these defects have been accurately replicated in the ES cell differentiation model (Weiss et al. 1994; Porcher et al. 1996; Wang et al. 1996; Lacaud et al. 2002). In addition to further validating the ES cell system as a model of hematopoietic development, the ability to analyze mutations in EBs provides a powerful model for structure/function studies as well as for the identification of molecular targets of the gene of interest.
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Lymphoid and HSC development from ES cells |
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; Cho et al. 1999). More recently Schmitt et al. (2004) demonstrated that expression of the Notch receptor ligand, delta-like 1, in the OP9 cells facilitates the differentiation of the developing lymphoid cells to a T-cell fate. These findings are encouraging as they indicate that populations similar to that of the P-Sp may be generated from differentiating ES cells and that signaling pathways, such as Notch, known to play a role in B-cell and T-cell fate in vivo may function in a similar manner in culture.
Two recent reports have provided evidence that cells with HSC properties can be generated from ES cells in culture. In the first, Kyba et al. (2002) transplanted recipient animals with ES-cell-derived hematopoietic cells that had been induced with forced expression of the HoxB4 gene. Donor cells were clearly evident in the transplanted animals up to 12 wk following repopulation in primary recipients and as long as 20 wk in secondary recipients. The majority of donor cells, however, appeared to be myeloid as the levels of lymphoid engraftment were very low. These patterns of repopulation differ from those generated by fetal liver or adult bone marrow HSCs that typically show extensive myeloid and lymphoid repopulation (Jordan et al. 1990; Kondo et al. 2003). Given these patterns of engraftment, it is unclear if the repopulation originates from the equivalent of a P-Sp multipotential HSC that is not sufficiently mature to display multilineage potential or from a yolk sac-like progenitor, whose ability to survive in vivo has been prolonged by the HoxB4 gene.
In the second study, CD45+c-kit+ cells isolated from EBs cultured for 7–10 d in the presence of c-kit ligand, IL-3, and IL-6 were transplanted into irradiated recipient animals (Burt et al. 2004). Even when transplanted into allogeneic recipients, these cells generated extensive hematopoietic chimerism and contributed to both the myeloid and lymphoid lineages. These findings are somewhat surprising, given that many different hematopoietic populations from ES cell differentiation cultures have been transplanted into different types of recipients with little evidence of repopulation. One possible reason for the success in this study is that the particular batch of FCS used may have contained factors that promote the development of HSCs. Establishment of serum-free conditions for the generation of these cells would enable other investigators to reproduce these findings. One interesting observation in this study was that the level of ES-cell-derived hematopoietic contribution was significantly higher in recipients in which the cells were transplanted directly into the femur rather than intravenously into the circulation. This observation suggests that repopulating cells generated in the ES cell differentiation cultures may not be fully differentiated and lack critical adhesion molecules that enable them to home to the bone marrow. A lack of homing potential may account for some of the failures in detecting HSCs in previous studies.
While these findings indicate that it is possible to generate cells that can persist and function in recipient animals, additional studies will be required to determine if these cells are comparable to HSCs found in the fetal liver and adult bone marrow. Given that the P-Sp is considered to be the site of HSC development in the early embryo, one important approach will be to establish conditions to generate populations comparable to the P-Sp in the ES cell differentiation cultures. Access to such cells should ultimately enable the routine generation of HSCs from ES cells.
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Establishment of the hematopoietic system: identification of the hemangioblast |
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; Nishikawa et al. 1998) and to test a long-standing hypothesis that the hematopoietic and endothelial lineages develop from a common progenitor, a cell known as the hemangioblast. The hemangioblast hypothesis was put forward many years ago, based on the observation that the blood cell and endothelial lineages develop in close proximity at the same time in the yolk sac blood islands (Sabin 1920; Murray 1932; Haar and Ackerman 1971). Circumstantial evidence supporting this hypothesis was provided by studies demonstrating that immature hematopoietic and vascular cells share the expression of a large number of genes (Orkin 1992; Watt et al. 1995; Young et al. 1995; Fong et al. 1996; Takakura et al. 1998) and that specific genes are essential for the proper development of both lineages (Dickson et al. 1995; Robb et al. 1995; Shalaby et al. 1995; Shivdasani et al. 1995). Formal proof that a progenitor with properties of the hemangioblast does exist was provided by studies using the ES differentiation model (Choi et al. 1998; Nishikawa et al. 1998). Analysis of early-stage, carefully timed EBs led to the identification of a progenitor known as the blast colony-forming cell (BL-CFC) that gives rise to blast colonies consisting of hematopoietic and vascular progenitors in methylcellulose cultures in the presence of vascular endothelial growth factor (VEGF) (Kennedy et al. 1997; Choi et al. 1998). The hematopoietic component of these colonies consisted of primitive erythroid progenitors and the subset of definitive hematopoietic lineages that is found in the yolk sac, while the vascular potential included both the endothelial and vascular smooth muscle lineages (VSM) (Kennedy et al. 1997; Choi et al. 1998; Ema et al. 2003). Initial studies identified the BL-CFC as a transient progenitor that develops early in EBs prior to the onset of primitive erythropoiesis. Subsequent experiments have shown that it expresses the receptor tyrosine kinase Flk-1 (Faloon et al. 2000), the transcription factor Runx1 (Lacaud et al. 2002), and the mesoderm gene brachyury (Fehling et al. 2003), suggesting that this progenitor represents a subpopulation of mesoderm undergoing commitment to the hematopoietic and vascular lineages (Fig. 3). The BL-CFC does not, however, express markers associated with hematopoietic and vascular development, including CD31, VE-Cadherin (VE-cad), CD34, or c-kit (Fehling et al. 2003; M. Kennedy and G. Keller, unpubl.), a finding consistent with the interpretation that this progenitor represents the earliest stage of hematopoietic commitment.
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). This progenitor arises in the posterior primitive streak of the embryo, coexpresses Flk-1 and brachyury, and displays the same developmental potential as the EB-derived BL-CFC (Fig. 3). Given these characteristics, this embryo-derived progenitor can be considered to be the illusive hemangioblast. The isolation and characterization of the embryo hemangioblast were made possible through the use of strategies developed for the identification of the BL-CFC in the ES/EB system, a clear demonstration that this in vitro model can provide important insights into early embryonic development.
The ES differentiation system has also been instrumental in characterizing the earliest stages of hematopoietic development, immediately following the appearance of the hemangioblast. Analyses of these ES-cellderived hematopoietic populations at different time points have revealed dynamic changes in the expression of cell surface proteins that likely reflect changes in the lineage composition of the system as well as maturation of the cells within a specific lineage (Kabrun et al. 1997; Nishikawa et al. 1998; Mikkola et al. 2003). Of particular interest is the observation that these early hematopoietic cells express markers that are not found on fetal liver and adult hematopoietic populations. Conversely, certain markers associated with fetal and adult hematopoietic cells are absent from embryonic hematopoietic cells. For instance, the earliest hematopoietic populations express the endothelial markers Flk-1 (Kabrun et al. 1997; Nishikawa et al. 1998), and VE-cad (Nishikawa et al. 1998) and the 
IIb (CD41) component of the platelet glycoprotein receptor IIb
3 (Mitjavila-Garcia et al. 2002; Ferkowicz et al. 2003; Mikkola et al. 2003). These markers are expressed prior to the onset of CD45, a hematopoietic-specific marker present on most fetal liver and adult bone marrow cells. In the fetal liver and adult, Flk-1 (Millauer et al. 1993; Yamaguchi et al. 1993) and VE-cad (Matsuyoshi et al. 1997) are restricted to the endothelial lineage, while CD41 is expressed exclusively in the megakaryocyte lineage (Phillips et al. 1988). The appearance of endothelial markers prior to CD45 has been interpreted by some as evidence that hematopoietic cells develop from a specialized population of endothelial cells, known as hemogenic endothelium. An equally plausible explanation is that the earliest embryonic hematopoietic progenitors that give rise to the later hematopoietic populations express Flk-1 and VE-cad.
With a more detailed understanding of the earliest stages of hematopoiesis, it has been possible to use the ES cell system to begin to investigate the regulation of hematopoietic commitment in a manner that could not be done in the embryo. Findings from such studies have demonstrated that the transcription factors Scl/tal-1 and Runx1 function early in development, specifically at the stage of hematopoietic commitment of the BL-CFC (Faloon et al. 2000; Robertson et al. 2000; Lacaud et al. 2002; D'Souza et al. 2005). With respect to induction and growth regulation, different groups have shown that the development of the BL-CFC and hematopoietic restricted progenitors is positively regulated, directly or indirectly by bFGF (Faloon et al. 2000), VEGF (Nakayama et al. 2000; Park et al. 2004), and Ephrin/Eph (Z. Wang et al. 2004) signaling together with serum-derived factors. Studies conducted in serum-free conditions revealed that BMP4 together with VEGF can support hematopoietic differentiation of ES cells (Nakayama et al. 2000; Park et al. 2004). These factors appear to act at specific developmental stages, with BMP4 functioning to induce Flk-1+ cells and VEGF playing a role in the generation of Scl/tal-1-expressing hematopoietic and vascular progenitors within this Flk-1+ population (Park et al. 2004). Molecular analysis revealed that the effects of BMP4 and Flk-1 are mediated through the activation of the SMAD1/5 and MAP kinase pathways, respectively (Park et al. 2004). The findings from these studies are significant as they demonstrate that hematopoiesis can be induced from ES cells with defined factors that are thought to function in a similar fashion in the early embryo. What is not resolved is the role of BMP4 as it could be functioning to induce mesoderm, to specify mesoderm to the hematopoietic program, or both. Further studies using approaches that enable one to monitor the earliest stages of germ layer induction will be required to define the precise role of such factors.
In addition to probing early stages of the hematopoietic system, the ES differentiation model offers the potential to generate large numbers of cells from specific hematopoietic lineages for both molecular and biochemical analyses as well as for transplantation for short-term lineage replacement therapy. To date, methods have been established for selectively expanding multipotential cell populations (Pinto do et al. 1998), neutrophils (Lieber et al. 2004), megakaryocytes (Eto et al. 2002), mast cells (Tsai et al. 2000), eosinophils (Hamaguchi-Tsuru et al. 2004), dendritic cells (Fairchild et al. 2003), and definitive erythroid cells (Carotta et al. 2004) from ES cells in culture.
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Hematopoietic development from hES cells |
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) or the generation of EBs (Chadwick et al. 2003; Cerdan et al. 2004). While differentiation was serum-induced, hematopoietic development was augmented in the EBs by the addition to the cultures of BMP4, VEGF, and a mixture of hematopoietic cytokines (Cerdan et al. 2004). Kinetic analysis revealed the development of definitive erythroid and myeloid progenitors following 2 wk of differentiation, a pattern that suggests that hematopoietic induction, under the conditions used, is slower than observed in mouse ES cell differentiation cultures. Distinct primitive and definitive erythroid populations have not yet been identified in the human cultures, although changes in patterns of hemoglobin expression within the ES-cell-derived erythroid lineages have been documented (Cerdan et al. 2004). These changes suggest that at least some aspects of globin switching are taking place, reflecting the changes found during normal fetal development (Stamatoyannopoulos and Grosveld 2001). The hematopoietic potential of hES cells has been recently extended to include the lymphoid lineages, with the observation that cells expressing B-cell markers develop from CD34+ cells following extensive culture on stromal cells (Vodyanik et al. 2005). Analysis of the earliest stages of hematopoietic development in the human system identified a CD45– Flk-1+ VE-cad+ CD31+ population at day 10 of differentiation that generated CD45+ hematopoietic cells following further culture (L. Wang et al. 2004). These findings suggest that human hematopoietic development within the EBs parallels that of the mouse in that the earliest hematopoietic progenitors express endothelial markers prior to their maturation to CD45+ cells. Together, the findings from this limited number of studies indicate that it is possible to generate hematopoietic cells from hES cells in culture and that the sequence of developmental events may reflect the onset of hematopoiesis in the early embryo. |
Vascular development |
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 TopAbstractMaintaining undifferentiated ES... |
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Abstract
Maintaining undifferentiated ES...
