While the early stages of EB differentiation do not give rise to lymphoid progeny, it has been possible to generate cells of both the T- and B-cell lineages following extended periods of time in culture. B-cell potential was demonstrated following the coculture of ES cells with OP9 stromal cells in medium containing lymphoid cytokines (Nakano et al. 1994; 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.
Establishment of the hematopoietic system: identification of the hemangioblast
One of the outstanding strengths of the ES cell differentiation model is that it provides access to early developmental stages that are difficult to access in the embryo. This unique advantage has been fully exploited to investigate the earliest stages of hematopoietic commitment (Choi et al. 1998; 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.
The BL-CFC is not an artifact of the ES cell model, as a similar progenitor has recently been identified in the early mouse embryo (Huber et al. 2004
). 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 IIb3 (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.