This study establishes that candidate HSC can be derived from hESC and can be functionally examined with a variety of in vitro and in vivo surrogate measures of stem cell and progenitor function. Given the difficulty of obtaining transplantable murine HSCs from mouse ESCs, it is interesting that, with the exception of ectopic HOXB4
), the repopulating function of hESC-derived hematopoietic cells could be detected here. The IBMT delivery method used seems essential for detecting hematopoietic potential from hESC-derived progeny, similar to that recently shown using mouse ESC-derived hematopoietic cells (43
). Although hematopoietic repopulating cells derived from hESC were observed in our study, the proliferative and migratory properties of these cells was significantly different from those of somatic HSCs. Insights into the molecular basis of these functional differences could be correlated to changes in gene expression of broad families of genes that are involved specific cellular processes (e.g., homing, adhesion, cell cycle). Because these differences between hESC-derived progeny and somatic cells may not be restricted to the hematopoietic lineage alone, our results have a number of important implications for future application of hESC for therapeutic purposes that extend beyond development of HSC from hESC lines. This notion is supported by years of studies in the murine model, in which mouse ESC progeny behave differently than would be expected from adult cells of the same lineage (44
Our study provides additional insights into hESC biology and strategies. First, comparisons must be made between hESC-derived stem cells and somatic stem cells. Importantly, the most appropriate surrogate stem cell assay must be used to gain an accurate picture. The in vitro progenitor assays did not predict the significant functional differences between the two sources of HSC. Indeed, even the reference standard repopulation assay based on i.v. transplantation was ineffective because of the unique cellular aggregation properties of the hESC-derived cells that resulted in lethality. Our study demonstrates that IBMT is an absolute requirement for functional in vivo assessment of candidate HSCs derived from hESC and demonstrates the power of this method to assess HSC activity of novel populations (8
Second, although HSCs were generated from hESC, the current methodology was not sufficient to induce a cellular and molecular program reflective of a somatic HSC. This result parallels the inability to reprogram accurately all types of nuclei by nuclear transfer (46
). The fact that several genes classes are differentially expressed among hESC-derived and somatic primitive hematopoietic cells reinforces the notions that single gene products do not define the molecular nature of HSCs and that a mosaic of combinatory factors are required to confer HSC function. This finding suggests that single-gene reconstitution strategies are unlikely to restore HSC function of hESC-derived hematopoietic cells. Recent evidence for the role of HOX gene products (47
) and cell cycle regulators (48
) in mammalian HSC behavior indicates that modulation of these classes of genes is liable to affect in vivo behavior of hESC-derived HSCs by affecting a broad range of target genes necessary to establish functional HSCs. Our study evaluates this issue directly by expressing HOXB4
, the best-known candidate for conferring HSC function on mammalian hematopoietic cells derived from ESC lines. We demonstrate that ectopic expression of HOXB4
induced proliferation but had no affect on HSC function of hESCs, thereby supporting our original supposition.
Third, this report provides the foundation for optimizing and improving differentiation methodology using extrinsic factors that mimic better the in utero orchestration of genetic programs required for generation of HSCs. These factors may include signaling pathways shown to be involved in embryonic specification of the hematopoietic lineage and also involved in somatic HSC self renewal, such as the Notch, Wnt, and hedgehog pathways. A strategy using both in vivo and molecular comparisons of hESC-derived cell types with their somatic counterparts will be instrumental in elucidating the fundamental principles required to generate tissue-specific stem cells from hESCs and to guide future applications of hESC-based regenerative therapies independent of lineage and disease target. We propose that similar preclinical modeling approaches will be required to develop responsibly appropriate and successful methods for using hESC in cell replacement.