Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopicHOXB4expression

Abstract

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.

A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification

“…Unexpectedly, MLL-AF4 expression led to an enhanced mature endothelial cell fate of the hemogenic precursors. Because hESC-derived hemogenic precursors are uniquely responsible for endothelial and hematopoietic development [15,21,22,30], these data indicate that MLL-AF4 does not block both endothelial and hematopoietic commitment of the hemogenic precursors but instead, it skews the hematoendothelial potential of these hemogenic precursors towards a pronounced endothelial cell fate.…”

“…They were then transferred to low-attachment plates (Corning, NY, USA) to allow hEB formation by overnight incubation in differentiation medium consisting of KO-DMEM supplemented with 20% fetal bovine serum (FBS), 1% nonessential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Medium was changed the next day (day 1) with the same differentiation medium supplemented with hematopoietic cytokines: 300 ng/ml SCF, 300 ng/ml Flt-3L, 10 ng/ml IL-3, 10 ng/ml IL-6, 50 ng/ml G-CSF and 25 ng/ml BMP-4 [14,30,37,43]. hEBs were dissociated using collagenase B (Roche Diagnostic, ON, Canada) for 2 h at 37 °C followed by 10 min incubation at 37 °C with Cell Dissociation Buffer (Invitrogen) at days 4, 7, 11 and 15 of development.…”

“…Hemogenic precursors with hemangioblastic properties were identified as CD34 + CD31 + CD45 − . Immature and mature blood cells were identified as CD45 + CD34 + and CD45 + CD34 − , respectively ( Figure 2B) [14,15,30,37].…”

Progress and prospects: gene transfer into embryonic stem cells

“…50 Another example is the ectopic expression of HOXB4, which has been reported by several independent groups to support the proliferation of engraftable hematopoietic progenitors in mESCs, 51 but not as robustly in hESCs. 52,53 Although hESCs have a stable and significant proliferative capacity, this may not be true of hESC-derived cells. Some evidence has suggested, for instance, that hESC-derived hematopoietic progenitors and keratinocytes 53,54 have much lower proliferative potential than their somatic counterparts.…”

“…52,53 Although hESCs have a stable and significant proliferative capacity, this may not be true of hESC-derived cells. Some evidence has suggested, for instance, that hESC-derived hematopoietic progenitors and keratinocytes 53,54 have much lower proliferative potential than their somatic counterparts. In the latter case, gene …”