Dynamic HoxB4-regulatory network during embryonic stem cell differentiation to hematopoietic cells.

Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies to treat hematologic diseases. To date, HoxB4 remains the most effective transcription factor (TF) the overexpression of which in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop more efficient protocols for in vitro derivation of HSCs. In the present study, we performed global gene-expression profiling and ChIP coupled with deep sequencing at 4 stages of the HoxB4-mediated ESC differentiation toward HSCs. Joint analyses of ChIP/deep sequencing and gene-expression profiling unveiled several global features of the HoxB4 regulatory network. First, it is highly dynamic and gradually expands during the differentiation process. Second, HoxB4 functions as a master regulator of hematopoiesis by regulating multiple hematopoietic TFs and chromatin-modification enzymes. Third, HoxB4 acts in different combinations with 4 other hematopoietic TFs (Fli1, Meis1, Runx1, and Scl) to regulate distinct sets of pathways. Finally, the results of our study suggest that down-regulation of mitochondria and lysosomal genes by HoxB4 plays a role in the impaired lymphoid lineage development from ESC-derived HSCs.

Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells.

Bone marrow transplantation is a curative treatment for many diseases, including leukemia, autoimmune diseases, and a number of immunodeficiencies. Recently, it was claimed that bone marrow cells transdifferentiate, a much desired property as bone marrow cells are abundant and therefore could be used in regenerative medicine to treat incurable chronic diseases. Using a Cre/loxP system, we studied cell fusion after bone marrow transplantation. Fused cells were chiefly Gr-1(+), a myeloid cell marker, and found predominantly in the bone marrow; in parenchymal tissues. Surprisingly, fused cells were most abundant in the kidney, Peyer's patches, and cardiac tissue. In contrast, after cell fusion with embryonic stem cells, bone marrow cells were reprogrammed into new tetraploid pluripotent stem cells that successfully differentiated into beating cardiomyocytes. Together, these data suggest that cell fusion is ubiquitous after cellular transplants and that the subsequent sharing of genetic material between the fusion partners affects cellular survival and function. Fusion between tumor cells and bone marrow cells could have consequences for tumor malignancy.

HOXB4-transduced embryonic stem cell-derived Lin-c-kit+ and Lin-Sca-1+ hematopoietic progenitors express H60 and are targeted by NK cells.

Embryonic stem (ES) cells are a novel source of cells, especially hematopoietic progenitor cells that can be used to treat degenerative diseases in humans. However, there is a need to determine how ES cell-derived progenitors are regulated by both the adaptive and innate immune systems post transplantation. In this study, we demonstrate that hematopoietic progenitor cells (HPCs) derived from mouse ES cells ectopically expressing HOXB4 fail to engraft long-term in the presence of NK cells. In particular, the H60-expressing Lin(-)c-kit(+) and Lin(-)Sca-1(+) subpopulations were preferentially deleted in Rag2(-/-), but not in Rag2(-/-)gamma(c)(-/-) mice. Up-regulation of class I expression on HPCs prevented their lysis by NK cells, and Ab-mediated depletion of NK cells restored long-term HPC engraftment. In contrast to the notion that ES-derived cells are immune-privileged, we show in this study that NK cells form a formidable barrier to the long-term engraftment of ES cell-derived hematopoietic progenitors.

HOXB4 but not BMP4 confers self-renewal properties to ES-derived hematopoietic progenitor cells.

BACKGROUND: Achieving transplantation tolerance remains an unresolved clinical challenge. Although bone marrow transplantation (BMT) induces mixed chimerism that establishes transplantation tolerance, the preconditioning regimens required for BMT to succeed are too prohibitive for routine use. Recently, embryonic stem (ES) cells have emerged as a potential alternative cell source to bone marrow cells. However, it remains difficult to efficiently differentiate these cells into hematopoietic cells. Here, we tested whether bone morphogenetic protein-4 (BMP-4)-treated or HOXB4-transduced ES-derived hematopoietic cells engraft permanently inducing long-term mixed chimerism. METHODS: Initially, 129 SvJ R1 ES cells (H-2) were treated with BMP-4 for 36 hr. The cells were phenotyped and polymerase chain reaction studies were performed. The robustness of mixed chimerism was tested using mixed lymphocyte cultures and skin grafts. Chimeric MRL (H-2) animals received grafts from 129SvJ (H-2), Balb/c (H-2) or class II (H-2) donor mice, and graft survival was monitored. Additionally, HOXB4-transduced ES cells were shown to more efficiently differentiate into hematopoietic progenitor cells that engrafted in allogenic and syngeneic recipient mice. RESULTS: BMP-4 treatment induced Sca-1 expression and up-regulated HOXB4, BMP-4, and BMP receptor gene expressions. The cells induced transient mixed chimerism, whereas cells derived from HOXB4-transduced ES cells engrafted long term (>100 days). CONCLUSIONS: Although BMP-4 promotes hematopoiesis of ES cells, its impact is only transient, whereas permanent ectopic expression of HOXB4 significantly confers self-renewal and long-term engraftment of ES-derived hematopoietic cells. This strategy could facilitate the establishment of an alternative source of hematopoietic cells that could induce transplantation tolerance.

ES-cell derived hematopoietic cells induce transplantation tolerance.

BACKGROUND: Bone marrow cells induce stable mixed chimerism under appropriate conditioning of the host, mediating the induction of transplantation tolerance. However, their strong immunogenicity precludes routine use in clinical transplantation due to the need for harsh preconditioning and the requirement for toxic immunosuppression to prevent rejection and graft-versus-host disease. Alternatively, embryonic stem (ES) cells have emerged as a potential source of less immunogenic hematopoietic progenitor cells (HPCs). Up till now, however, it has been difficult to generate stable hematopoietic cells from ES cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, we derived CD45(+) HPCs from HOXB4-transduced ES cells and showed that they poorly express MHC antigens. This property allowed their long-term engraftment in sublethally irradiated recipients across MHC barriers without the need for immunosuppressive agents. Although donor cells declined in peripheral blood over 2 months, low level chimerism was maintained in the bone marrow of these mice over 100 days. More importantly, chimeric animals were protected from rejection of donor-type cardiac allografts. CONCLUSIONS: Our data show, for the first time, the efficacy of ES-derived CD45(+) HPCs to engraft in allogenic recipients without the use of immunosuppressive agents, there by protecting cardiac allografts from rejection.

Hematopoiesis and immunity of HOXB4-transduced embryonic stem cell-derived hematopoietic progenitor cells.

The ability of embryonic stem (ES) cells to form cells and tissues from all 3 germ layers can be exploited to generate cells that can be used to treat diseases. In particular, successful generation of hematopoietic cells from ES cells could provide safer and less immunogenic cells than bone marrow cells, which require severe host preconditioning when transplanted across major histocompatibility complex barriers. Here, we exploited the self-renewal properties of ectopically expressed HOXB4, a homeobox transcription factor, to generate hematopoietic progenitor cells (HPCs) that successfully induce high-level mixed chimerism and long-term engraftment in recipient mice. The HPCs partially restored splenic architecture in Rag2(-/-)gamma(c)(-/-)-immunodeficient mice. In addition, HPC-derived newly generated T cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus and specifically secreted interleukin-2 and interferon-gamma upon CD3 stimulation. In addition, HPC-derived antigen presenting cells in chimeric mice efficiently presented viral antigen to wild-type T cells. These results demonstrate for the first time that leukocytes derived from ES cells ectopically expressing HOXB4 are immunologically functional, opening up new opportunities for the use of ES cell-derived HPCs in the treatment of hematologic and immunologic diseases.

Immunogenicity and engraftment of mouse embryonic stem cells in allogeneic recipients.

Embryonic stem cells (ESCs) are pluripotent and therefore able to differentiate both in vitro and in vivo into specialized tissues under appropriate conditions, a property that could be exploited for cellular therapies. However, the immunological nature of these cells in vivo has not been well understood. In vitro, mouse-derived ESCs fail to stimulate T cells, but they abrogate ongoing alloresponses by a process that requires cell-cell contact. We further show that despite a high expression of the NKG2D ligand retinoic acid early inducible-1 by mouse ESCs, they remain resistant to natural killer cell lysis. In vivo, allogeneic mouse ESCs populate the thymus, spleen, and liver of sublethally irradiated allogeneic host mice, inducing apoptosis to T cells and establishing multilineage mixed chimerism that significantly inhibits alloresponses to donor major histocompatibility complex antigens. Immunohistochemical imaging revealed a significant percentage of ESC-derived cells in the splenic marginal zones, but not in the follicles. Taken together, the data presented here reveal that nondifferentiated mouse embryonic stem cells are non-immunogenic and appear to populate lymphoid tissues in vivo, leading to T-cell deletion by apoptosis.

Induction of stable mixed chimerism by embryonic stem cells requires functional Fas/FasL engagement.

BACKGROUND: Recent data show the efficacy of embryonic stem cells (ESC) to engraft in allogeneic recipients without host pretreatment. This property is due to their low expression of major histocompatibility complex (MHC) class I antigens and lack of MHC class II expression. Here, we tested the hypothesis that the constitutive FasL expression by ESC is a requirement for their stable engraftment in allogeneic recipients. METHODS: MRL and MRL-lpr/lpr mice (H-2k) were infused allogeneic 129SvJ RW-4 (H-2b) ESC without host preconditioning. The development of mixed chimerism was monitored over 100 days by flow cytometry. RESULTS: Mixed chimerism was detectable by day 7. The amount of donor cells detected varied between 3-5.5% and were lymphoid, but nonmyeloid. Only 50% of lpr/lpr mice engrafted and lost donor cells by day 28 post-ESC infusion. In contrast, >80% wild type mice engrafted and maintained mixed chimerism up to day 100. CONCLUSIONS: These data suggest a critical role for Fas-FasL engagement in ESC engraftment. We conclude that ESC may induce clonal deletion of alloreactive T cells by Fas-induced apoptosis in recipient T cells, protecting them from rejection. The data provide a rationale for improved protocols for the achievement of robust ESC-induced mixed chimerism.