Helminth-Induced Production of TGF-ß and Suppression of Graft-versus-Host Disease Is Dependent on IL-4 Production by Host Cells.

Helminths stimulate the secretion of Th2 cytokines, like IL-4, and suppress lethal graft-versus-host disease (GVHD) after bone marrow transplantation. This suppression depends on the production of immune-modulatory TGF-ß and is associated with TGF-ß-dependent in vivo expansion of Foxp3+ regulatory T cells (Treg). In vivo expansion of Tregs is under investigation for its potential as a therapy for GVHD. Nonetheless, the mechanism of induced and TGF-ß-dependent in vivo expansion of Tregs, in a Th2 polarized environment after helminth infection, is unknown. In this study, we show that helminth-induced IL-4 production by host cells is critical to the induction and maintenance of TGF-ß secretion, TGF-ß-dependent expansion of Foxp3+ Tregs, and the suppression of GVHD. In mice with GVHD, the expanding donor Tregs express the Th2-driving transcription factor, GATA3, which is required for helminth-induced production of IL-4 and TGF-ß. In contrast, TGF-ß is not necessary for GATA3 expression by Foxp3+ Tregs or by Foxp3- CD4 T cells. Various cell types of innate or adaptive immune compartments produce high quantities of IL-4 after helminth infection. As a result, IL-4-mediated suppression of GVHD does not require invariant NKT cells of the host, a cell type known to produce IL-4 and suppress GVHD in other models. Thus, TGF-ß generation, in a manner dependent on IL-4 secretion by host cells and GATA3 expression, constitutes a critical effector arm of helminthic immune modulation that promotes the in vivo expansion of Tregs and suppresses GVHD.

Tracking embryonic hematopoietic stem cells to the bone marrow: nanoparticle options to evaluate transplantation efficiency.

BACKGROUND: As the prevalence of therapeutic approaches involving transplanted cells increases, so does the need to noninvasively track the cells to determine their homing patterns. Of particular interest is the fate of transplanted embryonic stem cell-derived hematopoietic progenitor cells (HPCs) used to restore the bone marrow pool following sublethal myeloablative irradiation. The early homing patterns of cell engraftment are not well understood at this time. Until now, longitudinal studies were hindered by the necessity to sacrifice several mice at various time points of study, with samples of the population of lymphoid compartments subsequently analyzed by flow cytometry or fluorescence microscopy. Thus, long-term study and serial analysis of the transplanted cells within the same animal was cumbersome, making difficult an accurate documentation of engraftment, functionality, and cell reconstitution patterns. METHODS: Here, we devised a noninvasive, nontoxic modality for tracking early HPC homing patterns in the same mice longitudinally over a period of 9 days using mesoporous silica nanoparticles (MSNs) and magnetic resonance imaging. RESULTS: This approach of potential translational importance helps to demonstrate efficient uptake of MSNs by the HPCs as well as retention of MSN labeling in vivo as the cells were traced through various organs, such as the spleen, bone marrow, and kidney. Altogether, early detection of the whereabouts and engraftment of transplanted stem cells may be important to the overall outcome. To accomplish this, there is a need for the development of new noninvasive tools. CONCLUSIONS: Our data suggest that multifunctional MSNs can label viably blood-borne HPCs and may help document the distribution and homing in the host followed by successful reconstitution.

Demethylation of induced pluripotent stem cells from type 1 diabetic patients enhances differentiation into functional pancreatic ß cells.

Type 1 diabetes (T1D) can be managed by transplanting either the whole pancreas or isolated pancreatic islets. However, cadaveric pancreas is scarcely available for clinical use, limiting this approach. As such, there is a great need to identify alternative sources of clinically usable pancreatic tissues. Here, we used induced pluripotent stem (iPS) cells derived from patients with T1D to generate glucose-responsive, insulin-producing cells (IPCs) via 3D culture. Initially, T1D iPS cells were resistant to differentiation, but transient demethylation treatment significantly enhanced IPC yield. The cells responded to high-glucose stimulation by secreting insulin in vitro The shape, size, and number of their granules, as observed by transmission electron microscopy, were identical to those found in cadaveric ß cells. When the IPCs were transplanted into immunodeficient mice that had developed streptozotocin-induced diabetes, they promoted a dramatic decrease in hyperglycemia, causing the mice to become normoglycemic within 28 days. None of the mice died or developed teratomas. Because the cells are derived from "self," immunosuppression is not required, providing a much safer and reliable treatment option for T1D patients. Moreover, these cells can be used for drug screening, thereby accelerating drug discovery. In conclusion, our approach eliminates the need for cadaveric pancreatic tissue.

Induced pluripotent stem cell-derived gamete-associated proteins incite rejection of induced pluripotent stem cells in syngeneic mice.

The safety of induced pluripotent stem cells (iPSCs) in autologous recipients has been questioned after iPSCs, but not embryonic stem cells (ESCs), were reported to be rejected in syngeneic mice. This important topic has remained controversial because there has not been a mechanistic explanation for this phenomenon. Here, we hypothesize that iPSCs, but not ESCs, readily differentiate into gamete-forming cells that express meiotic antigens normally found in immune-privileged gonads. Because peripheral blood T cells are not tolerized to these antigens in the thymus, gamete-associated-proteins (GAPs) sensitize T cells leading to rejection. Here, we provide evidence that GAPs expressed in iPSC teratomas, but not in ESC teratomas, are responsible for the immunological rejection of iPSCs. Furthermore, silencing the expression of Stra8, 'the master regulator of meiosis', in iPSCs, using short hairpin RNA led to significant abrogation of the rejection of iPSCs, supporting our central hypothesis that GAPs expressed after initiation of meiosis in iPSCs were responsible for rejection. In contrast to iPSCs, iPSC-derivatives, such as haematopoietic progenitor cells, are able to engraft long-term into syngeneic recipients because they no longer express GAPs. Our findings, for the first time, provide a unifying explanation of why iPSCs, but not ESCs, are rejected in syngeneic recipients, ending the current controversy on the safety of iPSCs and their derivatives.

Human iPS cell-derived insulin producing cells form vascularized organoids under the kidney capsules of diabetic mice.

Type 1 diabetes (T1D) is caused by autoimmune disease that leads to the destruction of pancreatic ß-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs) capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.

Progress toward establishing embryonic stem or induced pluripotent stem cell-based clinical translation.

PURPOSE OF REVIEW: Embryonic stem cells and induced pluripotent stem cells are pluripotent and therefore capable of differentiating into different cell types and tissues. However, their clinical potential, so far, has not been sufficiently probed. The major obstacle is the lack of protocols that allow efficient derivation of clinical grade cells or tissues. This review will address these questions and discuss the current state of the field. RECENT FINDINGS: I will address some of the ongoing clinical trials using stem cell-derived retinal pigment epithelial cells, cardiomyocytes, neurons and attempts to establish insulin-producing cells for the treatment of type 1 diabetes. SUMMARY: Are we there yet? The answer is clearly no. Progress in the different organs and tissues that are being generated is quite variable. Clearly, there has been more success in the derivation of retinal pigment epithelial cells, neuronal cells and cardiomyocytes than in any other tissues or organs. The derivation of insulin-producing cells and that of definitive hematopoietic progenitor cells in humans remains a challenge. Having said that the progress already made with other tissues is an encouraging sign that we may eventually see progress across the board.

Skin deep: from dermal fibroblasts to pancreatic beta cells.

Type I diabetes (T1D) is a chronic autoimmune disease caused by pancreatic ß-cell destruction induced by autoantibodies and autoreactive T cells. After significant reduction of the ß-cell mass, diabetes sets in and can cause significant complications. It is estimated that more than 3 million Americans have T1D, and its prevalence among young individuals is progressively rising; however, the reasons for this increase are not known. Islet transplantation is recognized as the ultimate cure for T1D, but unfortunately, the severe scarcity of available islets makes it necessary to establish alternative sources of ß-cells. Our lab seeks to establish human-induced pluripotent stem cells as an unlimited, novel source of insulin-producing cells (IPCs) that are patient-specific, obviating the requirement for immunosuppression. Although several reports have emerged demonstrating successful derivation of IPCs from human pluripotent stem cells, the efficiencies of derivation are inadequate and these IPCs do not respond to glucose stimulation in vitro. We reasoned that the use of a growth factor sequestering bioscaffold and promotion of cell-cell signaling through 3D clustering would enhance the generation of functionally superior IPCs compared to those derived by 2D differentiation. Here, we discuss a novel 3D platform for the generation of highly efficient human IPCs.

Embryonic stem cell-derived haematopoietic progenitor cells down-regulate the CD3 ξ chain on T cells, abrogating alloreactive T cells.

Murine embryonic stem (ES) cell-derived haematopoietic progenitor cells (HPCs) engraft and populate lymphoid organs. In vivo, HPCs engraft across MHC barriers protecting donor-type allografts from rejection. However, the underlying phenomenon remains elusive. Here, we sought to determine the mechanism by which ES cell-derived HPCs regulate alloreactive T cells. We used the 2C mouse, which expresses a transgenic T-cell receptor against H2-L(d) to determine whether HPCs are deleted by cytotoxic T lymphocytes (CTLs). Previously, we reported that HPCs express MHC class I antigens poorly and do not express class II antigens. In vitro stimulated 2C CTLs failed to lyse H2-L(d) HPCs in a standard 4-hr (51) chromium release assay. Similarly, when the HPCs were tested in an ELISPOT assay measuring the release of interferon-γ by CTLs, HPCs failed to induce CTL degranulation. In addition, mice that were injected with HPCs showed a marked decrease in T-cell responses to alloantigen and CD3 stimulation, but showed a normal response to PMA/ionomycin, suggesting that HPCs impaired T-cell signalling through the T-cell receptor/CD3 complex. Here, we show that HPCs secrete arginase, an enzyme that scavenges l-arginine, leading to metabolites that down-regulate CD3 ζ chain. Indeed an arginase inhibitor partially restored expression of the CD3 ζ chain, implicating arginase 1 in the down-regulation of T cells. This previously unrecognized property of ES cell-derived HPCs could positively enhance the engraftment of ES cell-derived HPCs across MHC barriers by preventing rejection.

Strategies to generate induced pluripotent stem cells.

The isolation of embryonic stem cells (ESCs) has furthered our understanding of normal embryonic development and fueled the progression of stem cell derived therapies. However, the generation of ESCs requires the destruction of an embryo, making the use of these cells ethically controversial. In 2006 the Yamanaka group overcame this ethical controversy when they described a protocol whereby somatic cells could be dedifferentiated into a pluripotent state following the transduction of a four transcription factor cocktail. Following this initial study numerous groups have described protocols to generate induced pluripotent stem cells (iPSCs). These protocols have simplified the reprogramming strategy by employing polycistronic reprogramming cassettes and flanking such polycistronic cassettes with loxP or piggyBac recognition sequences. Thus, these strategies allow for excision of the entire transgene cassette, limiting the potential for the integration of exogenous transgenes to have detrimental effect. Others have prevented the potentially deleterious effects of integrative reprogramming strategies by using non-integrating adenoviral vectors, traditional recombinant DNA transfection, transfection of minicircle DNA, or transfection of episomally maintained EBNA1/OriP plasmids. Interestingly, transfection of mRNA or miRNA has also been shown to be capable of reprogramming cells, and multiple groups have developed protocols using cell penetrating peptide tagged reprogramming factors to de-differentiate somatic cells in the absence of exogenous nucleic acid. Despite the numerous different reprogramming strategies that have been developed, the reprogramming process remains extremely inefficient. To overcome this inefficiency multiple groups have successfully used small molecules such as valproic acid, sodium butyrate, PD0325901, and others to generate iPSCs.The fast paced field of cellular reprogramming has recently produced protocols to generate iPSCs using non integrative techniques with an ever improving efficiency. These recent developments have brought us one step closer to developing a safe and efficient method to reprogram cells for clinical use. However, a lot of work is still needed before iPSCs can be implemented in a clinical setting.

Mouse ES cell-derived hematopoietic progenitor cells.

Future stem cell-based therapies will benefit from the new discoveries being made on pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (IPS) cells. Understanding the genes regulating pluripotency has opened new opportunities to generate patient-tailored therapies. However, protocols for deriving progenitor cells of therapeutic grade from these pluripotent stem cells are not yet worked out. In particular the potential of these cells in treating diseases when compared to their adult progenitor counterparts is unknown. This is crucial work that needs to be studied in detail because we will need to determine engraftment potential of these cells and their ability for multi-lineage engraftment in the in vivo setting before any clinical applications. The ability of these cells to engraft is dependent on their expression of cell surface markers which guide their homing patterns. In this review, I discuss murine hematopoietic progenitor cells derived from mouse ES cells. Stem cells in the bone marrow are found in the bone marrow niches. Our knowledge of the bone marrow niches is growing and will ultimately lead to improved clinical transplantation of bone marrow cells. We are, however, a long way in appreciating how hematopoietic progenitor cells migrate and populate lymphoid tissues. One of the variables in generating hematopoietic progenitor cells is that different labs use different approaches in generating progenitor cells. In some cases, the ES cell lines used show some variability as well. The cell culture media used by the different investigators highly influence the maturation level of the cells and their homing patterns. Here, mouse ES cell-derived progenitor cells are discussed.

Human iPS cell-derived hematopoietic progenitor cells induce T-cell anergy in in vitro-generated alloreactive CD8(+) T cells.

Human induced pluripotent stem cells (iPSCs) have emerged as an alternative source of pluripotent stem cells that can be used for tissue regeneration in place of the controversial human embryonic stem cells. However, immunologic knowledge about iPSC derivatives remains enigmatic. Here, we characterized human iPS-derived CD34(+) hematopoietic progenitor cells (HPCs). These HPCs poorly express major histocompatibility complex (MHC) I antigens and are MHC-II negative. Interestingly, they moderately express nonclassical HLA-G and HLA-E molecules. Consequently, alloreactive HLA-A2-specific cytotoxic T cells failed to recognize HLA-A2-expressing HPCs but became anergic. Subsequent upregulation of MHC-I using interferon-γ stimulation and provision of CD28 cosignaling led to T-cell activation, confirming that poor delivery of signals 1 and 2 by the HPCs mediated T-cell anergy. These data indicate for the first time that HPCs induce T-cell anergy, a unique characteristic of iPSC-derived cells that confers immunologic advantage for allogenic transplantation. Although iPSCs are ideal for patient-tailored treatments with the anticipation that no immunosuppression will be required, in cases of gene defects, their derivatives could be used to treat diseases in nonhistocompatible recipients.

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.

PDX1-engineered embryonic stem cell-derived insulin producing cells regulate hyperglycemia in diabetic mice.

BACKGROUND: Type 1 diabetes can be treated by the transplantation of cadaveric whole pancreata or isolated pancreatic islets. However, this form of treatment is hampered by the chronic shortage of cadaveric donors. Embryonic stem (ES) cell-derived insulin producing cells (IPCs) offer a potentially novel source of unlimited cells for transplantation to treat type 1 and possibly type 2 diabetes. However, thus far, the lack of a reliable protocol for efficient differentiation of ES cells into IPCs has hindered the clinical exploitation of these cells. METHODS: To efficiently generate IPCs using ES cells, we have developed a double transgenic ES cell line R1Pdx1AcGFP/RIP-Luc that constitutively expresses pancreatic ß-cell-specific transcription factor pancreatic and duodenal homeobox gene 1 (Pdx1) as well as rat insulin promoter (RIP) driven luciferase reporter. We have established several protocols for the reproducible differentiation of ES cells into IPCs. The differentiation of ES cells into IPCs was monitored by immunostaining as well as real-time quantitative RT-PCR for pancreatic ß-cell-specific markers. Pancreatic ß-cell specific RIP became transcriptionally active following the differentiation of ES cells into IPCs and induced the expression of the luciferase reporter. Glucose stimulated insulin secretion by the ES cell-derived IPCs was measured by ELISA. Further, we have investigated the therapeutic efficacy of ES cell-derived IPCs to correct hyperglycemia in syngeneic streptozotocin (STZ)-treated diabetic mice. The long term fate of the transplanted IPCs co-expressing luciferase in syngeneic STZ-induced diabetic mice was monitored by real time noninvasive in vivo bioluminescence imaging (BLI). RESULTS: We have recently demonstrated that spontaneous in vivo differentiation of R1Pdx1AcGFP/RIP-Luc ES cell-derived pancreatic endoderm-like cells (PELCs) into IPCs corrects hyperglycemia in diabetic mice. Here, we investigated whether R1Pdx1AcGFP/RIP-Luc ES cells can be efficiently differentiated in vitro into IPCs. Our new data suggest that R1Pdx1AcGFP/RIP-Luc ES cells efficiently differentiate into glucose responsive IPCs. The ES cell differentiation led to pancreatic lineage commitment and expression of pancreatic ß cell-specific genes, including Pax4, Pax6, Ngn3, Isl1, insulin 1, insulin 2 and PC2/3. Transplantation of the IPCs under the kidney capsule led to sustained long-term correction of hyperglycemia in diabetic mice. Although these newly generated IPCs effectively rescued hyperglycemic mice, an unexpected result was teratoma formation in 1 out of 12 mice. We attribute the development of the teratoma to the presence of either non-differentiated or partially differentiated stem cells. CONCLUSIONS: Our data show the potential of Pdx1-engineered ES cells to enhance pancreatic lineage commitment and to robustly drive the differentiation of ES cells into glucose responsive IPCs. However, there is an unmet need for eliminating the partially differentiated stem cells.

Immunological applications of stem cells in type 1 diabetes.

Current approaches aiming to cure type 1 diabetes (T1D) have made a negligible number of patients insulin-independent. In this review, we revisit the role of stem cell (SC)-based applications in curing T1D. The optimal therapeutic approach for T1D should ideally preserve the remaining ß-cells, restore ß-cell function, and protect the replaced insulin-producing cells from autoimmunity. SCs possess immunological and regenerative properties that could be harnessed to improve the treatment of T1D; indeed, SCs may reestablish peripheral tolerance toward ß-cells through reshaping of the immune response and inhibition of autoreactive T-cell function. Furthermore, SC-derived insulin-producing cells are capable of engrafting and reversing hyperglycemia in mice. Bone marrow mesenchymal SCs display a hypoimmunogenic phenotype as well as a broad range of immunomodulatory capabilities, they have been shown to cure newly diabetic nonobese diabetic (NOD) mice, and they are currently undergoing evaluation in two clinical trials. Cord blood SCs have been shown to facilitate the generation of regulatory T cells, thereby reverting hyperglycemia in NOD mice. T1D patients treated with cord blood SCs also did not show any adverse reaction in the absence of major effects on glycometabolic control. Although hematopoietic SCs rarely revert hyperglycemia in NOD mice, they exhibit profound immunomodulatory properties in humans; newly hyperglycemic T1D patients have been successfully reverted to normoglycemia with autologous nonmyeloablative hematopoietic SC transplantation. Finally, embryonic SCs also offer exciting prospects because they are able to generate glucose-responsive insulin-producing cells. Easy enthusiasm should be mitigated mainly because of the potential oncogenicity of SCs.

Immunity of embryonic stem cell-derived hematopoietic progenitor cells.

A number of medical conditions including hematopoietic stem cell malignancies, immunodeficiencies, and autoimmune diseases can be treated using bone marrow cells. However, the major hindrance to the routine use of bone marrow cells is their unparalleled immunogenicity, requiring the use of harsh and toxic preconditioning regimens that can be fatal. Thus, identification of a safer alternative source of hematopoietic stem cells that can be broadly used in such therapies is highly desirable. Despite the current limited number of human ES cell lines, we believe that the newer technology of reprogramming adult somatic cells into pluripotent cells will eventually lead to greater availability of stem cell lines. Even more compelling is the possibility to directly reprogram a somatic cell into another adult cell type of a different tissue without the need for generating pluripotent cells. Here, I will discuss the immunological properties of mouse ES cell-derived hematopoietic progenitor cells. These progenitor cells poorly express MHC class I antigens but are responsive to stimulation by IFN-γ and other cytokines. However, despite upregulating MHC class I antigens after stimulation, they do not express class II molecules, a consequence of their lack of expression of the critical class II transcription factor CIITA. In this overview, I will discuss some of the published data on antigenicity and immunogenicity of ES cell-derived tissues. As more cells and tissues derived from ES cells become available for transplantation, we will gain more insight and into their abilities to interact with immune cells.

Spontaneous in vivo differentiation of embryonic stem cell-derived pancreatic endoderm-like cells corrects hyperglycemia in diabetic mice.

BACKGROUND: Whole pancreas and islet transplantation are currently used for the treatment of type 1 diabetes. However, the major limitations of this potentially curative approach are an inadequate supply of cadaveric pancreata, lifelong immunosuppression, and chronic graft rejection. Therefore, there is an urgent need to develop new sources of insulin-producing cells (IPCs). Here, we investigated whether embryonic stem (ES) cells can be exploited for the derivation of IPCs, and whether their transplantation can correct hyperglycemia in diabetic mice. METHODS: ES cells engineered to express pancreatic and duodenal homeobox 1 (Pdx1), a critical pancreatic transcription factor, were differentiated into pancreatic endoderm-like cells (PELCs) and evaluated for their potential to correct hyperglycemia after transplantation in diabetic mice. RESULTS: After systemic injection, PELCs localized to the pancreas, liver, and kidney. They then spontaneously differentiated into IPCs that corrected hyperglycemia in diabetic mice. When transplanted under the kidney capsule, PELC-derived IPCs were equally efficient at correcting hyperglycemia. Real-time noninvasive in vivo bioluminescence imaging (BLI) of rat insulin promoter (RIP)-driven luciferase was used to monitor the fate of the transplanted PELCs. To confirm that the transplanted cells were responsible for the correction of hyperglycemia, kidneys containing the transplanted cells were nephrectomized, causing rapid hyperglycemia. Interestingly, none of the animals transplanted with PELCs developed tumors, a potential consequence of the differentiation and purification procedures. CONCLUSIONS: Our data suggest that Pdx1-expressing PELCs are capable of spontaneously undergoing differentiation in vivo into IPCs and leading to a sustained correction of hyperglycemia in diabetic mice.

Differentiation of human embryonic stem cells into hematopoietic cells in vitro.

The ability of embryonic stem cells (ES) cells to form cells and tissues from all three 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 cells, obviating the need for severe host preconditioning when transplanted across major histocompatibility complex barriers. To generate hematopoietic stem cells, protocols utilizing embryoid body (EB)-induced differentiation of human ES (hES) cells have been applied in the authors' laboratory. While this protocol results in targeted differentiation into hematopoietic cells, much remains to be done to improve these methods and make them more efficient.

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.