Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells

OBJECTIVE: We have previously shown the simultaneous generation of CD73(+) mesenchymal stromal cells (MSCs) along with CD34(+) hematopoietic cells from human embryonic stem cells (ESCs) when they are cocultured with OP9 murine stromal cells. We investigated whether MSCs can be derived from human ESCs without coculturing with OP9 cells, and if such cells exhibit immunological properties similar to MSCs derived from adult human bone marrow (BM). MATERIALS AND METHODS: Our starting populations were undifferentiated human ESCs cultured on Matrigel-coated plates without feeder cells. The differentiated fibroblast-looking cells were tested for expression of MSC markers and their potential for multilineage differentiation. We investigated surface expression of human leukocyte antigen (HLA) molecules on these MSCs before and after treatment with interferon-gamma (IFN-gamma). We also tested the proliferative response of T-lymphocytes toward MSCs and the effects of MSCs in mixed lymphocyte reaction (MLR) assays. RESULTS: We derived populations of MSCs from human ESCs with morphology, cell surface marker characteristics, and differentiation potential similar to adult BM-derived MSCs. Similar to BM-derived MSCs, human ESC-derived MSCs express cell surface HLA class I (HLA-ABC) but not HLA class II (HLA-DR) molecules. However, stimulation with IFN-gamma induced the expression of HLD-DR molecules. Human ESC-derived MSCs did not induce proliferation of T-lymphocytes when cocultured with peripheral blood mononuclear cells. Furthermore, ESC-derived MSCs suppressed proliferation of responder T-lymphocytes in MLR assays. CONCLUSIONS: MSCs can be derived from human ESCs without feeder cells. These human ESC-derived MSCs have cell surface markers, differentiation potentials, and immunological properties in vitro that are similar to adult BM-derived MSCs.     

Patterning definitive hematopoietic stem cells from embryonic stem cells

Derived from the inner cell mass of blastocysts, embryonic stem cells (ESCs) retain the pluripotent features of early embryonic epiblast cells. In vitro, ESCs undergo spontaneous differentiation into a multitude of tissues, and thus are a powerful tool for the study of early developmental processes and a promising resource for cell-based therapies. We have pursued the derivation of functional, multipotent and engraftable hematopoietic stem cells (HSCs) from ESCs in order to investigate the genetic pathways specifying blood formation, as well as to lay the foundation for hematopoietic cell replacement therapies based on engineered ESCs. Theoretically, the generation of HSCs from patient-specific ESCs derived by nuclear transfer could provide for autologous hematopoietic therapies for the treatment of malignant and genetic bone marrow disorders. Although significant progress has been made in achieving hematopoietic differentiation from both murine and human ESCs, we have only a primitive understanding of the underlying mechanisms that specify hematopoietic cell fate, and a very limited capacity to direct the differentiation of the definitive HSC that would be suitable for clinical engraftment studies. Here we will review the progress to date and the significant problems that remain, and outline a strategy to achieve the directed differentiation of HSCs under conditions that might be appropriate for clinical scale-up and disease applications.     

Towards hematopoietic reconstitution from embryonic stem cells: a sanguine future

PURPOSE OF REVIEW: To review recent progress towards the derivation of hematopoietic stem cells (HSCs) and blood lineages from embryonic stem cells (ESCs), and to highlight the hurdles that must be overcome in order to move the field closer to a clinical application. RECENT FINDINGS: Hematopoietic repopulating cells, red blood cells, and T cells have recently been derived from both murine and human ESCs. Although these results are encouraging, several outstanding issues remain to be addressed by the field before realizing clinical applicability: the phenotype of the ESC-derived HSC must be characterized, methods to purge residual teratoma-forming cells from differentiated populations must be established, and in-vivo models of human HSC function must be optimized to better assess the functionality of putative human ESC-derived HSCs. In addition, embryonic stem-cell derived progeny often represent primitive embryonic hematopoietic cells, rather than their definitive adult counterparts; this critical issue must also be addressed. SUMMARY: The literature firmly establishes that it is possible to isolate HSCs and certain mature blood lineages from both mouse and human ESCs. Although several issues remain to be addressed, these data demonstrate the value of ESCs as a potential source of transplantable HSCs.     

Hematopoietic potential of stem cells isolated from murine skeletal muscle

We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5-day in vitro culture were harvested, and 18 x 10(3) cells were introduced into each of six lethally irradiated recipients together with 200 x 10(3) distinguishable whole bone marrow cells. After 6 or 12 weeks, all recipients showed high-level engraftment of muscle-derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56 +/- 20% (SD), indicating that the cultured muscle cells generated approximately 10- to 14-fold more hematopoietic activity than whole bone marrow. When bone marrow from one mouse was harvested and transplanted into secondary recipients, all recipients showed high-level multilineage engraftment (mean 40%), establishing the extremely primitive nature of these stem cells. We also show that muscle contains a population of cells with several characteristics of bone marrow-derived hematopoietic stem cells, including high efflux of the fluorescent dye Hoechst 33342 and expression of the stem cell antigens Sca-1 and c-Kit, although the cells lack the hematopoietic marker CD45. We propose that this population accounts for the hematopoietic activity generated by cultured skeletal muscle. These putative stem cells may be identical to muscle satellite cells, some of which lack myogenic regulators and could be expected to respond to hematopoietic signals.     

Mouse gene targeting reveals an essential role of mTOR in hematopoietic stem cell engraftment and hematopoiesis

mTOR integrates signals from nutrients and growth factors to control protein synthesis, cell growth, and survival. Although mTOR has been established as a therapeutic target in hematologic malignancies, its physiological role in regulating hematopoiesis remains unclear. Here we show that conditional gene targeting of mTOR causes bone marrow failure and defects in multi-lineage hematopoiesis including myelopoiesis, erythropoiesis, thrombopoiesis, and lymphopoiesis. mTOR deficiency results in loss of quiescence of hematopoietic stem cells, leading to a transient increase but long-term exhaustion and defective engraftment of hematopoietic stem cells in lethally irradiated recipient mice. Furthermore, ablation of mTOR causes increased apoptosis in lineage-committed blood cells but not hematopoietic stem cells, indicating a differentiation stage-specific function. These results demonstrate that mTOR is essential for hematopoietic stem cell engraftment and multi-lineage hematopoiesis.     

Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34(+) cells in NOD/SCID mice

OBJECTIVE: Mesenchymal stem cells (MSC) have been implicated as playing an important role in hematopoietic stem cell engraftment. We identified and characterized a new population of MSC derived from human fetal lung. In cotransplantation experiments, we examined the homing of MSC as well as the effect on engraftment of human umbilical cord blood (UCB)-derived CD34(+) cells in NOD/SCID mice. MATERIALS AND METHODS: Culture-expanded fetal lung-derived CD34(+) cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. Irradiated (3.5 Gy) NOD/SCID mice (n = 51) were transplanted intravenously with 0.03 to 1.0 x 10(6) UCB CD34(+) cells in the presence or absence of 1 x 10(6) culture-expanded fetal lung-derived MSC, irradiated CD34(-) cells, B cells, or with cultured MSC only. RESULTS: Culture-expanded fetal lung CD34(+) cells were identified as MSC based on phenotype (CD105(+), SH3(+), SH4(+), CD160(+)) and their multilineage potential. Cotransplantation of low doses of UCB CD34(+) cells and MSC resulted in a three-fold to four-fold increase in bone marrow engraftment after 6 weeks, whereas no such effect was observed after cotransplantation of irradiated CD34(-) or B cells. Homing experiments indicated the presence of MSC in the lung, but not in the bone marrow, of NOD/SCID mice. CONCLUSIONS: We identified a population of MSC derived from human fetal lung. Upon cotransplantation, MSC, but not irradiated CD34(-) or B cells, promote engraftment of UCB CD34(+) cells in bone marrow, spleen, and blood by mechanisms that may not require homing of MSC to the bone marrow.     

Hematopoietic development from human embryonic stem cell lines

The most common human cell-based therapy applied today is hematopoietic stem cell (HSC) transplantation. Currently, human bone marrow, mobilized peripheral blood, and umbilical cord blood represent the major sources of transplantable HSCs, but their availability for use is limited by both compatibility between donor and recipient and required quantity. Although increasing evidence suggests that somatic HSCs can be expanded to meet current needs, their in vivo potential is concomitantly compromised after ex vivo culture. In contrast, human embryonic stem cells (hESC) possess indefinite proliferative capacity in vitro and have been shown to differentiate into the hematopoietic cell fate, giving rise to erythroid, myeloid, and lymphoid lineages using a variety of differentiation procedures. Human ESC-derived hematopoietic cells emerge from a subset of embryonic endothelium expressing PECAM-1, Flk-1, and VE-Cadherin, but lacking CD45 (CD45negPFV). These CD45negPFV precursors are exclusively responsible for hematopoietic potential of differentiated hESCs. hESC-derived hematopoietic cells show similar clonogenic capacity and primitive phenotype to somatic sources of hematopoietic progenitors and possess limited in vivo repopulating capacity in immunodeficient mice, suggestive of HSC function. Here, we will review current progress in studies of hESC-derived hematopoietic cells and discuss the potential precincts and applications.     

Progenipoietin-1: a multifunctional agonist of the granulocyte colony-stimulating factor receptor and fetal liver tyrosine kinase-3 is a potent mobilizer of hematopoietic stem cells

OBJECTIVE: Progenipoietin-1 is an agonist of both the granulocyte colony-stimulating factor and fetal liver tyrosine kinase-3 receptors capable of inducing the proliferation of multiple hematopoietic cell lineages. The potential of progenipoietin-1 to mobilize transplantable hematopoietic stem cells into the peripheral blood was evaluated. METHODS: Cohorts of donor mice were treated with either progenipoietin-1, fetal liver tyrosine kinase-3 ligand, granulocyte colony-stimulating factor, or a vehicle control. Hematopoietic progenitor/stem-cell activity in donor blood was assayed by radioprotection, multilineage reconstitution, secondary transplantation, and competitive repopulation. RESULTS: Only 1 microL of peripheral blood from progenipoietin-1-treated donors was required to protect 80% of lethally irradiated mice, while in contrast 1 microL of peripheral blood from granulocyte colony-stimulating factor-treated donors failed to protect any recipients. The radioprotected recipients of progenipoietin-1-treated donor cells showed donor-derived (Ly5.2) multilineage hematopoietic reconstitution for up to 6 months. Serial transplantation studies using bone marrow from radioprotected, chimeric recipients demonstrated long-term donor-derived hematopoiesis, indicating the successful transplantation of multipotent hematopoietic stem cells. The engraftment potential of progenipoietin-1 donor-derived cells was directly compared with donors treated with granulocyte colony-stimulating factor or fetal liver tyrosine kinase-3 ligand alone or in combination. Both spleen colony-forming activity and competitive repopulating activity was highest in the blood from progenipoietin-1-treated donors. CONCLUSIONS: These studies demonstrate that progenipoietin-1 is a potent mobilizer of transplantable hematopoietic stem cells and indicate that this dual-receptor agonist has greater biologic activity than its constituent molecules.     

Local irradiation enhances congenic donor pluripotent hematopoietic stem cell engraftment similarly in irradiated and nonirradiated sites

Long-term multilineage chimerism is achieved in CD45 congenic mice receiving high bone marrow doses with or without mediastinal irradiation (MI). Increased donor chimerism results in MI-treated compared with nonirradiated animals, suggesting that MI makes "space" for engraftment of donor pluripotent hematopoietic stem cells (PHSCs). We have now examined whether space is systemic or whether increased engraftment of donor marrow in locally irradiated mice is confined to the irradiated bones. While increased donor chimerism was observed in irradiated bones compared with nonirradiated bones of MI-treated animals 4 weeks following bone marrow transplantation (BMT), these differences were minimal by 40 weeks. MI-treated chimeras contained more adoptively transferable donor PHSCs in the marrow of both irradiated and distant bones compared with non-MI-treated chimeras. Similar proportions of donor PHSCs were present in irradiated and nonirradiated bones of locally irradiated mice at both 4 and 40 weeks. Irradiated bones contained more donor short-term repopulating cells than distant bones at 4 weeks, but not 40 weeks, after BMT. Our study suggests that local proliferation of donor PHSCs in mice receiving local irradiation rapidly leads to a systemic increase in donor PHSC engraftment.     

Activation of the G-CSF and Flt-3 receptors protects hematopoietic stem cells from lethal irradiation

OBJECTIVE: Synergy between Flt-3 ligand and G-CSF produces a marked expansion of hematopoietic progenitor cells and mobilizes large numbers of stem cells into the peripheral blood. To determine if the activation of the Flt-3 and G-CSF receptors enhances the regenerative capacity of the hematopoietic compartment, we evaluated whether activation of these receptors augments stem cell recovery following lethal doses of radiation. METHODS: C57BL/6 mice received a single injection of the bi-functional Flt-3 and G-GSF agonist progenipoietin-1, 24 hours prior to exposure to 1100 cGy of gamma radiation. Survival, hematopoietic reconstitution, and competitive repopulation potential were evaluated. RESULTS: All cytokine-treated mice survived for up to 9 months. Radioprotected recipients exhibited stable multilineage hematopoiesis and recovered normal numbers of T cells, B cells, and myelomonocytic cells in the blood, bone marrow, and thymus. Between 2 and 3 weeks following radiation, cytokine-treated mice demonstrated threefold higher serum hemoglobin levels, 10-fold higher nucleated blood cell counts, and 20-fold higher platelet counts compared to controls. Radioprotection of self-renewing hematopoietic stem cells was revealed by multilineage hematopoietic reconstitution following transplantation in a competitive repopulation assay. To further evaluate the extent of cytokine-induced radioprotective activity, a cohort of mice received a second cycle of cytokine treatment and a second exposure to radiation (1100 cGy). Survival of this serially irradiated group was 70% and analysis of the peripheral blood revealed sustained multilineage hematopoiesis. CONCLUSION: These results demonstrate that activation of both the Flt-3 and G-CSF receptors provides a high degree of radioprotection to the hematopoietic progenitor cell and stem cell compartment.     

Angiopoietin-1 promotes endothelial differentiation from embryonic stem cells and induced pluripotent stem cells

Angiopoietin-1 (Ang1) plays a crucial role in vascular and hematopoietic development, mainly through its cognate receptor Tie2. However, little is known about the precise role of Ang1 in embryonic stem cell (ESC) differentiation. In the present study, we used COMP-Ang1 (a soluble and potent variant of Ang1) to explore the effect of Ang1 on endothelial and hematopoietic differentiation of mouse ESCs in an OP9 coculture system and found that Ang1 promoted endothelial cell (EC) differentiation from Flk-1(+) mesodermal precursors. This effect mainly occurred through Tie2 signaling and was altered in the presence of soluble Tie2-Fc. We accounted for this Ang1-induced expansion of ECs as enhanced proliferation and survival. Ang1 also had an effect on CD41(+) cells, transient precursors that can differentiate into both endothelial and hematopoietic lineages. Intriguingly, Ang1 induced the preferential differentiation of CD41(+) cells toward ECs instead of hematopoietic cells. This EC expansion promoted by Ang1 was also recapitulated in induced pluripotent stem cells (iPSCs) and human ESCs. We successfully achieved in vivo neovascularization in mice by transplantation of ECs obtained from Ang1-stimulated ESCs. We conclude that Ang1/Tie2 signaling has a pivotal role in ESC-EC differentiation and that this effect can be exploited to expand EC populations.     

Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5

The signal transducer Stat5 plays a key role in the regulation of hematopoietic differentiation and hematopoietic stem cell function. To evaluate the effects of Stat5 signaling in the earliest hematopoietic progenitors, we have generated an embryonic stem cell line in which Stat5 signaling can be induced with doxycycline. Ectopic Stat5 activation at the point of origin of the hematopoietic lineage (from day 4 to day 6 of embryoid body differentiation) significantly enhances the number of hematopoietic progenitors with colony-forming potential. It does so without significantly altering total numbers or apoptosis of hematopoietic cells, suggesting a cell-intrinsic effect of Stat5 on either the developmental potential or clonogenicity of this population. From day-6 embryoid bodies, under the influence of Stat5 signaling, a population of semiadherent cells can be expanded on OP9 stromal cells that is comprised of primitive hematopoietic blast cells with ongoing, mainly myeloid, differentiation. When these cells are injected into lethally irradiated mice, they engraft transiently in a doxycycline-dependent manner. These results demonstrate that the hematopoietic commitment of embryonic stem cells may be augmented by a Stat5-mediated signal, and highlight the utility of manipulating individual components of signaling pathways for engineering tissue-specific differentiation of stem cells.     

A limited temporal window for the derivation of multilineage repopulating hematopoietic progenitors during embryonal stem cell differentiation in vitro

Embryonal stem cells have been shown to differentiate in vitro into all hematopoietic lineages. This has been used successfully as one approach to the study of genetic events occurring during haematopoiesis. However, studies on the commitment of mesodermal precursors to the hematopoietic lineage have been limited due to the inability to define a system in which embryonal stem (ES) cells will give rise to primitive hematopoietic stem cells in vitro. Using a colony forming assay (CFU- A), we determined that the earliest time point at which primitive multilineage hematopoietic precursors can be detected during ES cell differentiation in vitro in the absence of exogenous conditioned medium or stromal cell culture is 4 days. Lethally irradiated adult recipient mice that received differentiated ES cells from this time point survived for more than 3 weeks; and in two out three experiments, peripheral blood from these animals contained ES-derived progeny. Fluorescence activated cell sorting (FACS) found ES-derived CD45+ hematopoietic cells in both lymphoid and myeloid compartments at 12 weeks posttransplantation, suggesting that the population of day 4 differentiated ES cells contains primitive hematopoietic precursors. A preliminary RT-PCR analysis of gene expression around this time point suggests that there are very few hematopoietic cells present. This approach should prove useful in studies of genetic control of commitment to and maintenance of hematopoietic lineages in vitro and in vivo.     

Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice

OBJECTIVE: For approximately 5% of autologous transplant recipients and a higher proportion of allogeneic transplant recipients, low level and delayed platelet engraftment is an ongoing problem. Mesenchymal stem cells (MSC), which can be derived from bone marrow as well as other organs, are capable of differentiation into multiple cell types and also support hematopoiesis in vitro. Because cotransplantation of marrow-derived stromal cells has been shown to enhance engraftment of human hematopoietic stem cells, we hypothesized that cotransplantation of MSC could enhance platelet and myeloid cell development. MATERIALS AND METHODS: We tested this hypothesis by transplantation of CD34-selected mobilized human peripheral blood stem cells (PBSC) into sublethally irradiated NOD/SCID mice with or without culture-expanded human MSC and evaluated human myeloid, lymphoid, and megakaryocytic engraftment with flow cytometry and in vitro cultures. RESULTS: We find that MSC cotransplantation enhances human cell engraftment when a limiting dose (<1 x 10(6)) of CD34 cells is administered. This enhancement is characterized by a shift in the differentiation of human cells from predominantly B lymphocytes to predominantly CD13(+), CD14(+), and CD33(+) myeloid cells with a corresponding increase in myeloid CFU in the marrow. Megakaryocytopoiesis is enhanced by MSC cotransplantation as assessed by an increase in both marrow CFU-MK and circulating human platelets. In contrast, MSC do not affect the percentage of human bone marrow cells that expresses CD34(+). CONCLUSIONS: Cotransplantation of human mesenchymal stem cells with CD34(+)-selected hematopoietic stem cells enhances myelopoiesis and megakaryocytopoiesis.     

Long-term maintenance of human hematopoietic stem/progenitor cells by expression of BMI1

The polycomb group (PcG) gene BMI1 has been identified as one of the key epigenetic regulators of cell fates during different stages of development in multiple murine tissues. In a clinically relevant model, we demonstrate that enforced expression of BMI1 in cord blood CD34(+) cells results in long-term maintenance and self-renewal of human hematopoietic stem and progenitor cells. Long-term culture-initiating cell frequencies were increased upon stable expression of BMI1 and these cells engrafted more efficiently in NOD-SCID mice. Week 5 cobblestone area-forming cells (CAFCs) were replated to give rise to secondary CAFCs. Serial transplantation studies in NOD-SCID mice revealed that secondary engraftment was only achieved with cells overexpressing BMI1. Importantly, BMI1-transduced cells proliferated in stroma-free cytokine-dependent cultures for more than 20 weeks, while a stable population of approximately 1% to 5% of CD34(+) cells was preserved that retained colony-forming capacity. Whereas control cells lost most of their NOD-SCID engraftment potential after 10 days of ex vivo culturing in absence of stroma, NOD-SCID multilineage engraftment was retained by overexpression of BMI1. Thus, our data indicate that self-renewal of human hematopoietic stem cells is enhanced by BMI1, and we classify BMI1 as an intrinsic regulator of human stem/progenitor cell self-renewal.     

Contribution of alpha6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with alpha4 integrins

The laminin receptor integrin alpha6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. We have studied its role for homing of stem and progenitor cells to mouse hematopoietic tissues in vivo. A function-blocking anti-integrin alpha6 antibody significantly reduced progenitor cell homing to bone marrow (BM) of lethally irradiated mice, with a corresponding retention of progenitors in blood. Remarkably, the anti-integrin alpha6 antibody profoundly inhibited BM homing of long-term multilineage engrafting stem cells, studied by competitive repopulation assay and analysis of donor-derived lymphocytes and myeloid cells in blood 16 weeks after transplantation. A similar profound inhibition of long-term stem cell homing was obtained by using a function-blocking antibody against alpha4 integrin, studied in parallel. Furthermore, the anti-integrin alpha6 and alpha4 antibodies synergistically inhibited homing of short-term repopulating stem cells. Intravenous injection of anti-integrin alpha6 antibodies, in contrast to antibodies against alpha4 integrin, did not mobilize progenitors or enhance cytokine-induced mobilization by G-CSF. Our results provide the first evidence for a distinct functional role of integrin alpha6 receptor during hematopoietic stem and progenitor cell homing and collaboration of alpha6 integrin with alpha4 integrin receptors during homing of short-term stem cells.     

Hematopoietic differentiation of rhesus monkey embryonic stem cells

Several lines of embryonic stem cells (ESC) have been established from rhesus monkey blastocysts. We have examined two of these cell lines for their potential for generating hematopoietic progenitors in cell culture, and we identified culture conditions, including supplementation with bone morphogenetic proteins (BMP), that result in hematopoietic differentiation of rhesus ESC with high efficiency. We have also characterized the resulting hematopoietic progenitor cells for their patterns of gene expression, as compared to those of hematopoietic progenitor cells harvested from rhesus monkey bone marrow. Of more than 60 genes examined in this manner, CD34+/CD38- cells derived from embryonic stem cells and those obtained from bone marrow demonstrated very similar patterns of gene expression. However, with integrin alphaL, IL-6 receptor, and flt-3 gene expression was greatly diminished or absent in CD34+/CD38- cells derived from the ESC, whereas the bone marrow-derived progenitors showed substantial expression of all of these genes. When the same type of comparison was done with mouse (D3 and CCE) as well as human (H1) embryonic stem cells, in each case comparing ESC-derived hematopoietic progenitors with those harvested from bone marrow, the only consistent deficiency of gene expression was that of flt-3. In hematopoietic precursors derived from mouse ESC, globin-gene expression has previously been shown to be a useful index of the embryological maturity of the cells, and we also examined globin-gene expression in rhesus monkey ESC-derived hematopoietic precursor cells, using a semiquantitative technique. CD34+/CD38- cells demonstrated expression of the epsilon- and gamma-globin genes, but negligible levels of beta globin, suggesting that these cells were at the developmental stage in which the yolk sac and fetal liver are the primary sites of hematopoiesis.