Embryonic Stem Cell-derived Hematopoietic Progenitors Research Articles

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

BackgroundAs 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.MethodsHere, 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.ResultsThis 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.ConclusionsOur 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.

F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin− BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo. Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80+ macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80+ macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro. Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo.

Taming the lion: the challenge of immunity in regenerative medicine.

Taming the lion: the challenge of immunity in regenerative medicine.

Indirect immune recognition of mouse embryonic stem cell–derived hematopoietic progenitors in vitro

The clinical use of embryonic stem cell (ESC)-derived hematopoietic progenitors (ESHPs) requires the generation of ESHPs that produce mature hematopoietic cells and do not induce immune rejection after transplantation. We compared the developmental maturity and immunogenicity of ESHPs generated using two methods: embryoid body (EB) formation and culture of ESCs with the OP9 bone marrow stromal cell line (ESC-OP9). ESHPs derived from EBs displayed an immature hematopoietic phenotype and were devoid of immunogenicity marker expression. In contrast, ESHPs derived via ESC-OP9 displayed a mature phenotype and expressed high levels of some immunostimulatory molecules. ESHPs alone could not stimulate CD4(+) T lymphocyte proliferation directly. However, preferential phagocytosis of ESHPs and T cell proliferation were observed in the presence of antigen-presenting cells, consistent with a model of indirect immune recognition of ESHPs. These results suggest that depletion of host CD4(+) T lymphocytes or antigen-presenting cells may be necessary for successful ESHP transplantation.

Host macrophages are a barrier to the engraftment of embryonic stem cell-derived hematopoietic progenitors. (P4544)

Abstract Embryonic stem cell-derived hematopoietic progenitors (ES-HPs) have been proposed as an alternative bone marrow (BM) source. ES-HPs display features of adult hematopoietic progenitors, but do not achieve high levels of engraftment after transplantation, even in immunocompromised hosts. We hypothesized that host macrophages (Mϕ) are a barrier to ES-HP engraftment. To test this, we cultured and transplanted CD41+ ES-HPs into sublethally irradiated NOD.SCID.γc (NSG) mice that lack B, T and NK cells, but still possess Mϕ. Less than 5% ES-HP donor chimerism in the BM and spleen was observed in NSG recipients, in contrast to >90% chimerism in adult BM-transplanted controls. In addition, we noticed a 3.5-fold increase in spleen weight and a 1.75-fold increase in splenic host F4/80+ Mϕ in ES-HP recipients. In vitro phagocytosis assays confirmed that allogeneic Mϕ phagocytosed ES-HPs at higher levels compared to adult Lin- BM targets. Interestingly, syngeneic Mϕ also engulfed ES-HPs, but not adult Lin- BM. When Mϕ were eliminated with liposome-encapsulated clodronate, a 2.17-fold increase in ES-HP engraftment in the BM and 5.14-fold increase in the spleen was observed. Taken together, our data demonstrate that F4/80+ Mϕ are a barrier to ES-HP engraftment and suggest that cultured ES-HPs are unable to regulate host Mϕ phagocytosis. In line with this, ES-HPs expressed low levels of the Mϕ inhibitory ligand, CD47. Functional studies using CD47-Fc are in progress.

Host F4/80+ macrophages are increased after transplantation of embryonic stem cell derived hematopoietic progenitors (126.10)

Abstract Mice transplanted with embryonic stem cell (ESC)-derived hematopoietic progenitors (ES-HPs) historically have displayed low levels of engraftment. We hypothesized that the innate immune system was an obstacle to ES-HP engraftment. To investigate this, we derived ES-HPs in vitro by growing D3-ESCs (strain 129, CD45.2, H-2b) on the bone marrow (BM) stromal cell line, OP9. We transplanted purified CD41+ ES-HPs into sublethally irradiated NOD.SCID.γc knockout (NSG) mice hosts (CD45.1, H-2g7) which lack B, T, and NK cells, but have functional macrophages. Host and donor-derived hematopoiesis was observed. However, at Day 18 post-transplant, ES-HP recipients displayed enlarged spleens that were primarily composed of host macrophages. The absolute numbers of host-derived splenic F4/80+ macrophages were increased 1.81 fold compared to untransplanted controls and 1.7 fold compared to BM transplanted controls. In vitro phagocytosis assays confirmed that NSG macrophages specifically engulfed allogeneic ES-HPs, but not allogeneic adult BM cells, supporting our in vivo results. Taken together, these results suggest that host F4/80+ macrophages may pose a barrier to allogeneic ES-HP engraftment. Importantly, these data suggest that adult macrophages preferentially phagocytose ES-HPs, which could reveal a novel mechanism of rejection unique to ESC-derived cells. Experiments to evaluate the effects of host macrophage depletion on ES-HP engraftment in vivo are ongoing.

Ym1 is a stem cell secreted factor that augments NK cell killing of tumor cells (162.31)

Abstract We previously observed that embryonic stem cell-derived hematopoietic progenitor cells (HPCs) were resistant to NK cells in vitro despite their poor expression of MHC antigens. In contrast, HPCs were readily deleted by NK cells in vivo. This discrepancy led us to study protein expression by HPCs pre and post-transplantation using proteomics. The rationale was that in response to serum cytokines in vivo, HPCs likely upregulated certain proteins that enhanced NK cell cytotoxicity. Upon sequencing, most of these proteins were determined to be housekeeping proteins except for one protein, which we identified as Ym1, also known as chitinase 3-like protein 3. Subsequently, recombinant Ym1 was generated in E. coli. Ym1 strongly enhanced NK cell cytotoxicity to HPCs in vitro by activating NK cells into the highly cytotoxic Mac1hi subsets. This process was APC-dependent, suggesting cross-presentation of IL-15 to NK cells by APCs. In vivo, Ym1 induced the emergence of mature CD27loMac1hi peripheral blood NK cells, which have been found to be most effective at lysing tumor cells. Further, Ym1 caused a significant increase in the production of IFN-γ, perforin and granzyme B. Indeed, Ym1 remarkably enhanced target cell killing by 3-4 fold, which is a novel finding that has promising implications for the application of Ym1 in cancer treatment.

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.

Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Cells Fail to Elicit Allospecific Cytotoxic T Cell Responses In Vitro

Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Cells Fail to Elicit Allospecific Cytotoxic T Cell Responses In Vitro

Characterization of human embryonic stem cell-derived hematopoietic progenitor phenotype

Differentiation of human embryonic stem cells (hESCs) into hematopoietic lineages using various methods has been reported. However, the phenotype that precisely defines the hematopoietic progenitor compartment with clonogenic activities has yet to be determined. Here, we measured and characterized progenitor function of subfractions of cells prospectively isolated from human embryoid bodies (hEBs) during hematopoietic differentiation basing on surface markers CD45, CD34, CD43, and CD38. We report that hematopoietic progenitors predominantly resided in the CD45(+) subset. CD43(+) cells lacking CD45 expression were largely devoid of progenitor activity. However, progenitor activity and multipotentiality was more enriched in CD45(+) cells co-expressing CD43. CD45(+) subset co-expressing CD34 but lacking CD38 expression (CD45(+)CD34(+)CD38(-)) were further enriched for CFU capacity compared to the CD45(+)CD34(+)CD38(+) subset. Our study demonstrates a role of CD43 in enriching hematopoietic progenitors derived from hEBs and reveals a hierarchical organization of hESC-derived hematopoietic progenitor compartments defined by phenotypic markers.

Expression of migration-related genes is progressively upregulated in murine Lineage-Sca-1+c-Kit+ population from the fetal to adult stages of development

IntroductionHematopoietic stem cells (HSCs) follow a genetically programmed pattern of migration during development. Extracellular matrix and adhesion molecules, as well as chemokines and their receptors, are important in adult HSC migration. However, little is known about the role these molecules play at earlier developmental stages.MethodsWe have analyzed by quantitative polymerase chain reaction (qPCR) array the expression pattern of extracellular matrix and adhesion molecules as well as chemokines and chemokine receptors in Lineage-Sca-1+c-Kit+ (LSK) cells at different stages of development, in order to characterize the role played by these molecules in LSK. Data were represented by volcano plots to show the differences in expression pattern at the time points studied.ResultsOur results show marked changes in the expression pattern of extracellular matrix, adhesion molecules, chemokines and their receptors with developmental age, particularly in later stages of development. Ten molecules were significantly increased among the LSK populations studied. Our screen identified the upregulation of Col4a1, as well as molecules involved in its degradation (Mmp2, Timp2), with development. Other genes identified were Sell, Tgfbi, and Entpd1. Furthermore, we show that the expression of the chemokines Ccl4, Ccl9, Il18 and the chemokine receptor Cxcr4 increases in LSK cells during development.ConclusionsSeveral genes are upregulated in the LSK population in their transition to the bone marrow microenvironment, increasing at later stages of development. This gene pattern should be emulated by embryonic stem cell-derived hematopoietic progenitors in order to improve their properties for clinical applications such as engraftment.

Osteoclasts From Human Embryonic Stem Cell-Derived Hematopoietic Progenitors Support Development of An in Vitro Niche Microenvironment.

Abstract 2517Poster Board II-494Osteoclasts are bone resorbing cells located in the bone marrow that play a key role in hematopoiesis and formation of the hematopoietic niche. Previous studies demonstrate osteoclasts arise from the monocyte-macrophage lineage of cells within the bone marrow microenvironment. Osteoclast progenitor cells typically express the monocyte marker CD14, though relatively little is known about the developmental history of these osteoclastogeneic cells in humans. Osteoclast progenitors have been previously isolated from peripheral blood and bone marrow. Previous studies by our group and others demonstrate human embryonic stem cells (hESCs) can differentiate into CD34+CD45+ cells that can be supported to differentiate into many types of hematopoietic cells, though development of osteoclasts have not been previously demonstrated. To better evaluate osteoclastogenesis, hESCs were allowed to differentiate on M210 stromal cells for 19–21 days then sorted for CD34+CD45+ cells. These cells were placed into a secondary co-culture with M210 stromal cells using osteoclast differentiation factors macrophage colony-stimulating factor (hM-CSF) and receptor activator of nuclear factor–kB ligand (hRANKL) for 14–21 days. During this time, mononuclear hematopoietic cells expanded on top of the stromal cells, followed by appearance of multinucleated cells that became adherent and embedded within the stromal cell layer. These adherent multi-nucleated cells were TRAP-positive, consistent with osteoclasts. Q-RT-PCR analysis demonstrated expression of osteoclast genes NFATC1, TRAP, CATHEPSIN K AND MMP-9. Culturing these putative osteoclasts on dentin discs demonstrated dentin resorption and pit formation, consistent with osteoclasts cultured from peripheral blood. No pit formation observed when the same experiments were done without hRANKL and hMCSF. Most remarkable was the extensive proliferation of CD45+CD33+ myeloid cells around the periphery of the adherent, multinucleated osteoclasts in culture, resembling a multi-cellular hematopoietic niche environment. Further studies are underway to better evaluate the interaction of these osteoclast cells with other hESC-derived hematopoietic cells. Together, these studies demonstrate development of functional osteoclasts directly from hESC-derived CD34+CD45+ hematopoietic progenitor population. We also show development of a novel in vitro HSC niche microenvironment utilizing hESC-derived osteoclasts. This system has the potential to now incorporate other hESC-derived cell populations as a model to better define cellular and extracellular mechanisms that mediate human hematopoietic development. Disclosures:No relevant conflicts of interest to declare.

Long-term reconstitution of the mouse hematopoietic system by embryonic stem cell-derived fetal liver.

Murine embryonic stem (ES) cells are permanent blastocyst-derived cell lines capable of contributing to a wide variety of tissues, including the germ line, after injection into host blastocysts. Recently, we have shown that ES cells can produce all of the cells of the developing fetus after aggregation with developmentally compromised tetraploid embryos. Completely ES cell-derived embryos die perinatally, but the liver of these embryos is a source of entirely ES cell-derived hematopoietic progenitors. We have taken 14- to 15-day fetal liver cells from ES cell-tetraploid chimeras and reconstituted the hematopoietic system of lethally irradiated adult recipient mice. ES cell-derived hematopoietic stem cells were capable of long-term (greater than 6 months) repopulation of irradiated recipients, and all hematopoietic cell lineages analyzed (erythrocytes, T cells, mast cells, and macrophages) were derived exclusively from ES cells in such recipients. Thus, ES cells retain the capacity to differentiate into all hematopoietic cell types after prolonged passage in culture. This approach should provide a direct route to the production of mice whose hematopoietic cells carry genetic alterations that would be lethal if passed through the germ line.