Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.

Hemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential for enabling the investigation of the molecular determinants of HSC specification. Here, we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by a VE-cadherin(+)CD73(-)CD235a/CD43(-) phenotype. This phenotype discriminates true HEPs from VE-cadherin(+)CD73(+) non-HEPs and VE-cadherin(+)CD235a(+)CD41a(-) early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post-primitive-streak stage of differentiation directly from VE-cadherin-negative KDR(bright)APLNR(+)PDGFRα(low/-) hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR(+)PDGFRα(+) mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal of facilitating the generation of HSCs from hPSCs.

Efficient feeder-free episomal reprogramming with small molecules.

Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3ß inhibitor CHIR99021, TGF-ß/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.

A mesoderm-derived precursor for mesenchymal stem and endothelial cells.

Among the three embryonic germ layers, the mesoderm is a major source of the mesenchymal precursors giving rise to skeletal and connective tissues, but these precursors have not previously been identified and characterized. Using human embryonic stem cells directed toward mesendodermal differentiation, we show that mesenchymal stem/stromal cells (MSCs) originate from a population of mesodermal cells identified by expression of apelin receptor. In semisolid medium, these precursors form FGF2-dependent compact spheroid colonies containing mesenchymal cells with a transcriptional profile representative of mesoderm-derived embryonic mesenchyme. When transferred to adherent cultures, individual colonies give rise to MSC lines with chondro-, osteo-, and adipogenic differentiation potentials. Although the MSC lines lacked endothelial potential, endothelial cells could be derived from the mesenchymal colonies, suggesting that, similar to hematopoietic cells, MSCs arise from precursors with angiogenic potential. Together, these studies identified a common precursor of mesenchymal and endothelial cells, mesenchymoangioblast, as the source of mesoderm-derived MSCs.

Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin-CD34+CD43+CD45+ progenitors.

Basic research into human mature myelomonocytic cell function, myeloid lineage diversification and leukemic transformation, and assessment of myelotoxicity in preclinical drug development requires a constant supply of donor blood or bone marrow samples and laborious purification of mature myeloid cells or progenitors, which are present in very small quantities. To overcome these limitations, we have developed a protocol for efficient generation of neutrophils, eosinophils, macrophages, osteoclasts, DCs, and Langerhans cells from human embryonic stem cells (hESCs). As a first step, we generated lin-CD34+CD43+CD45+ hematopoietic cells highly enriched in myeloid progenitors through coculture of hESCs with OP9 feeder cells. After expansion in the presence of GM-CSF, these cells were directly differentiated with specific cytokine combinations toward mature cells of particular types. Morphologic, phenotypic, molecular, and functional analyses revealed that hESC-derived myelomonocytic cells were comparable to their corresponding somatic counterparts. In addition, we demonstrated that a similar protocol could be used to generate myelomonocytic cells from induced pluripotent stem cells (iPSCs). This technology offers an opportunity to generate large numbers of patient-specific myelomonocytic cells for in vitro studies of human disease mechanisms as well as for drug screening.

The response of human embryonic stem cell-derived endothelial cells to shear stress.

An important physiological function of vascular endothelial cells is to detect and respond to physical stimuli. While many efforts have been made to derive endothelial cells from human embryonic stem cells (hESCs), the ability of these derivatives to respond to mechanical forces has yet to be ascertained. hESC-derived endothelial cells (hEECs) were obtained by coculturing hESCs with OP9 stromal cells. Here we applied physiologic levels of shear stress to hEECs in a parallel plate flow chamber and observed changes in cell morphology and gene expression, comparing the response to that of human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs). Shear induced hEECs to elongate and align in the direction of flow, and their overall transcriptional response to shear was similar to the primary cells tested. In response to shear in hEECs, COX2 and MMP1 were upregulated four- and threefold, MCP1 and VCAM1 expression decreased over fivefold, and ICAM1 and TPA were downregulated almost threefold. TGFbeta1 and SOD2 transcription exhibited no change under the conditions tested. Additionally, preshearing of hEECs mitigated TNFalpha-induced VCAM1 surface expression. These findings suggest that hEECs are capable of functionally responding to changes in fluid shear stress by modulating gene expression and cell morphology.

Induced pluripotent stem cell lines derived from human somatic cells.

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

Directed differentiation of human embryonic stem cells to dendritic cells.

Embryonic stem cells represent a pluripotent population of cells capable of self-renewal, large-scale expansion, and differentiation in various cell lineages including cells of hematopoietic lineage. In this chapter, we describe a three-step cell culture method for directed differentiation of human embryonic stem cells (hESCs) to dendritic cells (DCs) that includes (1) hESC differentiation into hematopoietic progenitors by coculture with OP9 stromal cells, (2) expansion of myeloid DC precursors in suspension bulk cultures with granulocyte monocyte-colony stimulating factor (GM-CSF), and (3) differentiation of myeloid precursors to DCs in the serum-free medium with GM-CSF and interleukin-4 (IL-4). The method employs cell culture conditions selecting an almost pure population of myeloid DC precursors and does not require isolation of hematopoietic progenitors. With this method, hESCs can be differentiated to functional DCs within 30 days at an efficiency of at least four DCs per single undifferentiated hESC. Directed differentiation of DCs from hESCs could be useful for studying cellular and molecular mechanisms of DC development and potentially for the generation of antigen-presenting cells for cellular immunotherapy.

Hematoendothelial differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) represent a unique population of cells capable of self-renewal and differentiation into all types of somatic cells, including hematopoietic and endothelial cells. Since the pattern of hematopoietic and endothelial development observed in the embryo can be reproduced using ESCs differentiated in culture, hESCs can be used as a model for studies of specification and diversification of hematoendothelial progenitors. In addition, hESCs can be seen as a scalable source of hematopoietic and endothelial cells for in vitro studies. This unit describes a method for efficient differentiation of hESCs into hematopoietic progenitors and endothelial cells through coculture with mouse OP9 bone marrow stromal cells, as well as an approach for their analysis and isolation. Support protocols are provided for culture of mouse embryonic fibroblasts, evaluation of hematopoietic and endothelial differentiation by flow cytometry and colony-forming assay, removal of OP9 cells, and propagation of hESC-derived endothelial cells. Curr. Protoc.

Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures.

During hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile.

Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway.

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step, we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then, expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of Ag processing, triggering naive T cells in MLR, and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, because ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance.

Human embryonic stem cells reprogram myeloid precursors following cell-cell fusion.

Here, we examine the ability of undifferentiated human embryonic stem cells (hESCs) to reprogram the nuclei of hESC-derived myeloid precursors following cell-cell fusion. Using an OP9 coculture system, we produced CD45+ CD33+ myeloperoxidase+ myeloid precursors from an Oct4-enhanced green fluorescent protein (EGFP) knock-in hESC line and demonstrated that Oct4-EGFP expression was extinguished in these precursors. Upon fusion with undifferentiated hESCs, EGFP expression from the endogenous Oct4 promoter/regulatory region was re-established, ESC-specific surface antigens and marker genes were expressed, and myeloid precursor-specific antigens were no longer detectable. When the hybrid cells were formed into embryoid bodies, upregulation of genes characteristic of the three germ layers and extraembryonic tissues occurred, indicating that the hybrid cells had the potential to differentiate into multiple lineages. Interestingly, the hybrid cells were capable of redifferentiating into myeloid precursors with efficiency comparable with that of diploid hESCs despite their neartetraploid chromosome complement. These results indicate that hESCs are capable of reprogramming nuclei from differentiated cells and that hESC hybrid cells provide a new model system for studying the mechanisms of nuclear reprogramming.

Lack of soluble TNF-receptors in women with recurrent spontaneous abortion and possibility for its correction.

PROBLEM: Tumor necrosis factor (TNF) and soluble TNF receptors (sTNF-Rs) system related with Th1 and Th2 and activity of NF-kappaB/IkappaB regulatory system. This study was designed to compare sTNF-R1 and sTNF-R2 production (shedding) and levels of late activated CD8+ T-lymphocytes in non-pregnant (n = 30) and pregnant (n = 20) normal women and non-pregnant (n = 20) and pregnant (n = 30) RSA women. Effects of progesterone (natural structure) injections in RSA women were studied. METHODS OF STUDY: Levels of sTNF-R1, sTNF-R2, TNF in peripheral blood serum were detected by enzyme-linked immunosorbent assay. Lymphocyte subsets were estimated by multicolor flow cytometry. NK cell cytotoxic activity of peripheral blood lymphocytes (PBL) in whole blood against K562 targets was determined using Europium-release cytotoxicity assay. Mitogen-induced proliferative response of PBL to PHA-P, Con A and PWM were determined by standard 3H-thymidine incorporation assay. RESULTS: Levels of soluble TNF-R1 and TNF-R2 in normal pregnancy were elevated when compared with non-pregnant normal women and pregnant RSA women. Levels of late activated CD8+ T-lymphocytes in normal pregnancy were decreased but no changes were detected in RSA women. After progesterone therapy (i.m. injections of 2.5% oil solution) in RSA women elevation of sTNF-R1 and sTNF-R2 to normal pregnancy ranges was observed. No changes in levels of late activated CD8+ T-lymphocytes after progesterone treatment were detected. CONCLUSIONS: Elevation of levels of sTNF-R1, sTNF-R2 and decrease of late activated cytotoxic T-lymphocytes are pronounce markers of normal human pregnancy. In RSA women there are no elevation of sTNF-R1 and sTNF-R2 levels during pregnancy. This deficiency may be restored by progesterone treatment.

Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential.

Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 10(7) CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell-derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase-positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.

Up-regulation of interferon-gamma production by reduced glutathione, anthocyane and L-cysteine treatment in children with allergic asthma and recurrent respiratory diseases.

Negative correlation between serum IgE levels and production of IFN-gamma by lymphocytes and positive correlation between serum IgE levels and production of IL-4 by lymphocytes was detected in 12 children with allergic asthma and recurrent respiratory diseases. Deficiency of reduced glutathione in whole blood and some disorders in phagocytic and oxidative burst activity of monocytes were observed in these children. Use of reduced glutathione, L-cysteine and anthocyane (Recancostat, Clear Vision, Switzerland) resulted in elevation of IFN-gamma production, lymphocyte response to mitogens, NK cell activity, increase in percentage of naive CD4(+) T lymphocytes (refreshment effect) and improvement of clinical status. Positive clinical results were lasted during 6 months.