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1.
Stem Cell Reports ; 3(6): 1073-84, 2014 Dec 09.
Article in English | MEDLINE | ID: mdl-25448067

ABSTRACT

The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However, the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here, we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC), an extracellular matrix protein associated with HSC niches, strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders, including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential.


Subject(s)
Hemangioblasts/cytology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Precursor Cells, T-Lymphoid/cytology , Tenascin/genetics , Animals , Cell Culture Techniques , Cell Differentiation/genetics , Cell Lineage , Cells, Cultured , Cluster Analysis , Coculture Techniques , Culture Media , Gene Expression Profiling , Hemangioblasts/metabolism , Hematopoiesis/genetics , Humans , Mesoderm/cytology , Mesoderm/metabolism , Mice , Precursor Cells, T-Lymphoid/metabolism , Stromal Cells , Tenascin/metabolism , Transcriptome
2.
Nat Commun ; 5: 4372, 2014 Jul 14.
Article in English | MEDLINE | ID: mdl-25019369

ABSTRACT

Advancing pluripotent stem cell technologies for modelling haematopoietic stem cell development and blood therapies requires identifying key regulators of haematopoietic commitment from human pluripotent stem cells (hPSCs). Here, by screening the effect of 27 candidate factors, we reveal two groups of transcriptional regulators capable of inducing distinct haematopoietic programs from hPSCs: pan-myeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1). In both cases, these transcription factors directly convert hPSCs to endothelium, which subsequently transform into blood cells with pan-myeloid or erythro-megakaryocytic potential. These data demonstrate that two distinct genetic programs regulate the haematopoietic development from hPSCs and that both of these programs specify hPSCs directly to haemogenic endothelial cells. In addition, this study provides a novel method for the efficient induction of blood and endothelial cells from hPSCs via the overexpression of modified mRNA for the selected transcription factors.


Subject(s)
Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Animals , Cell Line , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/metabolism , Gene Expression Regulation , Hematopoiesis/physiology , Humans , Mice , Transcription Factors/metabolism
3.
Cell Rep ; 2(3): 553-67, 2012 Sep 27.
Article in English | MEDLINE | ID: mdl-22981233

ABSTRACT

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.


Subject(s)
Cell Differentiation/physiology , Hemangioblasts/metabolism , Pluripotent Stem Cells/metabolism , Antigens, CD/metabolism , Cadherins/metabolism , Cell Line , Fibroblast Growth Factor 2/metabolism , Hemangioblasts/cytology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Pluripotent Stem Cells/cytology
4.
Nat Protoc ; 6(3): 296-313, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21372811

ABSTRACT

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.


Subject(s)
Cell Differentiation , Myeloid Progenitor Cells/metabolism , Pluripotent Stem Cells/metabolism , Stromal Cells/metabolism , Coculture Techniques/methods , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Hematopoiesis/physiology , Humans , Myeloid Progenitor Cells/cytology , Pluripotent Stem Cells/cytology , Stromal Cells/cytology , Time Factors
5.
PLoS One ; 6(3): e17557, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21390254

ABSTRACT

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.


Subject(s)
Cell Culture Techniques/methods , Cellular Reprogramming/genetics , Enzyme Inhibitors/pharmacology , Plasmids/genetics , Animals , Cellular Reprogramming/drug effects , Culture Media/pharmacology , Genetic Vectors/genetics , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Mice , Plasmids/drug effects , Transgenes/genetics
6.
Stem Cells Dev ; 20(9): 1639-47, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21434814

ABSTRACT

Differentiation of human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into the erythroid lineage of cells offers a novel opportunity to study erythroid development, regulation of globin switching, drug testing, and modeling of red blood cell (RBC) diseases in vitro. Here we describe an approach for the efficient generation of RBCs from hiPSC/hESCs using an OP9 coculture system to induce hematopoietic differentiation followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. We showed that fibroblast-derived transgenic hiPSCs generated using lentivirus-based vectors and transgene-free hiPSCs generated using episomal vectors can be differentiated into RBCs with an efficiency similar to that of H1 hESCs. Erythroid cultures established with this approach consisted of an essentially pure population of CD235a(+)CD45(-) leukocyte-free RBCs with robust expansion potential and long life span (up to 90 days). Similar to hESCs, hiPSC-derived RBCs expressed predominately fetal γ and embryonic ɛ globins, indicating complete reprogramming of ß-globin locus following transition of fibroblasts to the pluripotent state. Although ß-globin expression was detected in hiPSC/hESC-derived erythroid cells, its expression was substantially lower than the embryonic and fetal globins. Overall, these results demonstrate the feasibility of large-scale production of erythroid cells from fibroblast-derived hiPSCs, as has been described for hESCs. Since RBCs generated from transgene-free hiPSCs lack genomic integration and background expression of reprogramming genes, they would be a preferable cell source for modeling of diseases and for gene function studies.


Subject(s)
Erythrocytes/cytology , Induced Pluripotent Stem Cells/physiology , Antigens, CD/metabolism , Cell Differentiation , Cell Line , Cell Proliferation , Cell Shape , Coculture Techniques , Embryonic Stem Cells/physiology , Erythrocytes/metabolism , Erythroid Cells/metabolism , Flow Cytometry , Hemoglobins/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism
7.
Cell Cycle ; 10(9): 1370-3, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21444996

ABSTRACT

Mesenchymal stem/stromal cells (MSCs) are fibroblastoid cells capable of long-term expansion and skeletogenic differentiation. While MSCs are known to originate from neural crest and mesoderm, immediate mesodermal precursors that give rise to MSCs have not been characterized. Recently, using human embryonic stem cells (hESCs), we demonstrated that mesodermal MSCs arise from APLNR+ precursors with angiogenic potential, mesenchymoangioblasts, which can be identified by FGF2-dependent colony-forming assay in serum-free semisolid medium. In this overview we provide additional insights on cellular pathways leading to MSC establishment from mesoderm, with special emphasis on endothelial-mesenchymal transition as a critical step in MSC formation. In addition, we highlight an essential role of FGF2 in induction of angiogenic cells with potential to transform into MSCs (mesenchymoangioblasts) or hematopoietic cells (hemangioblasts) from mesoderm, and discuss correlations of our in vitro findings with the course of angioblast development during embryogenesis.


Subject(s)
Cell Lineage/physiology , Endothelial Cells/cytology , Endothelial Cells/physiology , Epithelial-Mesenchymal Transition/physiology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Cell Differentiation/physiology , Humans
8.
Cell Stem Cell ; 7(6): 718-29, 2010 Dec 03.
Article in English | MEDLINE | ID: mdl-21112566

ABSTRACT

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.


Subject(s)
Cell Lineage , Endothelial Cells/cytology , Mesenchymal Stem Cells/cytology , Mesoderm/cytology , Animals , Bone Marrow Cells/cytology , Cell Differentiation , Endothelial Cells/metabolism , Fibroblast Growth Factor 2/metabolism , Humans , Mesenchymal Stem Cells/metabolism , Mice
9.
J Clin Invest ; 119(9): 2818-29, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19726877

ABSTRACT

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.


Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Myeloid Cells/cytology , Myeloid Cells/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Antigens, CD34/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Coculture Techniques , Embryonic Stem Cells/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Leukocyte Common Antigens/metabolism , Leukosialin/metabolism , Myeloid Cells/drug effects , Phenotype , Pluripotent Stem Cells/drug effects , Recombinant Proteins
10.
Stem Cells ; 27(3): 559-67, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19259936

ABSTRACT

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study, we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic), lin(-)CD34(+)CD43(+)CD45(-) (multipotent), and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal, or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks.


Subject(s)
Cell Differentiation/physiology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Hematopoietic System/cytology , Hematopoietic System/metabolism , Pluripotent Stem Cells/cytology , Antigens, CD34/metabolism , Cell Differentiation/genetics , Cell Line , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Flow Cytometry , Humans , Leukosialin/metabolism , Pluripotent Stem Cells/metabolism
11.
Exp Hematol ; 36(10): 1377-89, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18922365

ABSTRACT

OBJECTIVE: Cellular and molecular changes that occur during the genesis of the hematopoietic system and hematopoietic stem cells in the human embryo are mostly inaccessible to study and remain poorly understood. To address this gap we have exploited the human embryonic stem cell (hESC) system to molecularly characterize the global transcriptomes of the two functionally discreet and phenotypically separable populations of multipotent hematopoietic cells that first appear when hESCs are induced to differentiate on OP9 cells. MATERIALS AND METHODS: We prepared long serial analysis of gene expression libraries from lin-CD34+CD43+CD45- and lin-CD34+CD43+CD45+ subsets of primitive hematopoietic cells derived in vitro from hESCs, sequenced them to a depth of 200,000 tags and compared their content with similar libraries prepared from highly purified populations of very primitive human fetal liver and cord blood hematopoietic cells. RESULTS: Comparison of libraries obtained from hESC-derived lin-CD34+CD43+CD45- and lin-CD34+CD43+CD45+ revealed differences in their expression of genes associated with myeloid development, cellular biosynthetic processes, and cell-cycle regulation. Further comparisons with analogous data for primitive hematopoietic cells isolated from first-trimester human fetal liver and newborn cord blood showed an apparent similarity between the transcriptomes of the most primitive hESC- and in vivo-derived populations, with the main differences involving genes that regulate HSC self-renewal and homing, chromatin remodeling, AP1 transcription complex genes, and noncoding RNAs. CONCLUSION: These data suggest that primitive hematopoietic cells are generated from hESCs in vitro by processes similar to those operative during human embryogenesis in vivo, although some differences were also detected.


Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Gene Expression Profiling , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Antigens, CD/analysis , Antigens, CD/genetics , Cell Differentiation/physiology , Cell Division , Coculture Techniques , Computational Biology , Embryonic Development , Hematopoiesis/physiology , Humans , RNA/genetics , RNA/isolation & purification
12.
Biotechnol Bioeng ; 100(4): 830-7, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18306423

ABSTRACT

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.


Subject(s)
Adaptation, Biological/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Endothelial Cells/cytology , Endothelial Cells/physiology , Shear Strength , Animals , Cell Differentiation , Cell Line , Cell Shape , Coculture Techniques , Gene Expression , Humans , Mice , Rheology , Stress, Mechanical , Stromal Cells , Umbilical Veins
13.
Science ; 318(5858): 1917-20, 2007 Dec 21.
Article in English | MEDLINE | ID: mdl-18029452

ABSTRACT

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.


Subject(s)
Cell Line , Cellular Reprogramming , Fibroblasts/cytology , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation , Cell Proliferation , Cell Shape , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Embryonic Stem Cells/cytology , Fetus , HMGB Proteins/genetics , HMGB Proteins/physiology , Homeodomain Proteins/genetics , Homeodomain Proteins/physiology , Humans , Infant, Newborn , Karyotyping , Mice , Mice, SCID , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/physiology , Oligonucleotide Array Sequence Analysis , Pluripotent Stem Cells/physiology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/physiology , SOXB1 Transcription Factors , Stem Cell Transplantation , Teratoma/pathology , Transcription Factors/genetics , Transcription Factors/physiology , Transduction, Genetic , Transgenes
14.
Methods Mol Biol ; 407: 275-93, 2007.
Article in English | MEDLINE | ID: mdl-18453262

ABSTRACT

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.


Subject(s)
Cell Differentiation/physiology , Coculture Techniques/methods , Dendritic Cells/cytology , Embryonic Stem Cells/cytology , Flow Cytometry/methods , Cells, Cultured , Dendritic Cells/physiology , Embryonic Stem Cells/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Interleukin-4/metabolism , Myeloid Cells/cytology , Myeloid Cells/physiology
15.
Curr Protoc Cell Biol ; Chapter 23: Unit 23.6, 2007 Sep.
Article in English | MEDLINE | ID: mdl-18228507

ABSTRACT

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.


Subject(s)
Blood Cells/cytology , Cell Culture Techniques/methods , Embryonic Stem Cells/cytology , Endothelial Cells/cytology , Hematopoietic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Adult , Animals , Biomarkers , Cell Culture Techniques/instrumentation , Cell Differentiation , Cell Separation/methods , Cells, Cultured/cytology , Cells, Cultured/drug effects , Coculture Techniques , Colony-Forming Units Assay/methods , Culture Media/pharmacology , Embryonic Stem Cells/drug effects , Fibroblasts/cytology , Fibroblasts/metabolism , Flow Cytometry/methods , Hematopoietic Stem Cells/drug effects , Humans , Mice , Pluripotent Stem Cells/drug effects , Stromal Cells/cytology , Stromal Cells/metabolism
16.
Blood ; 108(6): 2095-105, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16757688

ABSTRACT

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.


Subject(s)
Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Leukosialin/metabolism , Antigens, CD34/metabolism , Cell Differentiation , Cell Line , Cells, Cultured , Coculture Techniques , Colony-Forming Units Assay , Gene Expression , Hematopoiesis , Hematopoietic Stem Cells/classification , Hematopoietic Stem Cells/metabolism , Humans , Models, Biological , Phenotype , Vascular Endothelial Growth Factor Receptor-2/metabolism
17.
J Immunol ; 176(5): 2924-32, 2006 Mar 01.
Article in English | MEDLINE | ID: mdl-16493050

ABSTRACT

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.


Subject(s)
Cell Differentiation/physiology , Dendritic Cells/cytology , Myeloid Cells/cytology , Myeloid Cells/physiology , Signal Transduction/immunology , Stem Cells/cytology , Animals , Cell Line , Cells, Cultured , Coculture Techniques , Dendritic Cells/immunology , Embryo, Mammalian , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Humans , Mice , Myeloid Cells/metabolism , T-Lymphocytes/immunology
18.
Stem Cells ; 24(1): 168-76, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16210403

ABSTRACT

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.


Subject(s)
Cell Fusion/methods , Hybrid Cells/physiology , Myeloid Progenitor Cells/physiology , Pluripotent Stem Cells/physiology , Stem Cells , Animals , Cell Culture Techniques/methods , Cell Differentiation , Cell Line , Coculture Techniques , Humans , Hyaluronan Receptors/metabolism , Mice , Mice, Transgenic , Octamer Transcription Factor-3/genetics , Peroxidase/metabolism
19.
Am J Reprod Immunol ; 54(5): 284-91, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16212650

ABSTRACT

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.


Subject(s)
Abortion, Habitual/blood , Receptors, Tumor Necrosis Factor, Type II/blood , Receptors, Tumor Necrosis Factor, Type I/blood , Abortion, Habitual/drug therapy , Abortion, Habitual/immunology , Adult , Cells, Cultured , Female , Humans , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Lymphocyte Count , Progesterone/administration & dosage , Receptors, Tumor Necrosis Factor, Type I/immunology , Receptors, Tumor Necrosis Factor, Type II/immunology , T-Lymphocyte Subsets/immunology
20.
Blood ; 105(2): 617-26, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15374881

ABSTRACT

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.


Subject(s)
Cell Communication/physiology , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Stromal Cells/cytology , Antigens, CD34/metabolism , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation/physiology , Cells, Cultured , Coculture Techniques , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , GATA2 Transcription Factor , Gene Expression/physiology , Hematopoietic Stem Cells/physiology , Humans , Phenotype , Proto-Oncogene Proteins/genetics , T-Cell Acute Lymphocytic Leukemia Protein 1 , Transcription Factors/genetics , Up-Regulation , Vascular Endothelial Growth Factor Receptor-2/genetics
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