Generation of T cells from human embryonic stem cell-derived hematopoietic zones.
ABSTRACT Human embryonic stem cells (hESC) are pluripotent stem cells. A major challenge in the field of hESC is the establishment of specific differentiation protocols that drives hESC down a particular lineage fate. So far, attempts to generate T cells from hESC in vitro were unsuccessful. In this study, we show that T cells can be generated in vitro from hESC-derived hematopoietic precursor cells present in hematopoietic zones (HZs). These zones are morphologically similar to blood islands during embryonic development, and are formed when hESC are cultured on OP9 stromal cells. Upon subsequent transfer of these HZs on OP9 cells expressing high levels of Delta-like 1 and in the presence of growth factors, cells expand and differentiate to T cells. Furthermore, we show that T cells derive exclusively from a CD34(high)CD43(low) population, further substantiating the notion that hESC-derived CD34(high)CD43(low) cells are formed in HZs and are the only population containing multipotent hematopoietic precursor cells. Differentiation to T cells sequentially passes through the physiological intermediates: CD34(+)CD7(+) T/NK committed, CD7(+)CD4(+)CD8(-) immature single positive, CD4(+)CD8(+) double positive, and finally CD3(+)CD1(-)CD27(+) mature T cell stages. TCRalphabeta(+) and TCRgammadelta(+) T cells are generated. Mature T cells are polyclonal, proliferate, and secrete cytokines in response to mitogens. This protocol for the de novo generation of T cells from hESC could be clinically and scientifically relevant.
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ABSTRACT: Stem cell-based strategies for treating HIV-infected individuals represent a novel approach toward reconstituting the ravaged immune system with the ultimate aim of clearing the virus from the body. Genetic modification of human hematopoietic stem cells to produce cells that are either resistant to infection, cells that produce lower amounts of infectious virus, or cells that specifically target the immune response against the virus are currently the dominant strategies under development. This review focuses on the understanding of stem cell-based approaches that are under investigation and the rationale behind such approaches. Significant progress has recently been made utilizing stem cell-based approaches to treat HIV infection. Ideally, a successful strategy would result in immune clearance of the virus from the body as well long-term restoration of overall immune responses to successfully combat everyday environmental antigens. Two recent clinical trails illustrate how new advances in stem cell-based approaches may propel this field forward to clinical reality: one that demonstrates that large-scale gene therapy trials can be performed in a conventional, reproducible manner; and one that demonstrates the utilization of a multipronged approach using lentiviral-based gene therapy vectors. These clinical trails serve as the foundation for the development of other technologies, discussed here, that are currently in preclinical development. Recent advances using stem cell-based approaches to treat HIV infection have provided the impetus for a renewed and expanded interest in the development of new cell-based strategies to treat HIV infection as well as a variety of other diseases.Current opinion in HIV and AIDS 01/2011; 6(1):68-73. · 4.75 Impact Factor
Article: Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells.[show abstract] [hide abstract]
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.Nature Protocol 02/2011; 6(3):296-313. · 8.36 Impact Factor
Article: Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.[show abstract] [hide abstract]
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.Cell reports. 09/2012; 2(3):553-67.