Retroviral transduction of hematopoietic progenitors derived from human embryonic stem cells.
Journal - Methods in molecular biology (Clifton, N.J.) (United States )
It has been recently identified that cytokines and BMP-4 promote hematopoiesis from human embryonic stem cells (hESC) and that, before hematopoietic commitment, a rare subpopulation of cells lacking CD45, but expressing PECAM-1, Flk-1, and VE-cadherin (hereinafter termed CD45(neg)PFV precursors), are exclusively responsible for hematopoietic cell fate on cytokine stimulation. Efficient strategies to stably transduce these hematopoietic precursors specifically generated from hESCs would provide a novel and desirable tool to study hematopoietic development through the introduction and characterization of candidate genes suspected to regulate self-renewal processes of hESC-derived hematopoietic cells or dynamically track hESC-derived hematopoietic stem cells in vivo. To date, only transient transfection and stable transduction using lentiviral vectors have been reported in undifferentiated hESC followed by random and spontaneous differentiation into different cell types. However, protocols for stable transduction of hematopoietic progenitors prospectively derived from hESC need to be developed yet. In the present chapter, we described detailed methods on the recently characterized and optimized GALV-pseudotyped retroviral gene transfer strategy to stably transduce the hematopoietic progenitor cells prospectively derived from CD45(neg)PFV hemogenic precursors as a vital tool to study hematopoietic development and to characterize candidate genes suspected to eventually confer robust and sustained repopulating ability to hESC-derived hematopoietic cells.
|ISSN : ||1064-3745|
|Mesh Heading : ||Antigens, CD Antigens, CD31 Antigens, CD45 Cadherins Cell Culture Techniques Cell Differentiation Cell Separation Flow Cytometry Gene Therapy Hematopoietic Stem Cells Humans Pluripotent Stem Cells Retroviridae Transduction, Genetic Vascular Endothelial Growth Factor Receptor-2 metabolism metabolism metabolism methods methods methods metabolism metabolism metabolism|
|Mesh Heading Relevant : ||cytology physiology cytology genetics methods|
Human embryonic stem cells: potential tool for achieving immunotolerance?
Journal - Stem cell reviews (United States )
The derivation of human embryonic stem cells (hESCs), whose in vitro differentiation might be directed toward different cell types, has raised the hope for cell replacement therapies. Despite the emerging reports to differentiate hESCs into specific lineages and then to distinct mature cell subsets, there are still several issues that need to be resolved before transplantation of these cells can be realized. In this context, immune rejection by the host immune system has been considered to be one of the greatest hurdles for cellular transplantation. However, recent data support the concept that hESCs and/or their differentiated derivatives possess immune-privileged properties, suggesting that cells derived from hESC may provide a potential tool for induction of immunetolerance. Currently, our understanding of the tolerogenic potential of hESCs is limited to assessment by in vitro assays or xenogenic transplantation approaches in vivo. Human ESCs express low levels of major histocompatability complex (MHC)-I antigens and lack expression of MHC-II antigens and costimulatory molecules, and are not recognized by natural killer cells and inhibit T-cell induced-stimulation by third-party antigen-presenting cells. Upon injection into immunocompetent mice, hESCs are unable to induce an immune response as demonstrated by their inability to induce an inflammatory response. Based on these initial observations, further studies in hESCs immunobiology are warranted and may reveal unique mechanisms that account for the immunological properties of hESCs. Here, we explore the prospect of using hESCs and their derivatives for immunomodulation and tolerance induction.
|ISSN : ||1550-8943|
|Mesh Heading : ||Animals Cell Differentiation Dendritic Cells Embryonic Stem Cells Humans Major Histocompatibility Complex T-Lymphocytes Transplantation Immunology cytology immunology cytology immunology cytology immunology|
|Mesh Heading Relevant : ||Immune Tolerance immunology|
Genetic manipulation of human embryonic stem cells: a system to study early human development and potential therapeutic applications.
Journal - Current gene therapy (Netherlands )
The successful derivation of human embryonic stem cell (hESC) lines by Thomson and colleagues [Thomson et al., 1998] provided a new area of investigation in both regenerative medicine and early human development. Fundamental study of the molecular and cellular mechanisms responsible for normal lineage development will rely on reproducible protocols to direct the differentiation of hESCs into specific lineages of interest and genetically manipulate both hESCs and their derivatives. Identifying standards for maintenance of hESCs, methods for controlled differentiation and genetic manipulation of hESCs and their derivatives will provide a foundation to explore their potential therapeutic use in cell and gene therapy. In the present review, our goal is to outline the latest advances in the field with particular focus on how hESCs and their derivatives can be genetically altered, how this may be useful in better understanding the cellular and molecular events of lineage differentiation, and how deregulation of these cellular processes may lead to abnormal development and disease.
|ISSN : ||1566-5232|
|Mesh Heading : ||Cell Differentiation Cells, Cultured Embryo, Mammalian Gene Expression Regulation, Developmental Gene Targeting Genes, Regulator Genetic Vectors Humans RNA Viruses Stem Cell Transplantation Stem Cells Transfection methods methods|
|Mesh Heading Relevant : ||Genetic Techniques Human Development cytology physiology physiology|
Retroviral transduction of hematopoietic cells differentiated from human embryonic stem cell-derived CD45(neg)PFV hemogenic precursors.
Journal - Molecular therapy : the journal of the American Society of Gene Therapy (United States )
Human embryonic stem cells (hESCs) provide a unique opportunity to study molecular mechanisms that regulate specification of the hematopoietic lineage in the human. Exploitation of this model using transgenic strategies depends on the ability to target cells of the hematopoietic lineage effectively and establish stable transgene expression. Here, a recently defined subpopulation of endothelial-like precursors derived from hESCs that is exclusively responsible for hematopoietic cell fate (CD45(neg)PFV) is shown to express GALVR-1 receptor and be efficiently transduced with GALV-pseudotyped retrovirus. Retroviral transduction, measured by enhanced green fluorescent protein, of hESC-derived CD45(+) cells differentiated from isolated CD45(neg)PFV precursors was 26.5 +/- 13% with 5.6 +/- 4% of these cells coexpressing CD34. An average of 17.5% of clonogenic hematopoietic progenitors derived from CD45(neg)PFV precursors expressed the retroviral transgene. Addition of serum to cultures after retroviral exposure supported transgene expression in resulting hematopoietic cells derived from hemogenic CD45(neg)PFV precursors. Our study represents the first report to demonstrate that retroviral transduction systems, similar to those used currently in clinical gene therapy protocols, are capable of efficient transduction of hematopoietic progenitors derived from hESCs.
|ISSN : ||1525-0016|
|Mesh Heading : ||Animals Antigens, CD45 Cattle Cell Line Cell Lineage Fetal Blood Gene Expression Hematopoietic Stem Cells Humans Kinetics Retroviridae Serum Transduction, Genetic drug effects drug effects drug effects physiology|
|Mesh Heading Relevant : ||Cell Differentiation analysis cytology metabolism cytology metabolism genetics|
Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression
Journal - Journal of Experimental Medicine
Despite the need for alternative sources of human hematopoieticstem cells (HSCs), the functional capacity of hematopoieticcells generated from human embryonic stem cells (hESCs) hasyet to be evaluated and compared with adult sources. Here, wereport that somatic and hESC-derived hematopoietic cells havesimilar phenotype and in vitro clonogenic progenitor activity.However, in contrast with somatic cells, hESC-derived hematopoieticcells failed to reconstitute intravenously transplanted recipientmice because of cellular aggregation causing fatal emboli formation.Direct femoral injection allowed recipient survival and resultedin multilineage hematopoietic repopulation, providing directevidence of HSC function. However, hESC-derived HSCs had limitedproliferative and migratory capacity compared with somatic HSCsthat correlated with a distinct gene expression pattern of hESC-derivedhematopoietic cells that included homeobox (HOX) A and B geneclusters. Ectopic expression of HOXB4 had no effect on repopulatingcapacity of hESC-derived cells. We suggest that limitationsin the ability of hESC-derived HSCs to activate a molecularprogram similar to somatic HSCs may contribute to their atypicalin vivo behavior. Our study demonstrates that HSCs can be derivedfrom hESCs and provides an in vivo system and molecular foundationto evaluate strategies for the generation of clinically transplantableHSC from hESC lines.
L. Wang and P. Menendez contributed equally to this work.