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In Vitro Generation of Human T Regulatory Cells: Generation, Culture, and Analysis of FOXP3-Transduced T Cells

T regulatory cells (Tregs) suppress immune responses and therefore have potential to be used in the clinic as a cellular therapy for autoimmune disease and to prevent rejection of transplanted organs. Obtaining sufficient numbers of these cells for therapeutic use is a challenge, however, ...

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Generation, Isolation, and Engraftment of In Vitro-Derived Human T Cell Progenitors

T cells typically differentiate via a series of coordinated steps within the highly specialized microenvironment of the thymus. Traditionally, human T-lymphopoiesis in vitro has been studied using the hybrid human/mouse fetal thymic organ culture system. Pioneering work by McCu ...

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The Development of T Lymphocytes in Fetal Thymus Organ Culture

Fetal thymus organ culture (FTOC) is a unique and powerful culture system that allows intrathymic T-lymphocyte development in vitro. T-cell development in FTOC well represents fetal thymocyte development in vivo. Here we describe the basic method for FTOC as well as several related techn ...

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Fluorescent In Situ Hybridization of DNA Probes in the Interphase and Metaphase Stages of the Cell Cycle

In the past decade, fluorescent in situ hybridization (FISH) has been used routinely in detecting molecular abnormalities in the interphase and metaphase stages of the cell cycle. Many of the molecular anomalies which are detected in this manner are diagnostic of a prenatal, postnatal, or ne ...

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Classical and Molecular Cytogenetic Analysis

Cytogenetic analysis is performed on cell cultures for several reasons, notably, to perform identity checks by verifying species of origin or the retention of key chromosome rearrangements in cell lines described previously. De novo chromosome analysis is usually performed when ch ...

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STR DNA Typing of Human Cell Lines: Detection of Intra- and Interspecies Cross-Contamination

Inter- and intraspecies cross-contaminations (CCs) of human and animal cells represent a chronic problem in cell cultures leading to false data. Microsatellite loci in the human genome harboring short tandem repeat (STR) DNA markers allow individualization of cell lines at the DNA level. ...

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Techniques in Embryoid Body Formation from Human Pluripotent Stem Cells

Embryoid bodies (EBs) can be generated by culturing human pluripotent stem cells in ultra-low attachment culture vessels, under conditions that are adverse to pluripotency and proliferation. EBs generated in suspension cultures are capable of differentiating into cells of the ect ...

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Formation of Embryoid Bodies from Human Pluripotent Stem Cells Using AggreWell Plates

Many human embryonic stem (hES) and induced pluripotent stem (hiPS) cell differentiation protocols begin with the formation of three-dimensional aggregates of cells called embryoid bodies (EBs). Traditional EB formation methods result in a heterogeneous population of EB sizes a ...

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Feeder-Independent Culture Systems for Human Pluripotent Stem Cells

The continued success of pluripotent stem cell research is ultimately dependent on access to reliable and defined reagents for the consistent culture and cryopreservation of undifferentiated, pluripotent cells. The development of defined and feeder-independent culture me ...

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Methods to Culture, Differentiate, and Characterize Neural Stem Cells from the Adult and Embryonic Mouse Central Nervous System

Since the discovery of neural stem cells (NSC) in the embryonic and adult mammalian central nervous system (CNS), there have been a growing numbers of tissue culture media and protocols to study and functionally characterize NSCs and its progeny in vitro. One of these culture systems introduced ...

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Eradication of Mycoplasma Contaminations

Mycoplasma contaminations have a multitude of effects on the cultured cell lines that may influence the results of experiments or pollute bioactive substances used in human medicine. The elimination of mycoplasma contaminations of cell cultures has become a practical alternative ...

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Hepatic Differentiation of Embryonic Stem Cells by Murine Fetal Liver Mesenchymal Cells

Hepatocytes derived from embryonic stem cells (ESCs) are a potential cell source for regenerative medicine. However, it has been technically difficult to differentiate ESCs into mature hepatocytes because the definitive growth factors and molecular mechanisms governing hep ...

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Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells

Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolat ...

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Cocultivation of Human Oral Keratinocytes and Human Osteoblast-Like Cells

Head and neck reconstruction transplants often require a bony structure but also tissue for the intraoral lining. This is why oral keratinocytes and osteoblast-like cells are essential cell types for combined tissue engineered transplants for defects in the field of craniomaxillof ...

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Isolation and Characterization of Human Hair Follicle Epithelial Cells

The mammalian hair follicle epithelial component contains various lineages of keratinocytes as well as their progenitor/stem cells. To characterize the subpopulations contained within this component and assess their functional capacity, the development of a feasible meth ...

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Enzymatic Dissociation, Flow Cytometric Analysis, and Culture of Normal Mouse Mammary Tissue

Evidence is emerging that the mouse mammary epithelium is arranged as a hierarchy that spans from stem cells to lineage-restricted progenitor cells to differentiated luminal and myoepithelial cells. The use of fluorescence-activated cell sorting (FACS) in combination with quant ...

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Human Prostate Epithelial Cell Cultures

Prostate cancer is the most common male cancer in the United States. Research on the mechanisms of prostate cancer progression has been limited by the lack of suitable in vitro systems. A hurdle in understanding the molecular genetic changes in prostate cancer has been the difficulty in establi ...

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In Vitro Methods to Culture Primary Human Breast Epithelial Cells

Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will pr ...

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Generation of a Pool of Human Platelet Lysate and Efficient Use in Cell Culture

Human platelets represent a promising source of bioactive substances as growth factors not just for in vivo wound healing and tissue repair, but also for the expansion of human stem and progenitor cells in vitro. The replacement of fetal bovine serum (FBS) as a standard culture supplement by human ...

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Isolation and Culture of Mesenchymal Stem Cells from Mouse Compact Bone

The bone marrow (BM) of numerous species, including rodents and man, contains a rare population of cells termed marrow stromal cells or mesenchymal stem cells (MSC). Given the ability of these cells to differentiate into cells of the osteogenic, chondrogenic and adipogenic lineages, there is ...

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