搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Adhesion properties of hematopoietic stem cells (HSCs) in the bone marrow (BM) niches control their migration and affect their cell-cycle dynamics. The serum response factor (Srf) regulates growth factor-inducible genes and genes controlling cytoskeleton structures involved in cell spreading,adhesion,and migration. We identified a role for Srf in HSC adhesion and steady-state hematopoiesis. Conditional deletion of Srf in BM cells resulted in a 3-fold expansion of the long- and short-term HSCs and multipotent progenitors (MPPs),which occurs without long-term modification of cell-cycle dynamics. Early differentiation steps to myeloid and lymphoid lineages were normal,but Srf loss results in alterations in mature-cell production and severe thrombocytopenia. Srf-null BM cells also displayed compromised engraftment properties in transplantation assays. Gene expression analysis identified Srf target genes expressed in HSCs,including a network of genes associated with cell migration and adhesion. Srf-null stem cells and MPPs displayed impair expression of the integrin network and decreased adherence in vitro. In addition,Srf-null mice showed increase numbers of circulating stem and progenitor cells,which likely reflect their reduced retention in the BM. Altogether,our results demonstrate that Srf is an essential regulator of stem cells and MPP adhesion,and suggest that Srf acts mainly through cell-matrix interactions and integrin signaling. View Publication

In the CNS,oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust,fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined,growth factor-rich medium. Within 8 d,PSCs differentiate into paired box 6-positive (PAX6(+)) neural stem cells,which give rise to OLIG2(+) progenitors by day 12. Oligodendrocyte lineage transcription factor 2-positive (OLIG2(+)) cells begin to express the transcription factor NKX2.2 around day 18,followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4(+)) appear around day 50 and reach,on average,43% of the cell population after 75 d of differentiation. O4(+) OPCs can be isolated by cell sorting for myelination studies,or they can be terminally differentiated to myelin basic protein-positive (MBP(+)) oligodendrocytes. This protocol also describes an alternative strategy for markedly reducing the length and the costs of the differentiation and generating ∼30% O4(+) cells after only 55 d of culture. View Publication

Transplantation of ex vivo expanded corneal limbal stem cells (LSCs) has been the main treatment for limbal stem cell deficiency,although the shortage of donor corneal tissues remains a major concern for its wide application. Due to the development of tissue engineering,embryonic stem cells (ESCs)-derived corneal epithelial-like cells (ESC-CECs) become a new direction for this issue. However,the immunogenicity of ESC-CECs is a critical matter to be solved. In the present study,we explored the immunological properties of ESC-CECs,which were differentiated from ESCs. The results showed that ESC-CECs had a similar character and function with LSCs both in vitro and in vivo. In ESC-CECs,a large number of genes related with immune response were down-regulated. The expressions of MHC-I,MHC-II,and co-stimulatory molecules were low,but the expression of HLA-G was high. The ESC-CECs were less responsible for T cell proliferation and NK cell lysis in vitro,and there was less immune cell infiltration after transplantation in vivo compared with LSCs. Moreover,the immunological properties were not affected by interferon-$$. All these results indicated a low immunogenicity of ESC-CECs,and they can be promising in clinical use. View Publication

Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article,we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule,E-cadherin,to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin,but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase,suggesting that E-cadherin promotes rapid neuronal differentiation. Further,these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin,neurofilament,and neural cell adhesion molecule. Moreover,blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation,we are able to utilize a specific cell-cell adhesion molecule,E-cadherin,to influence the nature and degree of neural specialization. View Publication

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently,differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately,differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether,our results provide a new platform for the study of kidney diseases and lineage commitment,and open new avenues for the future application of regenerative strategies in the clinic. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. View Publication

NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases,their role in immunity to Trypanosoma congolense has not been investigated. NK cells are vital sources of IFN-gamma and TNF-alpha; two key cytokines that are known to play important roles in resistance to African trypanosomes. In this article,we show that infection with T. congolense leads to increased levels of activated and functional NK cells in multiple tissue compartments. Systemic depletion of NK cells with anti-NK1.1 mAb led to increased parasitemia,which was accompanied by significant reduction in IFN-gamma production by immune cells in the spleens and liver of infected mice. Strikingly,infected NFIL3-/- mice (which genetically lack NK cell development and function) on the normally resistant background were highly susceptible to T. congolense infection. These mice developed fulminating and uncontrolled parasitemia and died significantly earlier (13 ± 1 d) than their wild-type control mice (106 ± 26 d). The enhanced susceptibility of NFIL3-/- mice to infection was accompanied by significantly impaired cytokine (IFN-gamma and TNF-alpha) response by CD3+ T cells in the spleens and liver. Adoptive transfer of NK cells into NFIL3-/- mice before infection rescued them from acute death in a perforin-dependent manner. Collectively,these studies show that NK cells are critical for optimal resistance to T. congolense,and its deficiency leads to enhanced susceptibility in infected mice. View Publication

X-ALD is an inherited neurodegenerative disorder where mutations in the ABCD1 gene result in clinically diverse phenotypes: the fatal disorder of cerebral childhood ALD (cALD) or a milder disorder of adrenomyeloneuropathy (AMN). The various models used to study the pathobiology of X-ALD disease lack the appropriate presentation for different phenotypes of cALD vs AMN. This study demonstrates that induced pluripotent stem cells (IPSC) derived brain cells astrocytes (Ast),neurons and oligodendrocytes (OLs) express morphological and functional activities of the respective brain cell types. The excessive accumulation of saturated VLCFA,a hallmark" of X-ALD� View Publication

Human pluripotent stem cells (PSCs),which include human embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs),represent an important source of cellular therapies in regenerative medicine and the study of early human development. As such,it is becoming increasingly important to develop methods for the large-scale banking of human PSC lines. There are several well-established methods for the propagation of human PSCs. The key to development of a good manufacturing practice (GMP) bank is to determine a manufacturing method that is amenable to large-scale production using materials that are fully documented. We have developed several banks of hESCs using animal feeder cells,animal-based matrices,or animal-free matrices. Protocols for growing hESCs on mouse embryonic fibroblasts (MEFs) are well established and are very helpful for producing research grade banks of cells. As most human ESCs cultured by research laboratories have been exposed to xenogeneic reagents,it is not imperative that all materials used in the production of a master cell bank be animal-free in origin. Nevertheless,as the field develops,it will no doubt become increasingly important to produce a bank of cells for clinical use without xenogeneic reagents,particularly nonhuman feeder cells which might harbor viruses with potential risk to human health or cell product integrity. Thus,even for cell lines previously exposed to xenogeneic reagents,it is important to minimize any subsequent exposure of the cell lines to additional adventitious agents. We have specifically described procedures for the growth of hESCs on Matrigel,an animal-matrix,and CELLstart,an animal-free matrix,and these can be used to produce hESCs as part of a clinical manufacturing process. View Publication