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

During an immune response,activated antigen (Ag)-specific T cells condition dendritic cells (DCs) to enhance DC function and survival within the inflamed draining lymph node (LN). It has been difficult to ascertain the role of the tumor necrosis factor (TNF) superfamily member lymphotoxin-alphabeta (LTalphabeta) in this process because signaling through the LTbeta-receptor (LTbetaR) controls multiple aspects of lymphoid tissue organization. To resolve this,we have used an in vivo system where the expression of TNF family ligands is manipulated only on the Ag-specific T cells that interact with and condition Ag-bearing DCs. We report that LTalphabeta is a critical participant required for optimal DC function,independent of its described role in maintaining lymphoid tissue organization. In the absence of LTalphabeta or CD40L on Ag-specific T cells,DC dysfunction could be rescued in vivo via CD40 or LTbetaR stimulation,respectively,suggesting that these two pathways cooperate for optimal DC conditioning. View Publication

The neural crest is a source to produce multipotent neural crest stem cells that have a potential to differentiate into diverse cell types. The transcription factor SOX10 is expressed through early neural crest progenitors and stem cells in vertebrates. Here we report the generation of SOX10-Nano-lantern (NL) reporter human induced pluripotent stem cells (hiPS) by using CRISPR/Cas9 systems,that are beneficial to investigate the generation and maintenance of neural crest progenitor cells. SOX10-NL positive cells are produced transiently from hiPS cells by treatment with TGFβ inhibitor SB431542 and GSK3 inhibitor CHIR99021. We found that all SOX10-NL-positive cells expressed an early neural crest marker NGFR,however SOX10-NL-positive cells purified from differentiated hiPS cells progressively attenuate their NL-expression under proliferation. We therefore attempted to maintain SOX10-NL-positive cells with additional signaling on the plane and sphere culture conditions. These SOX10-NL cells provide us to investigate mass culture with neural crest cells for stem cell research. View Publication

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung,liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells,hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2,SOX17,HNF1B,members of the GATA family,and the surface receptor CXCR4. However,differentiation protocols are rarely 100% efficient. Here,we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations. View Publication

BACKGROUND AIMS: We evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte-colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m²) once daily,intravenously on day 1). We also investigated the relationship between the number of SSC(lo) CD45(dim) CD34(hi) cells,SSC(lo) ALDH(br) cells and engraftment. METHODS: Thirty patients (20 males and 10 females),who were candidates for autologous peripheral blood stem cell transplantation,were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n = 14),Hodgkin's lymphoma (n = 7),non-Hodgkin's lymphoma (n = 2),acute myloid leukemia (n = 2),chronic lymphocytic leukemia (n = 1) and germ cell testis tumor (n = 1). RESULTS: Numbers of SSC(lo) CD45(dim) CD34(hi) cells and SSC(lo) ALDH(br) cells were highly correlated in both peripheral blood and apheresis products (P textless 0.001). We could not find a relationship between the transplanted SSC(lo) CD45(dim) CD34(hi) cell dose or SSC(lo) ALDH(br) cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC(lo) CD45(dim) CD34(hi) cells were 5.40 × 10�?�/kg for neutrophil recovery and 7.22 x 10�?�/kg for platelet recovery. The optimal thresholds for SSC(lo) ALDH(br) cells were 6.53 x 10�?�/kg for neutrophil recovery and 8.72 x 10�?�/kg platelet recovery. CONCLUSIONS: According to our data,numbers of SSC(lo) ALDH(br) cells are in very good agreement with numbers of SSC(lo) CD45(dim) CD34(hi) cells and can be a predictor of stem cell mobilization. View Publication

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells,and overexpression of Hoxa-9 markedly expands hematopoietic stem cells,suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However,sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia,indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays,Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability,a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures,Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation,with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo. 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

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening. View Publication

BACKGROUND: Mesenchymal stromal cells are multipotent cells considered to be of great promise for use in regenerative medicine. However,the cell dose may be a critical factor in many clinical conditions and the yield resulting from the ex vivo expansion of mesenchymal stromal cells derived from bone marrow may be insufficient. Thus,alternative sources of mesenchymal stromal cells need to be explored. In this study,mesenchymal stromal cells were successfully isolated from second trimester amniotic fluid and analyzed for chromosomal stability to validate their safety for potential utilization as a cell therapy product. DESIGN AND METHODS: Mesenchymal stromal cells were expanded up to the sixth passage starting from amniotic fluid using different culture conditions to optimize large-scale production. RESULTS: The highest number of mesenchymal stromal cells derived from amniotic fluid was reached at a low plating density; in these conditions the expansion of mesenchymal stromal cells from amniotic fluid was significantly greater than that of adult bone marrow-derived mesenchymal stromal cells. Mesenchymal stromal cells from amniotic fluid represent a relatively homogeneous population of immature cells with immunosuppressive properties and extensive proliferative potential. Despite their high proliferative capacity in culture,we did not observe any karyotypic abnormalities or transformation potential in vitro nor any tumorigenic effect in vivo. CONCLUSIONS: Fetal mesenchymal stromal cells can be extensively expanded from amniotic fluid,showing no karyotypic abnormalities or transformation potential in vitro and no tumorigenic effect in vivo. They represent a relatively homogeneous population of immature mesenchymal stromal cells with long telomeres,immunosuppressive properties and extensive proliferative potential. Our results indicate that amniotic fluid represents a rich source of mesenchymal stromal cells suitable for banking to be used when large amounts of cells are required. View Publication

Current approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous secondary" somatic cells� View Publication

Breast cancer stem cells are a group of undifferentiated cells with self-renewal and multidifferentiation potential. Chemotherapeutic and radiotherapeutic resistance,hypoxic resistance,high tumorigenicity,high cell invasion,and metastatic abilities are characteristics of these cells,which are responsible for breast cancer recurrence. Therefore,the correct sorting and identification of breast cancer stem cells is a primary step for research in this field. This article briefly describes the recent progress on sorting and identification technologies for breast cancer stem cells. Sorting technologies include the side population technique,technologies that depend on cell surface markers,ALDEFLUOR assays,and in situ detection. Identification technologies include mammosphere cultures,limited dilution in vitro,and in-vivo animal models. This review provides an important reference for breast cancer stem cell research,which will explore new methods for the treatment of patients with breast cancer. View Publication

Natural T regulatory cells (nTregs) play a key role in inducing and maintaining immunological tolerance. Cell-based therapy using purified nTregs is under consideration for several conditions,but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells. We have established a method for isolation and ex vivo expansion of human nTregs from healthy blood donors for cellular therapy aimed at preventing allograft rejection in organ transplants. The Robosep instrument was used for initial nTreg isolation and rapamycin was included in the expansion phase of cell cultures. The resulting cell population exhibited a stable CD4(+)CD25(++bright)Foxp3(+) phenotype,had potent functional ability to suppress CD4(+)CD25(negative) T cells without evidence of conversion to effector T cells including TH17 cells,and manifested little to no production of pro-inflammatory cytokines upon in vitro stimulation. Boolean gating analysis of cytokine-expressing cells by flow cytometry for 32 possible profile end points revealed that 96% of expanded nTregs did not express any cytokine. From a single buffy coat,approximately 80 million pure nTregs were harvested after expansion under cGMP conditions; these cell numbers are adequate for infusion of approximately one million cells kg�?�¹ for cell therapy in clinical trials. View Publication

NK cells are key regulators of innate defense against mouse CMV (MCMV). Like NK cells,NKT cells also produce high levels of IFN-γ rapidly after MCMV infection. However,whether similar mechanisms govern activation of these two cell types,as well as the significance of NKT cells for host resistance,remain unknown. In this article,we show that,although both NKT and NK cells are activated via cytokines,their particular cytokine requirements differ significantly in vitro and in vivo. IL-12 is required for NKT cell activation in vitro but is not sufficient,whereas NK cells have the capacity to be activated more promiscuously in response to individual cytokines from innate cells. In line with these results,GM-CSF-derived dendritic cells activated only NK cells upon MCMV infection,consistent with their virtual lack of IL-12 production,whereas Flt3 ligand-derived dendritic cells produced IL-12 and activated both NK and NKT cells. In vivo,NKT cell activation was abolished in IL-12(-/-) mice infected with MCMV,whereas NK cells were still activated. In turn,splenic NK cell activation was more IL-18 dependent. The differential requirements for IL-12 and IL-18 correlated with the levels of cytokine receptor expression by NK and NKT cells. Finally,mice lacking NKT cells showed reduced control of MCMV,and depleting NK cells further enhanced viral replication. Taken together,our results show that NKT and NK cells have differing requirements for cytokine-mediated activation,and both can contribute nonredundantly to MCMV defense,revealing that these two innate lymphocyte subsets function together to fine-tune antiviral responses. View Publication