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

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR™1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel™-coated substrates. Mechanistically,it was resolved that bovine serum albumin (BSA) in the mTeSR™1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g.,peptides,proteins,or Matrigel™) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion. View Publication

Cell culture is one of the most common methods used to recapitulate a human disease environment in a laboratory setting. Cell culture techniques are used to grow and maintain cells of various types including those derived from primary tissues,such as stem cells and cancer tumors. However,a major confounding factor with cell culture is the use of serum and animal (xeno) products in the media. The addition of animal products introduces batch and lot variations that lead to experimental variability,confounds studies with therapeutic outcomes for cultured cells,and represents a major cost associated with cell culture. Here we report a commercially available serum-free,albumin-free,and xeno free (XF) media (Neuro-Pure(TM)) that is more cost-effective than other commercial medias. Neuro-Pure was used to maintain and differentiate various cells of neuronal lineages,fibroblasts,as well as specific cancer cell lines; without the use of contaminants such serum,albumin,and animal products. Neuro-Pure allows for a controlled and reproducible cell culture environment that is applicable to translational medicine and general tissue culture. View Publication

AIMS The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro,and test the effect of in vivo environments on the further maturation of hESCs derived cells. MAIN METHODS Embryoid bodies (EBs) formed from hESCs,with serum-free KSR-based medium and mesodermal specification related factors,CHIR,and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB,TGF β3,BMP4 in sequence for 24days. The morphology,FACS,histological staining as well as the expression of chondrogenic specific genes were detected in each stage,and further in vivo experiments,cell injections and tissue transplantations,further verified the formation of chondrocytes. KEY FINDINGS We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule,their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation. SIGNIFICANCE The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes,and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease. View Publication

Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk,a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability,clonogenic hematopoietic potential,and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells,together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel,the supportive cells self-assemble into a hypoxic stromal network,stimulating CD34+ CD38+ cell formation,while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks,in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly,the primary in vitro niche model supports HSPCs with no cytokine addition. Overall,the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches,in the context of normal hematopoiesis or blood-related diseases. View Publication

Retroviral overexpression of NF-Ya,the regulatory subunit of the transcription factor NF-Y,activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer,more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4,a target gene of NF-Ya,using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction,the proliferation of human CD34(+) cells in the presence of myeloid cytokines was increased 4-fold. Moreover,TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45(+) cells recovered from the bone marrow of sublethally irradiated,transplanted NOD-Scid IL2Rγ(null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy. View Publication

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells,although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need,we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and,in some cases,lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL-3),IL-6,and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean,27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony-forming unit-granulocyte-macrophage),as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418,averaged 11% and 12%,respectively,in 6 experiments. These values could be increased to 100% and 77%,respectively,by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%,respectively,in the CD34+HSA- cells. In addition,the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony-forming cells in the unsorted population was textless or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells,ie,those expressing little or no CD38,could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition,such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry. View Publication