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

Natural killer T (NKT) cells are innate-like lymphocytes that recognize lipid antigens and have been shown to enhance B-cell activation and antibody production. B cells typically recruit T-cell help by presenting internalized antigens recognized by their surface antigen receptor. Here,we demonstrate a highly efficient means whereby human B cells present lipid antigens to NKT cells,capturing the antigen using apolipoprotein E (apoE) and the low-density lipoprotein receptor (LDL-R). ApoE dramatically enhances B-cell presentation of alpha-galactosylceramide (alphaGalCer),an exogenous CD1d presented antigen,inducing activation of NKT cells and the subsequent activation of B cells. B cells express the LDL-R on activation,and the activation of NKT cells by B cells is completely LDL-R dependent,as shown by blocking experiments and the complete lack of presentation when using apoE2,an isoform of apoE incapable of LDL-R binding. The dependence on apoE and the LDL-R is much more pronounced in B cells than we had previously seen in dendritic cells,which can apparently use alternate pathways of lipid antigen uptake. Thus,B cells use an apolipoprotein-mediated pathway of lipid antigen presentation,which constitutes a form of innate help for B cells by NKT cells. View Publication

In this study,we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells,in serum-free medium,supplemented only with fibroblast growth factor (FGF)-1,FGF-2,or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays,high levels of stem cell activity were detectable at 1,3,and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However,cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant,showing that HSC activity originated from LSK cells. Subsequently,we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules,stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response,inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly,although HSC activity is typically rapidly lost after short-term culture in vitro,our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks. View Publication

Culturing human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) is one of the most costly and labor-intensive tissue cultures,as media containing expensive factors/cytokines should be changed every day to maintain and propagate undifferentiated hESCs/iPSCs in vitro. We recently reported that doxycycline,an anti-bacterial agent,had dramatic effects on hESC/iPSC survival and promoted self-renewal. In this study,we extended the effects of doxycycline to a more practical issue to save cost and labor in hESC/iPSC cultures. Regardless of cultured cell conditions,hESCs/iPSCs in doxycycline-supplemented media were viable and proliferating for at least 3 days without media change,while none or few viable cells were detected in the absence of doxycycline in the same conditions. Thus,hESCs/iPSCs supplemented with doxycycline can be cultured for a long period of time with media changes at 3-day intervals without altering their self-renewal and pluripotent properties,indicating that doxycycline supplementation can reduce the frequency of media changes and the amount of media required by 1/3. These findings strongly encourage the use of doxycycline to save cost and labor in culturing hESCs/iPSCs. View Publication

BCR-ABL and v-ABL are oncogenic forms of the Abl tyrosine kinase that can cause leukemias in mice and humans. ABL oncogenes trigger multiple signaling pathways whose contribution to transformation varies among cell types. Activation of phosphoinositide 3-kinase (PI3K) is essential for ABL-dependent proliferation and survival in some cell types,and global PI3K inhibitors can enhance the antileukemia effects of the Abl kinase inhibitor imatinib. Although a significant fraction of BCR-ABL-induced human leukemias are of B-cell origin,little is known about PI3K signaling mechanisms in B-lineage cells transformed by ABL oncogenes. Here we show that activation of class I(A) PI3K and downstream inactivation of FOXO transcription factors are essential for survival of murine pro/pre-B cells transformed by v-ABL or BCR-ABL. In addition,analysis of mice lacking individual PI3K genes indicates that products of the Pik3r1 gene contribute to transformation efficiency by BCR-ABL. These findings establish a role for PI3K signaling in B-lineage transformation by ABL oncogenes. View Publication

Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However,these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation,thereby limiting their range of applications. In this protocol,we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA). View Publication

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

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

OBJECTIVES Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney,stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources,pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However,little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study,we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system. MATERIALS AND METHODS We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak,intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR,real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility. RESULTS After modification of culture period and concentration of exogenous factors,hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2,GDNF,HOXD11,WT1 and CITED1 in addition to OSR1,PAX2,SALL1 and EYA1. Moreover,NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular,approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems. CONCLUSIONS Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases. View Publication

Pluripotent stem cells can be induced from somatic cells,providing an unlimited cell resource,with potential for studying disease and use in regenerative medicine. However,genetic manipulation and technically challenging strategies such as nuclear transfer used in reprogramming limit their clinical applications. Here,we show that pluripotent stem cells can be generated from mouse somatic cells at a frequency up to 0.2% using a combination of seven small-molecule compounds. The chemically induced pluripotent stem cells resemble embryonic stem cells in terms of their gene expression profiles,epigenetic status,and potential for differentiation and germline transmission. By using small molecules,exogenous master genes" are dispensable for cell fate reprogramming. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications." 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

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly,the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins,tamoxifen can efficiently induce Cre-mediated recombination,thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen,recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein,the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals. View Publication

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes,for drug discovery,and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional,defined and highly efficient protocol that avoids the use of feeder cells,serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells. View Publication