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

A method for the generation of human induced pluripotent stem (iPS) cells was established. This method employs adenovirus carrying the ecotropic retrovirus receptor mCAT1 and Moloney murine leukemia virus (MMLV)-based retroviral vectors carrying the four transcription factors POU5F1 (OCT3/4),KLF4,SOX2,and MYC (c-Myc) (Masaki H & Ishikawa T Stem Cell Res 1:105-15,2007). The differentiation of human iPS cells into hepatic cells was performed by a stepwise protocol (Song Z et al. Cell Res 19:1233-42,2009). These cells have potential as patient-specific in vitro models for studying disease etiology and could be used in drug discovery programs tailored to deal with genetic variations in drug efficacy and toxicity. View Publication

Human induced pluripotent stem cells (HIPSC) have tremendous value as a source of autologous cells for cellular transplantation in the treatment of degenerative diseases. The protocols described here address methods for large-scale genetic modification of HIPSCs. The first is an optimized method for transfecting HIPSCs cultured in feeder-free conditions. The second method allows nucleofection of trypsinized HIPSCs at an optimal cell density. Both methods enable robust generation of stable HIPSC transfectants within two weeks. Our protocols are highly reproducible and do not require optimization for individual HIPSC and human embryonic stem cell (HESC) lines. View Publication

Human mesenchymal stem cells are thought to be multipotent cells,which are present in adult marrow,that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues,including bone,cartilage,fat,tendon,muscle,and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic,chondrocytic,or osteocytic lineages. Individual stem cells were identified that,when expanded to colonies,retained their multilineage potential. View Publication

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system,hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1,which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype,stable proliferation rate,and high telomerase activity. Similar to cells cultured on feeders,hES cells maintained under feeder-free conditions expressed OCT-4,hTERT,alkaline phosphatase,and surface markers including SSEA-4,Tra 1-60,and Tra 1-81. In addition,hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus,the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production. View Publication

BACKGROUND Emerging evidence of antibody-independent functions,as well as the clinical efficacy of anti-CD20 depleting therapies,helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis. OBJECTIVE To investigate whether CD19+ B cells may share expression of the serine-protease granzyme-B (GzmB),resembling classical cytotoxic CD8+ T lymphocytes,in the peripheral blood from relapsing-remitting MS (RRMS) patients. METHODS In this study,104 RRMS patients during different treatments and 58 healthy donors were included. CD8,CD19,Runx3,and GzmB expression was assessed by flow cytometry analyses. RESULTS RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8+GzmB+ T lymphocytes when compared to healthy volunteers. An increase in circulating CD19+GzmB+ B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA),untreated RRMS patients,and healthy donors but not when compared to interferon-$\beta$ (IFN). Moreover,regarding Runx3,the transcriptional factor classically associated with cytotoxicity in CD8+ T lymphocytes,the expression of GzmB was significantly higher in CD19+Runx3+-expressing B cells when compared to CD19+Runx3- counterparts in RRMS patients. CONCLUSIONS CD19+ B cells may exhibit cytotoxic behavior resembling CD8+ T lymphocytes in MS patients during different treatments. In the future,monitoring cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions." View Publication

The origin and function of human double negative (DN) TCR-alphabeta+ T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. In this study,we provide evidence that human TCR-alphabeta+ CD4- CD8- DN T cells can derive from activated CD8+ T cells. Freshly isolated TCR-alphabeta+ DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells produce a defined array of proinflammatory mediators that includes IL-1beta,IL-17,IFN-gamma,CXCL3,and CXCL2. These results indicate that,upon activation,CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity. View Publication

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC),we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34(+) cells. Treatment of PMF CD34(+) cells with chromatin-modifying agents (CMAs) but not hydroxyurea,Janus kinase 2 (JAK2) inhibitors,or low doses of interferon-α led to the generation of greater numbers of CD34(+) chemokine (C-X-C motif) receptor (CXCR)4(+) cells,which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F(+). Furthermore,sequential treatment of PMF CD34(+) cells but not normal CD34(+) cells with decitabine (5-aza-2'-deoxycytidine [5azaD]),followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA),or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F(+) PMF CD34(+) cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγ(null) mice,the percentage of JAK2V617F/JAK2(total) in human CD45(+) marrow cells was dramatically reduced. These findings suggest that both PMF HPCs,short-term and long-term SCID repopulating cells (SRCs),are JAK2V617F(+) and that JAK2V617F(+) HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs,therefore,represents a possible effective means of treating PMF at the level of the malignant SRC. View Publication

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However,the well-known limitations of current reprogramming technologies include low efficiency,slow kinetics,and transgene integration and residual expression. In the present study,we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient,occurs 1-3 weeks faster compared with the reprogramming of fibroblasts,and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR,indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9,blood-derived iPSCs could be differentiated back to the blood cells,albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods. View Publication

Transcripts expressed in cytotoxic T lymphocytes (CTL) have mechanistic and diagnostic importance in transplantation. We used microarrays to select CTL-associated transcripts (CATs) expressed in human CD4(+) CTL,CD8(+) CTL and NK cells,excluding transcripts expressed in B cells,monocytes and kidney. This generated three transcript sets: CD4(+)-associated,CD8(+)-associated and NK-associated. Surprisingly,many CATs were expressed in effector memory cells e.g. granzyme B/GZMB,interferon-gamma/IFNG. Transcript expression was very similar between CD4(+) and CD8(+) CTL. There were no transcripts highly selective for CD4(+) CTL or CD8(+) CTL: for example,cytotoxic molecule transcripts (perforin,granzymes,granulysin) were shared between CD8(+) CTL and CD4(+) CTL although expression remained higher in CD8(+) CTL. Transcripts that differentiated between CD8(+) CTL and CD4(+) CTL were primarily those shared between CD8(+) CTL and NK cells (e.g. NK receptors KLRC1,KLRC3,KLRD1,KLRK1). No transcripts could differentiate CD4(+) CTL from CD8(+) CTL but NK cell-associated transcripts could differentiate NK cells from CTL. This study serves as a foundation for the interpretation of CATs in rejecting allografts and highlights the extensive sharing of CATs among CD4(+) CTL,CD8(+) CTL and effector memory T cells. View Publication

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here,we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA,12.5% of cell colonies demonstrated CCR5 editing,of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells,we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells,including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication,macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro,and generation of HIV-resistant cells for potential therapeutic applications. View Publication

The existence of a hyaluronic acid-rich node and duct system (HAR-NDS) within the lymphatic and blood vessels was demonstrated previously. The HAR-NDS was enriched with small (3.0-5.0 μm in diameter),adult stem cells with properties similar to those of the very small embryonic-like stem cells (VSELs). Sca-1(+)Lin(-)CD45(-) cells were enriched approximately 100-fold in the intravascular HAR-NDS compared with the bone marrow. We named these adult stem cells node and duct stem cells (NDSCs)." NDSCs formed colonies on C2C12 feeder layers were positive for fetal alkaline phosphatase and could be subcultured on the feeder layers. NDSCs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(+) while VSELs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(-). NDSCs had higher sphere-forming efficiency and proliferative potential than VSELs and they were found to differentiate into neuronal cells in vitro. Injection of NDSCs into mice partially repaired ischemic brain damage. Thus we report the discovery of potential adult stem cells that may be involved in tissue regeneration. The intravascular HAR-NDS may serve as a route that delivers these stem cells to their target tissues. View Publication

It is widely accepted that canonical Wnt (cWnt) signaling is required for the differentiation of osteoprogenitors into osteoblasts. Furthermore,tumor-derived secretion of the cWnt-antagonist Dickkopf-1 (Dkk-1) is known to cause bone destruction,inhibition of repair and metastasis in many bone malignancies,but its role in osteosarcoma (OS) is still under debate. In this study,we examined the role of Dkk-1in OS by engineering its overexpression in the osteochondral sarcoma line MOS-J. Consistent with the known role of Dkk-1 in osteoblast differentiation,Dkk-1 inhibited osteogenesis by the MOSJ cells themselves and also in surrounding tissue when implanted in vivo. Surprisingly,Dkk-1 also had unexpected effects on MOSJ cells in that it increased proliferation and resistance to metabolic stress in vitro and caused the formation of larger and more destructive tumors than controls upon orthotopic implantation. These effects were attributed in part to upregulation of the stress response enzyme and cancer stem cell marker aldehyde-dehydrogenase-1 (ALDH1). Direct inhibition of ALDH1 reduced viability under stressful culture conditions,whereas pharmacological inhibition of cWnt or overexpression of ALDH1 had a protective effect. Furthermore,we observed that ALDH1 was transcriptionally activated in a c-Jun-dependent manner through a pathway consisting of RhoA,MAP-kinase-kinase-4 and Jun N-terminal Kinase (JNK),indicating that noncanonical planar cell polarity-like Wnt signaling was the mechanism responsible. Together,our results therefore demonstrate that Dkk-1 enhances resistance of OS cells to stress by tipping the balance of Wnt signaling in favor of the non-canonical Jun-mediated Wnt pathways. In turn,this results in transcriptional activation of ALDH1 through Jun-responsive promoter elements. This is the first report linking Dkk-1 to tumor stress resistance,further supporting the targeting of Dkk-1 not only to prevent and treat osteolytic bone lesions but also to reduce numbers of stress-resistant tumor cells. View Publication