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

The discovery of induced pluripotent stem cells(iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however,the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here,we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore,we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells' propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests,including comprehensive tumorigenicity assays and genomic integrity validation,should be rigorously executed before the clinical application of HiPSCs. In addition,HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability. View Publication

Human tumor vessels express tumor vascular markers (TVM),proteins that are not expressed in normal blood vessels. Antibodies targeting TVMs could act as potent therapeutics. Unfortunately,preclinical in vivo studies testing anti-human TVM therapies have been difficult to do due to a lack of in vivo models with confirmed expression of human TVMs. We therefore evaluated TVM expression in a human embryonic stem cell-derived teratoma (hESCT) tumor model previously shown to have human vessels. We now report that in the presence of tumor cells,hESCT tumor vessels express human TVMs. The addition of mouse embryonic fibroblasts and human tumor endothelial cells significantly increases the number of human tumor vessels. TVM induction is mostly tumor-type-specific with ovarian cancer cells inducing primarily ovarian TVMs,whereas breast cancer cells induce breast cancer specific TVMs. We show the use of this model to test an anti-human specific TVM immunotherapeutics; anti-human Thy1 TVM immunotherapy results in central tumor necrosis and a three-fold reduction in human tumor vascular density. Finally,we tested the ability of the hESCT model,with human tumor vascular niche,to enhance the engraftment rate of primary human ovarian cancer stem-like cells (CSC). ALDH(+) CSC from patients (n = 6) engrafted in hESCT within 4 to 12 weeks whereas none engrafted in the flank. ALDH(-) ovarian cancer cells showed no engraftment in the hESCT or flank (n = 3). Thus,this model represents a useful tool to test anti-human TVM therapy and evaluate in vivo human CSC tumor biology. View Publication

Stem cell-derived ?-cells (SC-BCs) represent a potential source for curing diabetes. To date,in vitro generated SC-BCs display an immature phenotype and lack important features in comparison to their bona-fide counterparts. Transplantation into a living animal promotes SC-BCs maturation,indicating that components of the in vivo microenvironment trigger final SC-BCs development. Here,we investigated whether cues of the pancreas specific extracellular matrix (ECM) can improve the differentiation of human induced pluripotent stem cells (hiPSCs) towards ?-cells in vitro. To this aim,a pancreas specific ECM (PanMa) hydrogel was generated from decellularized porcine pancreas and its effect on the differentiation of hiPSC-derived pancreatic hormone expressing cells (HECs) was tested. The hydrogel solidified upon neutralization at 37 °C with gelation kinetics similar to Matrigel. Cytocompatibility of the PanMa hydrogel was demonstrated for a culture duration of 21 days. Encapsulation and culture of HECs in the PanMa hydrogel over 7 days resulted in a stable gene and protein expression of most ?-cell markers,but did not improve ?-cell identity. In conclusion,the study describes the production of a PanMa hydrogel,which provides the basis for the development of ECM hydrogels that are more adapted to the demands of SC-BCs. View Publication

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study,we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4,hSox2,hKlf4,and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci,a hallmark of DM1,were detected in DM1 iPSCs,neural stem cells (NSCs),and terminally differentiated neurons and astrocytes. In conclusion,we have successfully established disease-specific human DM1 iPSC lines,NSCs,and neuronal lineages with pathognomonic intranuclear RNA foci,which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development. View Publication

Dominant inhibitory receptors for HLA class I (HLA-I) endow NK cells with high intrinsic responsiveness,a process termed licensing or education,but hinder their ability to kill HLA-I+ tumor cells. Cancer immunotherapy with adoptive transfer of NK cells must overcome inhibitory signals by such receptors to promote elimination of HLA-I+ tumor cells. As proof of concept,we show here that a chimeric antigen receptor (CAR) can be engineered to overcome inhibition by receptors for HLA-I and to promote lysis of HLA-I+ tumor cells by CAR-NK cells. The design of this NK-tailored CAR (NK-CAR) relied on the potent NK cell activation induced by the synergistic combination of NK receptors CD28H (CD28 homolog,TMIGD2) and 2B4 (CD244,SLAMF4). An NK-CAR consisting of the single-chain fragment variable (scFv) of a CD19 antibody,the CD28H transmembrane domain,and the fusion of CD28H,2B4,and TCR$\zeta$ signaling domains was compared to a third-generation T-cell CAR with a CD28-41BB-TCR$\zeta$ signaling domain. The NK-CAR delivered stronger activation signals to NK cells and induced more robust tumor cell lysis. Furthermore,such CAR-NK cells could overcome inhibition by HLA-E or HLA-C expressed on tumor cells. Therefore,engineering of CAR-NK cells that could override inhibition by HLA-I in patients undergoing cancer immunotherapy is feasible. This approach offers an attractive alternative to more complex strategies,such as genetic editing of inhibitory receptors in CAR-NK cells or treatment of patients with a combination of CAR-NK cells and checkpoint blockade with antibodies to inhibitory receptors. A significant benefit of inhibition-resistant NK-CARs is that NK cell inhibition would be overcome only during contact with targeted tumor cells and that HLA-I on healthy cells would continue to maintain NK cell responsiveness through licensing. View Publication

BACKGROUND: Persistent mixed chimerism represents a state in which recipient and donor cells stably co-exist after hematopoietic stem cell transplantation. However,since in most of the studies reported in literature the engraftment state was observed in the nucleated cells,in this study we determined the donor origin of the mature erythrocytes of patients with persistent mixed chimerism after transplantation for hemoglobinopathies. Results were compared with the engraftment state observed in singly picked out burst-forming unit - erythroid colonies and in the nucleated cells collected from the peripheral blood and from the bone marrow. DESIGN AND METHODS: The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocyte suspension after incubation with anti-ABO and/or anti-C,-c,-D,-E and -e monoclonal antibodies by a flow cytometer. Analysis of short tandem repeats was used to determine the donor origin of nucleated cells and burst-forming unit - erythroid colonies singly picked out after 14 days of incubation. RESULTS: The proportions of donor-derived nucleated cells in four transplanted patients affected by hemoglobinopathies were 71%,46%,15% and 25% at day 1364,1385,1314 and 932,respectively. Similar results were obtained for the erythroid precursors,analyzing the donor/recipient origin of the burst-forming unit - erythroid colonies. In contrast,on the same days of observation,the proportions of donor-derived erythrocytes in the four patients with persistent mixed chimerism were 100%,100%,73% and 90%. Conclusions Our results showed that most of the erythrocytes present in four long-term transplanted patients affected by hemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient for clinical control of the disease in patients affected by hemoglobinopathies is relevant,although the biological mechanisms underlying these observations need further investigation. View Publication

It has been shown that genetic inhibition of p53 leads to enhanced proliferation of hematopoietic stem cells (HSCs). This could,in theory,contribute to the increased frequency of tumor development observed in p53-deficient mice and humans. In our previous work,we identified chemical p53 inhibitors (PFTs) that suppress the transactivation function of p53 and protect cultured cells and mice from death induced by gamma irradiation (IR). Here we found that when applied to bone marrow cells in vitro or injected into mice,PFTb impeded IR-induced reduction of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) population sizes. In addition,we showed that PFTb stimulated HSC and HPC proliferation in the absence of IR in vitro and in vivo and mobilized HSCs to the peripheral blood. Importantly,however,PFTb treatment did not affect the timing or frequency of tumor development in irradiated p53 heterozygous mice used as a model for determination of carcinogenicity. Thus,although PFTb administration led to increased numbers of HSCs and HPCs,it was not carcinogenic in mice. These findings suggest that chemical p53 inhibitors may be clinically useful as safe and effective stimulators of hematopoiesis. View Publication

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study,we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of {\textgreater}50 small molecules were developed using structure-activity-relationship studies of SB203580,a known p38-MAPK inhibitor. A particular analog,C7,resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p {\textless} .001). In depth phenotypic analysis revealed {\textgreater}600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p {\textless} .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p {\textless} .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion,a small molecule,C7,could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393. View Publication

Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here,we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks,enhancing the reconstitution capacity of edited HSPCs. However,this results in lower HDR efficiency,rendering ex vivo culture necessary yet detrimental. Mechanistically,ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation,which generates mitogenic reactive oxygen species (ROS),promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus,p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs,ultimately endowing edited cells with superior multi-lineage differentiation,persistence throughout serial transplantation,enhanced polyclonal repertoire,and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications. View Publication

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB-HSCs) in transplantation,however,is the low numbers of HSCs in a unit of cord blood. To overcome this limitation,various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study,we investigated a synergistic effect of the combination of HIL-6,SR1,and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34+ cells. These results suggest that the combination of SR1,UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus,SR1,UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation. View Publication

Serum response factor (Srf) is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1,a cofactor of Srf,is part of the t(1;22) translocation in acute megakaryoblastic leukemia,and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development,we crossed Pf4-Cre mice,which express Cre recombinase in cells committed to the megakaryocytic lineage,to Srf(F/F) mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/Srf(F/F) knockout (KO) mice are born with normal Mendelian frequency,but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast,the BM has increased number and percentage of CD41(+) megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation,and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus,Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes. View Publication

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support,but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study,five separate laboratories,each with experience in human embryonic stem cell culture,used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods,with propagation in the presence of Knockout Serum Replacer,FGF-2,and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment,death,and differentiated morphology by phase contrast microscopy,for growth by serial cell counts,and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems,only the control and those based on two commercial media,mTeSR1 and STEMPRO,supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment,cell death,or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study,and the lack of success with other formulations from academic groups compared to previously published results,include: the complex combination of growth factors present in the commercial preparations; improved development,manufacture,and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. View Publication