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

Gelatin coatings are effective in increasing the retention of MSCs injected into the heart and minimizing the damage from acute myocardial infarction (AMI),but early studies suffered from low fractions of the MSCs coated with gelatin. Biotinylation of the MSC surface is a critical first step in the gelatin coating process,and in this study,we evaluated the use of biotinylated cholesterol lipid insertion" anchors as a substitute for the covalent NHS-biotin anchors to the cell surface. Streptavidin-eosin molecules where eosin is our photoinitiator can then be bound to the cell surface through biotin-streptavidin affinity. The use of cholesterol anchors increased streptavidin density on the surface of MSCs further driving polymerization and allowing for an increased fraction of MSCs coated with gelatin (83{\%}) when compared to NHS-biotin (52{\%}). Additionally the cholesterol anchors increased the uniformity of the coating on the MSC surface and supported greater numbers of coated MSCs even when the streptavidin density was slightly lower than that of an NHS-biotin anchoring strategy. Critically this improvement in gelatin coating efficiency did not impact cytokine secretion and other critical MSC functions. Proper selection of the cholesterol anchor and the biotinylation conditions supports cellular function and densities of streptavidin on the MSC surface of up to {\~{}}105 streptavidin molecules/$\mu$m2 . In all these cholesterol anchors offer an effective path towards the formation of conformal coatings on the majority of MSCs to improve the retention of MSCs in the heart following AMI." View Publication

Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research,particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y),termed conditionally reprogrammed cell (CRC) technology,enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings,we then created six CF cell CRC populations,three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted,and cells were subcultured,without irradiated feeders plus Y,into air-liquid interface conditions in nonproprietary and proprietary Ultroser G-containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y,dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air-liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage,but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus,CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers. View Publication

The concept that bone marrow (BM)-derived cells participate in neural regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. We recently reported that the BM contains a highly mobile population of CXCR4+ cells that express mRNA for various markers of early tissue-committed stem cells (TCSCs),including neural TCSCs. Here,we report that these cells not only express neural lineage markers (beta-III-tubulin,Nestin,NeuN,and GFAP),but more importantly form neurospheres in vitro. These neural TCSCs are present in significant amounts in BM harvested from young mice but their abundance and responsiveness to gradients of motomorphogens,such as SDF-1,HGF,and LIF,decreases with age. FACS analysis,combined with analysis of neural markers at the mRNA and protein levels,revealed that these cells reside in the nonhematopoietic CXCR4+/Sca-1+/lin-/CD45 BM mononuclear cell fraction. Neural TCSCs are mobilized into the peripheral-blood following stroke and chemoattracted to the damaged neural tissue in an SDF-1-CXCR4-,HGF-c-Met-,and LIF-LIF-R-dependent manner. Based on these data,we hypothesize that the postnatal BM harbors a nonhematopoietic population of cells that express markers of neural TCSCs that may account for the beneficial effects of BM-derived cells in neural regeneration. View Publication

Human pluripotent stem cells (hPSCs) possess great value in the aspect of cellular therapies due to its self-renewal and potential to differentiate into all somatic cell types. A few defined synthetic surfaces such as polymers and adhesive biological materials conjugated substrata were established for the self-renewal of hPSCs. However,none of them was effective in the generation of human induced pluripotent stem cells (hiPSCs) and long-term maintenance of multiple hPSCs,and most of them required complicated manufacturing processes. Polydopamine has good biocompatibility,is able to form a stable film on nearly all solid substrates surface,and can immobilize adhesive biomolecules. In this manuscript,a polydopamine-mediated surface was developed,which not only supported the reprogramming of human somatic cells into hiPSCs under defined conditions,but also sustained the growth of hiPSCs on diverse substrates. Moreover,the proliferation and pluripotency of hPSCs cultured on the surface were comparable to Matrigel for more than 20 passages. Besides,hPSCs were able to differentiate to cardiomyocytes and neural cells on the surface. This polydopamine-based synthetic surface represents a chemically-defined surface extensively applicable both for fundamental research and cell therapies of hPSCs. View Publication

Transplantation of human neural progenitor cells (NPCs) into the brain or spinal cord to replace lost cells,modulate the injury environment,or create a permissive milieu to protect and regenerate host neurons is a promising therapeutic strategy for neurological diseases. Deriving NPCs from human fetal tissue is feasible,although problematic issues include limited sources and ethical concerns. Here we describe a new and abundant source of NPCs derived from human induced pluripotent stem cells (iPSCs). A novel chopping technique was used to transform adherent iPSCs into free-floating spheres that were easy to maintain and were expandable (EZ spheres) (Ebert et al. [2013] Stem Cell Res 10:417–427). These EZ spheres could be differentiated towards NPC spheres with a spinal cord phenotype using a combination of all-trans retinoic acid (RA) and epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) mitogens. Suspension cultures of NPCs derived from human iPSCs or fetal tissue have similar characteristics,although they were not similar when grown as adherent cells. In addition,iPSC-derived NPCs (iNPCs) survived grafting into the spinal cord of athymic nude rats with no signs of overgrowth and with a very similar profile to human fetal-derived NPCs (fNPCs). These results suggest that human iNPCs behave like fNPCs and could thus be a valuable alternative for cellular regenerative therapies of neurological diseases. J. Comp. Neurol. 522:2707–2728,2014. textcopyright 2014 Wiley Periodicals,Inc. View Publication

BACKGROUND: Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy,although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. METHODOLOGY/FINDINGS: Treatment of lung tumor cells with doxorubicin,cisplatin,or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133,CD117,SSEA-3,TRA1-81,Oct-4,and nuclear beta-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres,maintain self-renewal capacity,and differentiate. Differentiated progenitors lost expression of CD133,gained CK 8/18 and acquired drug sensitivity. In the presence of drugs,differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice,which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF,bFGF,IL-6,IL-8,HGF,PDGF-BB,G-CSF,and SCGF-beta). CSCs also showed elevated levels of expression of human VEGFR2,FGFR2,CXCR1,2 and 4 receptors. Moreover,human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy. View Publication

BACKGROUND: Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells,termed cancer stem cells (CSC),appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed,therefore,that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA(+)CD44(+) phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents,then residual tumors might be expected to contain a higher frequency of CoCSC. METHODS AND FINDINGS: Xenogeneic tumors initiated with CoCSC were allowed to reach approximately 400 mm(3),at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover,the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones,we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. CONCLUSIONS: CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC,major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes,thereby suggesting avenues for improving cancer therapy. View Publication