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

The authors surveyed whole-exome and RNA-sequencing data from 252 unique pluripotent stem cell lines,some of which are in the pipeline for clinical use,and found that approximately 5{\%} of cell lines had acquired mutations in the TP53 gene that allow mutant cells to rapidly outcompete non-mutant cells,but do not prevent differentiation. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

Pluripotency of embryonic stem cells (ESCs) is defined by their ability to differentiate into three germ layers and derivative cell types and is established by an interactive network of proteins including OCT4 (also known as POU5F1; ref. ),NANOG (refs ,),SOX2 (ref. ) and their binding partners. The forkhead box O (FoxO) transcription factors are evolutionarily conserved regulators of longevity and stress response whose function is inhibited by AKT protein kinase. FoxO proteins are required for the maintenance of somatic and cancer stem cells; however,their function in ESCs is unknown. We show that FOXO1 is essential for the maintenance of human ESC pluripotency,and that an orthologue of FOXO1 (Foxo1) exerts a similar function in mouse ESCs. This function is probably mediated through direct control by FOXO1 of OCT4 and SOX2 gene expression through occupation and activation of their respective promoters. Finally,AKT is not the predominant regulator of FOXO1 in human ESCs. Together these results indicate that FOXO1 is a component of the circuitry of human ESC pluripotency. These findings have critical implications for stem cell biology,development,longevity and reprogramming,with potentially important ramifications for therapy. View Publication

The supply of human hepatic stem cells (hHpSCs) and other hepatic progenitors has been constrained by the limited availability of liver tissues from surgical resections,the rejected organs from organ donation programs,and the need to use cells immediately. To facilitate accessibility to these precious tissue resources,we have established an effective method for serum-free cryopreservation of the cells,allowing them to be stockpiled and stored for use as an off-the-shelf product for experimental or clinical programs. The method involves use of buffers,some serum-free,designed for cryopreservation and further supplemented with hyaluronans (HA) that preserve adhesion mechanisms facilitating postthaw culturing of the cells and preservation of functions. Multiple cryopreservation buffers were found to yield high viabilities (80-90%) of cells on thawing of the progenitor cells. Serum-free CS10 supplemented with 0.05% hyaluronan proved the most effective,both in terms of viabilities of cells on thawing and in yielding cell attachment and formation of expanding colonies of cells that stably maintain the stem/progenitor cell phenotype. Buffers to which 0.05 or 0.1% HAs were added showed cells postthaw to be phenotypically stable as stem/progenitors,as well as having a high efficiency of attachment and expansion in culture. Success correlated with improved expression of adhesion molecules,particularly CD44,the hyaluronan receptor,E-cadherin,β4 integrin in hHpSCs,and β1 integrins in hepatoblasts. The improved methods in cryopreservation offer more efficient strategies for stem cell banking in both research and potential therapy applications. View Publication

Perhaps one of the most significant achievements in modern science is the discovery of human induced pluripotent stem cells (hiPSCs),which have paved the way for regeneration therapy using patients' own cells. Cardiomyocytes differentiated from hiPSCs (hiPSC-CMs) could be used for modelling patients with heart failure,for testing new drugs,and for cellular therapy in the future. However,the present cardiomyocyte differentiation protocols exhibit variable differentiation efficiency across different hiPSC lines,which inhibit the application of this technology significantly. Here,we demonstrate a novel myocyte differentiation protocol that can yield a significant,high percentage of cardiac myocyte differentiation (backslashtextgreater85%) in 2 hiPSC lines,which makes the fabrication of a human cardiac muscle patch possible. The established hiPSCs cell lines being examined include the transgene integrated UCBiPS7 derived from cord blood cells and non-integrated PCBC16iPS from skin fibroblasts. The results indicate that hiPSC-CMs derived from established hiPSC lines respond to adrenergic or acetylcholine stimulation and beat regularly for greater than 60 days. This data also demonstrates that this novel differentiation protocol can efficiently generate hiPSC-CMs from iPSC lines that are derived not only from fibroblasts,but also from blood mononuclear cells. View Publication

Importance Human motor neurons may be reliably derived from induced pluripotent stem cells (iPSCs). In vivo transplant studies of human iPSCs and their cellular derivatives are essential to gauging their clinical utility. Objective To determine whether human iPSC-derived motor neurons can engraft in an immunodeficient mouse model of sciatic nerve injury. Design,Setting,and Subjects This nonblinded interventional study with negative controls was performed at a biomedical research institute using an immunodeficient,transgenic mouse model. Induced pluripotent stem cell-derived motor neurons were cultured and differentiated. Cells were transplanted into 32 immunodeficient mice with sciatic nerve injury aged 6 to 15 weeks. Tissue analysis was performed at predetermined points after the mice were killed humanely. Animal experiments were performed from February 24,2015,to May 2,2016,and data were analyzed from April 7,2015,to May 27,2016. Interventions Human iPSCs were used to derive motor neurons in vitro before transplant. Main Outcomes and Measures Evidence of engraftment based on immunohistochemical analysis (primary outcome measure); evidence of neurite outgrowth and neuromuscular junction formation (secondary outcome measure); therapeutic effect based on wet muscle mass preservation and/or electrophysiological evidence of nerve and muscle function (exploratory end point). Results In 13 of the 32 mice undergoing the experiment,human iPSC-derived motor neurons successfully engrafted and extended neurites to target denervated muscle. Human iPSC-derived motor neurons reduced denervation-induced muscular atrophy (mean [SD] muscle mass preservation,54.2% [4.0%]) compared with negative controls (mean [SD] muscle mass preservation,33.4% [2.3%]) (P = .04). No electrophysiological evidence of muscle recovery was found. Conclusions and Relevance Human iPSC-derived motor neurons may have future use in the treatment of peripheral motor nerve injury,including facial paralysis. Level of Evidence NA. View Publication

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs),but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing,we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel,we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally,to demonstrate the scalable potential of these procedures,we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging,we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. View Publication

The purpose of this study was to examine the in vitro and in vivo osteogenic potential of human induced pluripotent stem cells (hiPSCs) against that of human bone marrow mesenchymal stem cells (hBMMSCs). Embryoid bodies (EBs),which were formed from undifferentiated hiPSCs,were dissociated into single cells and underwent osteogenic differentiation using the same medium as hBMMSCs for 14 days. Osteoinduced hiPSCs were implanted on the critical-size calvarial defects and long bone segmental defects in rats. The healing of defects was evaluated after 8 weeks and 12 weeks of implantation,respectively. Osteoinduced hiPSCs showed relatively lower and delayed in vitro expressions of the osteogenic marker COL1A1 and bone sialoprotein,as well as a weaker osteogenic differentiation through alkaline phosphatase staining and mineralization through Alizarin red staining compared with hBMMSCs. Calvarial defects treated with osteoinduced hiPSCs had comparable quality of new bone formation,including full restoration of bone width and robust formation of trabeculae,to those treated with hBMMSCs. Both osteoinduced hiPSCs and hBMMSCs persisted in regenerated bone after 8 weeks of implantation. In critical-size long bone segmental defects,osteoinduced hiPSC treatment also led to healing of segmental defects comparable to osteoinduced hBMMSC treatment after 12 weeks. In conclusion,despite delayed in vitro osteogenesis,hiPSCs have an in vivo osteogenic potential as good as hBMMSCs. View Publication

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II,the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature,fetal liver-like erythroblasts,that are smaller,express mostly fetal hemoglobin,and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated,and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary,because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA. View Publication