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

Myocardial infarction is a leading cause of mortality and morbidity worldwide,and current treatments fail to address the underlying scarring and cell loss,which is a major cause of heart failure after infarction. The novel strategy,therapeutic angiogenesis and/or vasculogenesis with endothelial progenitor cells transplantation holds great promise to increase blood flow in ischemic areas,thus rebuild the injured heart and reverse the heart failure. Given the potential of self-renewal and differentiation into virtually all cell types,human embryonic stem cells (hESCs) may provide an alternate source of therapeutic cells by allowing the derivation of large numbers of endothelial cells for therapeutic angiogenesis and/or vasculogenesis of ischemic heart diseases. Moreover,to fully understand the fate of implanted hESCs or hESC derivatives,investigators need to monitor the motility of cells in living animals over time. In this chapter,we describe the application of bioluminescence reporter gene imaging to track the transplanted hESC-derived endothelial cells for treatment of myocardial infarction. The technology of inducing endothelial cells from hESCs will also be discussed. View Publication

Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120h after tooth extraction,and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC,ending with development of procedures for banking. First,we aimed to optimize cryopreservation of established DPSC cultures,with regards to optimizing the cryoprotective agent (CPA),the CPA concentration,the concentration of cells frozen,and storage temperatures. Secondly,we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly,we evaluated the growth,surface markers and differentiation properties of DPSC obtained from intact teeth and undigested,whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me(2)SO at a concentration between 1 and 1.5M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen,at least up to 2x10(6) cells/mL. It was further established that DPSC can be stored at -85 degrees C or -196 degrees C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues,with digestion and culture performed post-thaw. A recovery of cells from textgreater85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free,defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions. View Publication

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies-containing from 3 to textgreater 100 Mk,detectable as glycoprotein IIb/IIIa+ cells in APAAP-stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions,the smallest Mk-containing colonies (CFC-Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC-Mk at 18-21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL-3 alone would support the formation of large numbers of CFC-Mk. However,optimal yields of colonies containing cells of both Mk and non-Mk lineages required the addition of other growth factors,of which a combination of IL-3,IL-6,GM-CSF and Steel factor (SF) +/- TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large pure' Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colonies became detectable. Flt3-ligand alone was unable to support the growth of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FACS-sorted sub-populations of CD34+ marrow cells in both serum-free agarose and methylcellulose assays demonstrated that most CFC-Mk are generated from CD34+ cells that are CD45RA- and CD71+� View Publication

Human embryonic stem cells (hESCs) have two properties of interest for the development of cell therapies: self-renewal and the potential to differentiate into all major lineages of somatic cells in the human body. Widespread clinical application of hESC-derived cells will require culture methods that are low-cost,robust,scalable and use chemically defined raw materials. Here we describe synthetic peptide-acrylate surfaces (PAS) that support self-renewal of hESCs in chemically defined,xeno-free medium. H1 and H7 hESCs were successfully maintained on PAS for over ten passages. Cell morphology and phenotypic marker expression were similar for cells cultured on PAS or Matrigel. Cells on PAS retained normal karyotype and pluripotency and were able to differentiate to functional cardiomyocytes on PAS. Finally,PAS were scaled up to large culture-vessel formats. Synthetic,xeno-free,scalable surfaces that support the self-renewal and differentiation of hESCs will be useful for both research purposes and development of cell therapies. View Publication

The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far,the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here,we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system,and utilized this approach to genotype mice from F0 to F2 generation,which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus,PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN. View Publication

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently,the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed,respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last,screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together,our results highlight the potential of hiPSCs for studying human tumourigenesis. View Publication

Human induced pluripotent stem cells (iPSCs) have great potential in research,but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here,we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD,NANOG,SPP1),endoderm (CER1,EOMES,GATA6),mesoderm (APLNR,HAND1,HOXB7),and ectoderm (HES5,PAMR1,PAX6). Using these genes,we develop a machine learning-based scoring system,“hiPSCore”,trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time,subjectivity,and resource use,thus enhancing iPSC quality for scientific and medical applications. Quality control,including pluripotency testing of human iPSCs lacks standardization. Here,authors identify and validate gene markers to develop the machine learning-based hiPSCore to streamline pluripotency testing and elevate iPSC quality. View Publication

IntroductionPlasmacytoid dendritic cells (pDCs) are capable of triggering broad immune responses,yet,their scarcity in blood coupled to their reduced functionality in cancer,makes their therapeutic use for in situ activation or vaccination challenging. MethodsWe designed an in vitro differentiation protocol tailored for human pDCs from cord blood (CB) hematopoietic stem cells (HSCs) with StemRegenin 1 (SR-1) and GM-CSF supplementation. Next,we evaluated the identity and function of CB-pDCs compared to human primary pDCs. Furthermore,we tested the potential of CB-pDCs to support anti-tumor immune responses in co-culture with tumor explants from CRC patients. ResultsHere,we report an in vitro differentiation protocol enabling the generation of 200 pDCs per HSC and highlight the role of GM-CSF and SR-1 in CB-pDC differentiation and function. CB-pDCs exhibited a robust resemblance to primary pDCs phenotypically and functionally. Transcriptomic analysis confirmed strong homology at both,baseline and upon TLR9 or TLR7 stimulation. Further,we could confirm the potential of CB-pDCs to promote inflammation in the tumor microenvironment by eliciting cytokines associated with NK and T cell recruitment and function upon TLR7 stimulation ex vivo in patient tumor explants. DiscussionThis study highlights CB-pDCs as surrogates for primary pDCs to investigate their biology and for their potential use as cell therapy in cancer. View Publication

Conventional human embryonic stem cells (hESCs) are capable of self-renewal and simultaneously poised for differentiation. But the mechanisms underlying this primed pluripotent state,which endows them with elevated responsiveness to differentiation cues,remain largely underexplored. Especially,little is known about the pivotal transcription factors (TFs) that orchestrate hESCs towards primed pluripotency. Here,we report a function of TF ZNF263 in pluripotency priming. Genetic and functional assays reveal that ZNF263 directly initiates the incipient expression of early differentiation genes and concurrently dampens the core pluripotency circuitry in hESCs,greatly tilting the balance from pluripotency maintenance to lineage priming. Importantly,ZNF263 deficiency markedly impairs pluripotency dissolution and multi-lineage differentiation in hESCs,particularly toward ectoderm. Moreover,single-cell transcriptomic profiling reveals that ZNF263 promotes the priming of cell fate commitment in hESCs,suggesting its indispensable requirement for pluripotency priming and lineage commitment continuum. Together,we demonstrate the role of ZNF263 in establishing the primed pluripotent state in hESCs and facilitating their differentiation into primary germ layer lineages. Human embryonic stem cells are simultaneously capable of self-renewal and poised for differentiation. Here,the authors show a role for the ZNF263 transcription factor promotes primed pluripotency and facilitates differentiation into primary germ layer lineages. View Publication