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

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However,techniques to generate cell type-specific lineage reporters,as well as reliable tools to disrupt,repair or overexpress genes by gene targeting,are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First,using ZFNs specific for the OCT4 (POU5F1) locus,we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second,we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally,we targeted the PITX3 gene,demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. View Publication

Mitochondria are critical to neurogenesis,but the mechanisms of mitochondria in neurogenesis have not been well explored. We fully characterized mitochondrial alterations and function in relation to the development of human induced pluripotent stem cell (hiPSC)-derived dopaminergic (DA) neurons. Following directed differentiation of hiPSCs to DA neurons,mitochondria in these neurons exhibit pronounced changes during differentiation,including mature neurophysiology characterization and functional synaptic network formation. Inhibition of mitochondrial respiratory chains via application of complex IV inhibitor KCN (potassium cyanide) or complex I inhibitor rotenone restricted neurogenesis of DA neurons. These results demonstrated the direct importance of mitochondrial development and bioenergetics in DA neuronal differentiation. Our study also provides a neurophysiologic model of mitochondrial involvement in neurogenesis,which will enhance our understanding of the role of mitochondrial dysfunctions in neurodegenerative diseases. View Publication

Coxsackie virus and adenovirus receptor,CXADR (CAR),is present during embryogenesis and is involved in tissue regeneration,cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells,from the cleavage stage up to the precursor epiblast,and corresponded with the presence of soluble CXADR3/7 splice variant. CAR was displayed on the membrane,involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells,and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts,CAR was reduced in the membrane and concentrated in the nucleus,which correlated with the switch in RNA expression to the CXADR4/7 and CXADR2/7 splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus,corresponding with the transmembrane CXADR and CXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage,the expression of CXADR was downregulated. We concluded that CXADR is differentially expressed during human preimplantation development. We described various CAR expressions: i) soluble CXADR marking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types,such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated. View Publication

For diseases of the brain,the pig (Sus scrofa) is increasingly being used as a model organism that shares many anatomical and biological similarities with humans. We report that pig induced pluripotent stem cells (iPSC) can recapitulate events in early mammalian neural development. Pig iPSC line (POU5F1(high)/SSEA4(low)) had a higher potential to form neural rosettes (NR) containing neuroepithelial cells than either POU5F1(low)/SSEA4(low) or POU5F1(low)/SSEA4(high) lines. Thus,POU5F1 and SSEA4 pluripotency marker profiles in starting porcine iPSC populations can predict their propensity to form more robust NR populations in culture. The NR were isolated and expanded in vitro,retaining their NR morphology and neuroepithelial molecular properties. These cells expressed anterior central nervous system fate markers OTX2 and GBX2 through at least seven passages,and responded to retinoic acid,promoting a more posterior fate (HOXB4+,OTX2-,and GBX2-). These findings offer insight into pig iPSC development,which parallels the human iPSC in both anterior and posterior neural cell fates. These in vitro similarities in early neural differentiation processes support the use of pig iPSC and differentiated neural cells as a cell therapy in allogeneic porcine neural injury and degeneration models,providing relevant translational data for eventual human neural cell therapies. View Publication

Environmental influences and insults by reproductive toxicant exposure can lead to impaired spermatogenesis or infertility. Understanding how toxicants disrupt spermatogenesis is critical for determining how environmental factors contribute to impaired fertility. While current animal models are available,understanding of the reproductive toxic effects on human fertility requires a more robust model system. We recently demonstrated that human pluripotent stem cells can differentiate into spermatogonial stem cells/spermatogonia,primary and secondary spermatocytes,and haploid spermatids; a model that mimics many aspects of human spermatogenesis. Here,using this model system,we examine the effects of 2-bromopropane (2-BP) and 1,2,dibromo-3-chloropropane (DBCP) on in vitro human spermatogenesis. 2-BP and DBCP are non-endocrine disrupting toxicants that are known to impact male fertility. We show that acute treatment with either 2-BP or DBCP induces a reduction in germ cell viability through apoptosis. 2-BP and DBCP affect viability of different cell populations as 2-BP primarily reduces spermatocyte viability,whereas DBCP exerts a much greater effect on spermatogonia. Acute treatment with 2-BP or DBCP also reduces the percentage of haploid spermatids. Both 2-BP and DBCP induce reactive oxygen species (ROS) formation leading to an oxidized cellular environment. Taken together,these results suggest that acute exposure with 2-BP or DBCP causes human germ cell death in vitro by inducing ROS formation. This system represents a unique platform for assessing human reproductive toxicity potential of various environmental toxicants in a rapid,efficient,and unbiased format. View Publication

Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation,physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation,cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons. View Publication

Events defining the progression to human type 1 diabetes (T1D) have remained elusive owing to the complex interaction between genetics,the immune system,and the environment. Type 1 interferons (T1-IFN) are known to be a constituent of the autoinflammatory milieu within the pancreas of patients with T1D. However,the capacity of IFNα/β to modulate human activated autoreactive CD8+ T-cell (cytotoxic T lymphocyte) responses within the islets of patients with T1D has not been investigated. Here,we engineer human β-cell-specific cytotoxic T lymphocytes and demonstrate that T1-IFN augments cytotoxicity by inducing rapid phosphorylation of STAT4,resulting in direct binding at the granzyme B promoter within 2 h of exposure. The current findings provide novel insights concerning the regulation of effector function by T1-IFN in human antigen-experienced CD8+ T cells and provide a mechanism by which the presence of T1-IFN potentiates diabetogenicity within the autoimmune islet. View Publication

The embryonic neuroepithelium gives rise to the entire central nervous system in vivo,making it an important tissue for developmental studies and a prospective cell source for regenerative applications. Current protocols for deriving homogenous neuroepithelial cultures from human pluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followed by a manual isolation step or adherent differentiation using small molecule inhibitors. Here,we report that hPSCs maintained under chemically defined,feeder-independent,and xeno-free conditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax6(+)/N-cadherin(+) with widespread rosette formation) within 6 days under adherent conditions,without small molecule inhibitors,and using only minimalistic medium consisting of Dulbecco's modified Eagle's medium/F-12,sodium bicarbonate,selenium,ascorbic acid,transferrin,and insulin (i.e.,E6 medium). Furthermore,we provide evidence that the defined culture conditions enable this high level of neural conversion in contrast to hPSCs maintained on mouse embryonic fibroblasts (MEFs). In addition,hPSCs previously maintained on MEFs could be rapidly converted to a neural compliant state upon transfer to these defined conditions while still maintaining their ability to generate all three germ layers. Overall,this fully defined and scalable protocol should be broadly useful for generating therapeutic neural cells for regenerative applications. View Publication

Proteomic analysis of human cerebral organoids may reveal how psychedelics regulate biological processes,shedding light on drug-induced changes in the brain. This study elucidates the proteomic alterations induced by lysergic acid diethylamide (LSD) in human cerebral organoids. By employing high-resolution mass spectrometry-based proteomics,we quantitatively analyzed the differential abundance of proteins in cerebral organoids exposed to LSD. Our findings indicate changes in proteostasis,energy metabolism,and neuroplasticity-related pathways. Specifically,LSD exposure led to alterations in protein synthesis,folding,autophagy,and proteasomal degradation,suggesting a complex interplay in the regulation of neural cell function. Additionally,we observed modulation in glycolysis and oxidative phosphorylation,crucial for cellular energy management and synaptic function. In support of the proteomic data,complementary experiments demonstrated LSD’s potential to enhance neurite outgrowth in vitro,confirming its impact on neuroplasticity. Collectively,our results provide a comprehensive insight into the molecular mechanisms through which LSD may affect neuroplasticity and potentially contribute to therapeutic effects for neuropsychiatric disorders. View Publication

BackgroundAbnormalities in T cell activation play an important role in the pathogenesis of myocarditis,and persistent T cell responses can lead to autoimmunity and chronic cardiac inflammation,as well as even dilated cardiomyopathy. Although previous work has examined the role of T cells in myocarditis in animal models,the specific mechanism for human cardiomyocytes has not been investigated.MethodsIn this study,we constructed the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and established the T cell-mediated cardiac injury model by co-culturing with activated CD4 + T or CD8 + T cells that were isolated from peripheral mononuclear blood to elucidate the pathogenesis of myocardial cell injury caused by inflammation.ResultsBy combination of quantitative proteomics with tissue and cell immunofluorescence examination,we established a proteome profile of inflammatory myocardia from hiPSC-CMs with obvious cardiomyocyte injury and increased levels of lactate dehydrogenase content,creatine kinase isoenzyme MB and cardiac troponin. A series of molecular dysfunctions of hiPSC-CMs was observed and indicated that CD4 + cells could produce direct cardiomyocyte injury by activating the NOD-like receptor signals pathway.ConclusionsThe data presented in our study established a proteome map of inflammatory myocardial based on hiPSC-CMs injury model. These results can provide guidance in the discovery of improved clinical treatments for myocarditis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03791-4. View Publication

AbstractDespite the success of combination antiretroviral therapy (ART) for individuals living with HIV,mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation,neuronal dysfunction,and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia,we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1,ISG15,ISG20,IFI27,IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART,consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However,S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells,mature and immature ChP cells,neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons,accompanied by increased expression of genes promoting cellular senescence and cell death. Together,these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons. View Publication