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

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and,therefore,are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex,a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover,increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality. View Publication

Angelman syndrome (AS) and Prader-Willi syndrome (PWS),two distinct neurodevelopmental disorders,result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele,respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless,gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences,reprogramming methods,and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here,we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally,these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS. View Publication

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease,and death rates have remained at approximately 50% for decades. New tumor-targeting strategies are desperately needed,and a previous report indicated the triggered differentiation of HPV-negative HNSCC cells to confer therapeutic benefits. Using patient-derived tumor cells,we created a similar HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions,including irregularly enlarged cell morphology,cell cycle arrest with downregulation of Ki67,and reduced cell viability. RNA-Seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover,immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly,HPV surrogate marker p16,and stress-associated basal keratinocyte marker KRT17,indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. We are the first to report that carcinoma cells can undergo a triggered myocyte-like differentiation,and our study suggests that the targeted differentiation of HPV+ HNSCCs might be therapeutically valuable. Subject terms: Oral cancer,Mechanisms of disease,Cell death View Publication

Prenatal alcohol exposure (PAE) affects embryonic development,causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neurodevelopmental disorders and birth defects. To explore the effects of PAE on gastrulation,we used an in vitro model with subchronic moderate (20 mM) and severe (70 mM) ethanol exposures during the differentiation of human embryonic stem cells into germ layer cells. We analyzed genome-wide gene expression (mRNA sequencing),DNA methylation (EPIC Illumina microarrays) and metabolome (non-targeted LC-MS) of the endodermal,mesodermal and ectodermal cells. The largest number of ethanol-induced alterations were observed in endodermal cells,whereas the most prominent changes were in ectodermal cells. Methionine metabolism and genes of the main signaling pathways involved in gastrulation and body patterning were affected by ethanol in all germ layers. Many of the altered genes,including BMP4,FGF8,SIX3 and LHX2,have previously been associated with PAE and phenotypes of FASD,like defects in heart and corpus callosum development as well as holoprosencephaly. Our findings support the early origin of alcohol-induced developmental disorders and strengthen the role of methionine cycle in the etiology of FASD. View Publication

Human airways contain specialized rare epithelial cells including CFTR-rich ionocytes that regulate airway surface physiology and chemosensory tuft cells that produce asthma-associated inflammatory mediators. Here,using a lung cell atlas of 311,748 single cell RNA-Seq profiles,we identify 687 ionocytes (0.45%). In contrast to prior reports claiming a lack of ionocytes in the small airways,we demonstrate that ionocytes are present in small and large airways in similar proportions. Surprisingly,we find only 3 mature tuft cells (0.002%),and demonstrate that previously annotated tuft-like cells are instead highly replicative progenitor cells. These tuft-ionocyte progenitor (TIP) cells produce ionocytes as a default lineage. However,Type 2 and Type 17 cytokines divert TIP cell lineage in vitro,resulting in the production of mature tuft cells at the expense of ionocyte differentiation. Our dataset thus provides an updated understanding of airway rare cell composition,and further suggests that clinically relevant cytokines may skew the composition of disease-relevant rare cells. Subject terms: Interleukins,Systems analysis,Differentiation,Sequencing View Publication

Human induced pluripotent stem cells (hiPSC) provide an attractive tool to study disease mechanisms of neurodevelopmental disorders such as schizophrenia. A pertinent problem is the development of hiPSC-based assays to discriminate schizophrenia (SZ) from autism spectrum disorder (ASD) models. Healthy control individuals as well as patients with SZ and ASD were examined by a panel of diagnostic tests. Subsequently,skin biopsies were taken for the generation,differentiation,and testing of hiPSC-derived neurons from all individuals. SZ and ASD neurons share a reduced capacity for cortical differentiation as shown by quantitative analysis of the synaptic marker PSD95 and neurite outgrowth. By contrast,pattern analysis of calcium signals turned out to discriminate among healthy control,schizophrenia,and autism samples. Schizophrenia neurons displayed decreased peak frequency accompanied by increased peak areas,while autism neurons showed a slight decrease in peak amplitudes. For further analysis of the schizophrenia phenotype,transcriptome analyses revealed a clear discrimination among schizophrenia,autism,and healthy controls based on differentially expressed genes. However,considerable differences were still evident among schizophrenia patients under inspection. For one individual with schizophrenia,expression analysis revealed deregulation of genes associated with the major histocompatibility complex class II (MHC class II) presentation pathway. Interestingly,antipsychotic treatment of healthy control neurons also increased MHC class II expression. In conclusion,transcriptome analysis combined with pattern analysis of calcium signals appeared as a tool to discriminate between SZ and ASD phenotypes in vitro. View Publication

DNA repair mechanisms in human primary cells,including error-free repair,and,recurrent nuclease cleavage events,remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR),an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs,small indels,large deletions,and other aberrations in absolute terms in clinically relevant edited cells,including HSPCs,iPSCs,and T-cells. By quantifying up to 90% of loci with unresolved DSBs,CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore,we accurately quantify DNA repair precision,revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics,paving the way for mechanistic studies to advance gene therapy,designer editors,and small molecule discovery. Quantifying genomic aberrations resulting from designer nucleases activity is essential for gene therapy clinical translation. Here,the authors present a modular digital PCR technique that profiles DNA repair precision and cut-repair cycles at the edited loci,exposing current evaluation biases. View Publication

The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system,but it involutes with age,resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro,but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However,these approaches require access to primary human tissues or murine bone marrow stromal cells,are allogeneic,and do not support negative selection. Recently,we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here,we combined hPSC-derived TEPs,hematopoietic progenitor cells (HPCs),and mesenchymal cells,differentiated from the same hPSC line,and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic,patient-specific thymic organoids in vitro. View Publication

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates,its activity in combination with other antiretrovirals,and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L,T215Y/F,plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V,Y115F,and M184V plus one other NAM) or stavudine (V75T/M,M41L,T215F/Y,and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold,respectively (these changes gave values comparable to or less than the corresponding values for zidovudine,lamivudine,abacavir,and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades,including A/G,B,C,D,A(E),D/F,F,and H. SPD754 showed additive effects in combination with other NRTIs,tenofovir,nevirapine,or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro,SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity. View Publication

The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner,mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However,the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover,we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects,and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells,inducing a dose-dependent increase in SOX2,OCT4 and Nanog proteins,leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells. View Publication