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

Although since 1998 more than 1,200 different hESC lines have been established worldwide,there is still a recognized interest in the establishment of new lines of hESC,particularly from HLA types and ethnic groups underrepresented among the currently available lines. The methodology of hESC derivation has evolved significantly since the initial derivations using human LIF (hLIF) for maintenance of pluripotency. However,there are still a number of alternative strategies for the different steps involved in establishing a new line of hESC. We have analyzed the different strategies/parameters used between 1998 and 2010 for the derivation of the 375 hESC lines able to form teratomas in immunocompromised mice deposited in two international stem cell registries. Here we describe some trends in the methodology for establishing hESC lines,discussing the developments in the field. Nevertheless,we describe a much greater heterogeneity of strategies for hESCs derivation than what is used for murine ESC lines,indicating that optimum conditions have not been identified yet,and thus,hESC establishment is still an evolving field of research. View Publication

Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors,but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue,we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct,which inhibits signaling through all Notch receptors,and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease,but not a complete abrogation,of these cells in dnMAML-expressing cells. Interestingly,when assessed in secondary assays in vitro or in vivo,there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool,which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population. View Publication

Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However,the low efficiency of iPSC derived from somatic cells (0.01-0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal,proliferation,and maintenance of embryonic stem cells (ESCs),but the contribution of this pathway or its well-known negative regulator,phosphatase,and tensin homolog deleted on chromosome ten (Pten),to somatic cell reprogramming remains largely unknown. Here,we show that activation of the PI3K pathway by the Pten inhibitor,dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate,improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs. View Publication

Mast cells are unique hematopoietic cells that are richly distributed in the skin and mucosal surfaces of the respiratory and gastrointestinal tract. They play a key role in allergic inflammation by releasing a cocktail of granular constituents,including histamine,serine proteases,and various eicosanoids and cytokines. As such,a number of drugs target either inhibition of mast cell degranulation or the products of degranulation. To identify potential novel drugs and mechanisms in mast cell biology,assays were developed to identify inhibitors of mast cell degranulation and activation in a phenotypic screen. Due to the challenges associated with obtaining primary mast cells,cord blood-derived mononuclear cells were reproducibly differentiated to mast cells and assays developed to monitor tryptase release and prostaglandin D2 generation. The tryptase assay was particularly sensitive,requiring only 500 cells per data point,which permitted a set of approximately 12,000 compounds to be screened robustly and cost-effectively. Active compounds were tested for concomitant inhibition of prostaglandin D2 generation. This study demonstrates the robustness and effectiveness of this approach in the identification of potential novel compounds and mechanisms targeting mast cell-driven inflammation,to enable innovative drug discovery efforts to be prosecuted. View Publication

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay,we identify TRAIL to be associated with NK cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms,we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFN$\gamma$ production. Moreover,TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I,adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings,we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multifunctional role beyond receptor-mediated induction of apoptosis,acting as a regulator for the induction of different effector functions. View Publication

Recent studies suggest that highly activated,polyfunctional CD4+ T cells are incredibly effective in strengthening and sustaining overall host antitumor immunity,promoting tumor-specific CD4+ T-cell responses and effectively enhancing antitumor immunity by immunotherapy. Previously,we developed a novel cryo-thermal therapy for local tumor ablation and achieved long-term survival rates in several tumor models. It was discovered that cryo-thermal therapy remodeled the tumor microenvironment and induced an antigen-specific CD4+ T-cell response,which mediated stronger antitumor immunity in vivo. In this study,the phenotype of bulk T cells in spleen was analyzed by flow cytometry after cryo-thermal therapy and both CD4+ Th1 and CD8+ CTL were activated. In addition,by using T-cell depletion,isolation,and adoptive T-cell therapy,it was found that cryo-thermal therapy induced Th1-dominant CD4+ T cells that directly inhibited the growth of tumor cells,promoted the maturation of MDSCs via CD4+ T-cell-derived IFN-? and enhanced the cytotoxic effector function of NK cells and CD8+ T cells,and promoted the maturation of APCs via cell-cell contact and CD4+ T-cell-derived IFN-?. Considering the multiple roles of cryo-thermal-induced Th1-dominant CD4+ T cells in augmenting antitumor immune memory,we suggest that local cryo-thermal therapy is an attractive thermo-immunotherapy strategy to harness host antitumor immunity and has great potential for clinical application. View Publication

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies,efficient and scalable expansion procedures must be developed. For other adherent human cell types,the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study,a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings,indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( N s 1 u ),thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics,computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner,improved process understanding allowed an expansion factor of ≈ 26 to be achieved,yielding more than 3 × 10 9 cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability,identity,and differentiation potential throughout cultivation. • N s 1 u can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs The online version contains supplementary material available at 10.1007/s00253-024-13372-3. View Publication

Understanding the interaction between genetic and epigenetic variation remains a challenge due to confounding environmental factors. We propose that human induced Pluripotent Stem Cells (iPSCs) are an excellent model to study the relationship between genetic and epigenetic variation while controlling for environmental factors. In this study,we have created a comprehensive resource of high-quality genomic,epigenomic,and transcriptomic data from iPSC lines and three iPSC-derived cell types (neural stem cell (NSC),motor neuron,monocyte) from three healthy donors. We find that epigenetic variation is most strongly associated with genetic variation at the iPSC stage,and that relationship weakens as epigenetic variation increases in differentiated cells. Additionally,cell type is a stronger source of epigenetic variation than genetic variation. Further,we elucidate a utility of studying epigenetic variation in iPSCs and their derivatives for identifying important loci for GWAS studies and the cell types in which they may be acting. Subject terms: Epigenomics,Genomics,Transcriptomics View Publication

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence,this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes,Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution,the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay,which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2{\%} vs. 9.9{\%},p {\textgreater} 0.05). However,when 100 mM Z-VAD-FMK was added to the post-thaw culture media,there was a significant enhancement in the survival rate from 9.9{\%} to 14.4{\%} (p {\textless} 0.05),which was further increased to 18.7{\%} when Z-VAD-FMK was also added to the freezing solution as well (p {\textless} 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy,and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture. View Publication

BackgroundCertain structural variants (SVs) including large-scale genetic copy number variants,as well as copy number-neutral inversions and translocations may not all be resolved by chromosome karyotype studies. The identification of genetic risk factors for Parkinson’s disease (PD) has been primarily focused on the gene-disruptive single nucleotide variants. In contrast,larger SVs,which may significantly influence human phenotypes,have been largely underexplored. Optical genomic mapping (OGM) represents a novel approach that offers greater sensitivity and resolution for detecting SVs. In this study,we used induced pluripotent stem cell (iPSC) lines of patients with PD-linked SNCA and PRKN variants as a proof of concept to (i) show the detection of pathogenic SVs in PD with OGM and (ii) provide a comprehensive screening of genetic abnormalities in iPSCs.ResultsOGM detected SNCA gene triplication and duplication in patient-derived iPSC lines,which were not identified by long-read sequencing. Additionally,various exon deletions were confirmed by OGM in the PRKN gene of iPSCs,of which exon 3–5 and exon 2 deletions were unable to phase with conventional multiplex-ligation-dependent probe amplification. In terms of chromosomal abnormalities in iPSCs,no gene fusions,no aneuploidy but two balanced inter-chromosomal translocations were detected in one line that were absent in the parental fibroblasts and not identified by routine single nucleotide variant karyotyping.ConclusionsIn summary,OGM can detect pathogenic SVs in PD-linked genes as well as reveal genomic abnormalities for iPSCs that were not identified by other techniques,which is supportive for OGM’s future use in gene discovery and iPSC line screening.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-024-10902-1. View Publication

Differentiation of induced pluripotent stem cells (iPSCs) into specialized cell types is essential for uncovering cell-type specific molecular mechanisms and interrogating cellular function. Transcription factor screens have enabled efficient production of a few cell types; however,engineering cell types that require complex transcription factor combinations remains challenging. Here,we report an iterative,high-throughput single-cell transcription factor screening method that enables the identification of transcription factor combinations for specialized cell differentiation,which we validated by differentiating human microglia-like cells. We found that the expression of six transcription factors,SPI1,CEBPA,FLI1,MEF2C,CEBPB,and IRF8,is sufficient to differentiate human iPSC into cells with transcriptional and functional similarity to primary human microglia within 4 days. Through this screening method,we also describe a novel computational method allowing the exploration of single-cell RNA sequencing data derived from transcription factor perturbation assays to construct causal gene regulatory networks for future cell fate engineering. Liu et al. developed a platform to identify transcription factors (TFs) that turn stem cells into desired cell types. They discovered six key TFs that produce microglia efficiently,enhancing cell differentiation methods. View Publication