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

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models,genomic health analyses,and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small,clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (TiPS") retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology View Publication

We asked this question: in normal humans,is either a normal dietary intake or normal serum concentration of phosphorus a determinant of the serum concentration of 1,25(OH)2D? In seven normal men whose dietary phosphorus was decreased from 2,300 to 625 mg/d,each intake for 8-9 d,under strictly controlled,normal metabolic conditions,we measured serum concentrations of 1,25(OH)2D daily,and concentrations of phosphorus hourly throughout a 24-h period,before and after restriction. Decreasing dietary phosphorus induced: (a) a 58% increase in serum levels of 1,25(OH)2D; (b) a 35% decrease in serum levels of phosphorus measured in the afternoon; (c) a 12% decrease in the 24-h mean serum level of phosphorus; but,(d) no decrease in morning fasting levels of phosphorus. Serum concentrations of 1,25(OH)2D varied inversely and significantly with 24-h mean concentrations of phosphorus (r = -0.77,P less than 0.001). When these data are combined with those of our prior study in which dietary phosphorus was varied over an extreme range,the relationship between serum levels of 1,25(OH)2D and 24-h mean serum levels of phosphorus is even stronger (r = -0.90,P less than 0.001). In the aggregate,the results demonstrate that in normal men,dietary phosphorus throughout a normal range and beyond,can finely regulate the renal production and serum concentration of 1,25(OH)2D,and provide evidence that this regulation is mediated by fine modulation of the serum concentration of phosphorus. View Publication

The response to programmed death-1 (PD-1) blockade varies in hepatocellular carcinoma (HCC). We utilize a panel of 16 serum factors to show that a circulating level of serum amyloid A (SAA) > 20.0 mg/L has the highest accuracy in predicting anti-PD-1 resistance in HCC. Further experiments show a correlation between peritumoral SAA expression and circulating SAA levels in patients with progressive disease after PD-1 inhibition. In vitro experiments demonstrate that SAA induces neutrophils to express PD-L1 through glycolytic activation via an LDHA/STAT3 pathway and to release oncostatin M,thereby attenuating cytotoxic T cell function. In vivo,genetic or pharmacological inhibition of STAT3 or SAA eliminates neutrophil-mediated immunosuppression and enhances antitumor efficacy of anti-PD-1 treatment. This study indicates that SAA may be a critical inflammatory cytokine implicated in anti-PD-1 resistance in HCC. Targeting SAA-induced PD-L1+ neutrophils through STAT3 or SAA inhibition may present a potential approach for overcoming anti-PD1 resistance. The reasons for why hepatocellular carcinoma (HCC) is unresponsive to anti-PD-1 inhibition in some patients is not fully understood. Here the authors use human samples and mice tumour models to implicate serum amyloid A and STAT3 signalling involvement in the resistance to anti-PD1 immunotherapy in HCC. View Publication

Natural killer (NK) cells are cytotoxic immune cells that can eliminate target cells without prior stimulation. Human induced pluripotent stem cells (iPSCs) provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers. In this in vitro study,a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells,and distinct maturational stages of iPSC-NK were characterized. Mature cells of CD56bright CD16bright phenotype showed upregulation of CD56,CD16,and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure,while exposure to aggressive atypical teratoid/rhabdoid tumor (ATRT) cell lines enhanced NKG2D and NKp46 expression. Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-? secretion in activated NK cells. CD56bright CD16bright iPSC-NK cells showed a ratio-dependent killing of ATRT cells,and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT. The iPSC-NK cells were also cytotoxic against other brain,kidney,and lung cancer cell lines. Further NK maturation yielded CD56?ve CD16bright cells,which lacked activation markers even after exposure to interleukins or ATRT cells - indicating diminished cytotoxicity. Generation and characterization of different NK phenotypes from iPSCs,coupled with their promising anti-tumor activity against ATRT in vitro,offer valuable insights into potential immunotherapeutic strategies for brain tumors. Graphical abstractImage 1 Highlights•Natural killer (NK) cells were derived from human induced pluripotent stem cells (iPSCs) in the absence of feeder cells.•Various maturational subtypes of iPSC-NK cells were characterized,and the phenotypic and functional properties were studied.•iPSC-NK cells of CD56bright CD16bright phenotype expressed activation markers in response to interleukin stimuli.•iPSC-NK cells were cytotoxic toward human atypical teratoid and rhabdoid tumor (ATRT) cells and other human cancer cells.•The cytotoxicity of iPSC-NK cells against various cancer cells in vitro might be translated into an in vivo immunotherapy. View Publication

Schwann cells (SCs) play a crucial role in peripheral nerve repair by supporting axonal regeneration and remyelination. While extensive research has been conducted using rodent SCs,increasing attention is being directed toward human SCs due to species-specific differences in phenotypical and functional properties,and accessibility of human SCs derived from diverse sources. A major challenge in translating SC-based therapies for nerve repair lies in the inability to replicate human SC myelination in vitro,posing a significant obstacle to drug discovery and preclinical research. In this study,we compared the myelination capacity of human,rodent,and porcine SCs in various co-culture conditions,including species-matched and cross-species neuronal environments in a serum-free medium. Our results confirmed that rodent and porcine SCs readily myelinate neurites under standard culture conditions after treatment with ascorbic acid for two weeks,whereas human SCs,at least within the four-week observation period,failed to show myelin staining in all co-cultures. Furthermore,we investigated whether cell culture manipulation impairs human SC myelination by transplanting freshly harvested and predegenerated human nerve segments into NOD-SCID mice for four weeks. Despite supporting host axonal regeneration into the grafts,human SCs exhibited very limited myelination,suggesting an intrinsic species-specific restriction rather than a cell culture-induced defect. These observations suggest fundamental differences between human and rodent SCs and highlight the need for human-specific models and protocols to advance our understanding of SC myelination. View Publication

FMS-related tyrosine kinase 3 ligand (FLT3L),encoded by FLT3LG,is a hematopoietic factor essential for the development of natural killer (NK),B cells,and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant,with a history of various recurrent infections,including severe cutaneous warts. The patients’ bone marrow was hypoplastic,with low levels of hematopoietic progenitors,particularly myeloid and B-cell precursors. Counts of B cells,monocytes,and DCs were low in the patients’ blood,whereas the other blood subsets,including NK cells,were affected only moderately,if at all. The patients had normal counts of Langerhans cells and dermal macrophages in the skin but lacked dermal DCs. Thus,FLT3L is required for B-cell and DC development in mice and humans. However,unlike its murine counterpart,human FLT3L is required for the development of monocytes but not NK cells. View Publication

In bone marrow,cell numbers are balanced between production and loss. After chemotherapy,blood cell counts decrease initially but later recover as hematopoietic progenitor cells expand,although the mechanisms underlying this recovery are still unclear. We investigated the influence of red blood cells (RBCs) on hematopoietic stem cell (HSC) function during bone marrow recovery. Following chemotherapy,RBC concentrations in bone marrow peaked on day 5 posttreatment,coinciding with the recovery of hematopoiesis. Coculture of HSCs with RBCs resulted in a significant increase in hematopoiesis. Direct contact between RBCs and HSCs was essential for enhancement of hematopoiesis,and HSCs precultured with RBCs resulted in greater numbers of donor‐derived mature hematopoietic cells after transplantation. RNA‐sequencing analysis showed that Hes1 was the most significantly upregulated transcription factor in RBC coculture,and the response to RBC‐induced hematopoiesis of Hes1‐deficient HSCs was reduced. These findings imply a role of RBCs and Hes1 in the enhancement of hematopoietic recovery following bone marrow stress. View Publication

Plp1 gene expression occurs very early in development,well before the onset of myelination,creating a conundrum with regard to the function of myelin proteolipid protein (PLP),one of the major proteins in compact myelin. Using PLP-EGFP mice to investigate Plp1 promoter activity,we found that,at very early time points,PLP-EGFP was expressed in Sox2+ undifferentiated precursors in the spinal cord ventricular zone (VZ),as well as in the progenitors of both neuronal and glial lineages. As development progressed,most PLP-EGFP-expressing cells gave rise to oligodendrocyte progenitor cells (OPCs). The expression of PLP-EGFP in the spinal cord was quite dynamic during development. PLP-EGFP was highly expressed as cells delaminated from the VZ. Expression was downregulated as cells moved laterally through the cord,and then robustly upregulated as OPCs differentiated into mature myelinating oligodendrocytes. The presence of PLP-EGFP expression in OPCs raises the question of its role in this migratory population. We crossed PLP-EGFP reporter mice into a Plp1-null background to investigate the role of PLP in early OPC development. In the absence of PLP,normal numbers of OPCs were generated and their distribution throughout the spinal cord was unaffected. However,the orientation and length of OPC processes during migration was abnormal in Plp1-null mice,suggesting that PLP plays a role either in the structural integrity of OPC processes or in their response to extracellular cues that orient process outgrowth. View Publication

Mesenchymal stem cells (MSCs) are defined as non-hematopoietic,plastic-adherent,self-renewing cells that are capable of tri-lineage differentiation into bone,cartilage or fat in vitro. Thus,MSCs are promising candidates for cell-based medicine. However,classifications of MSCs have been defined retrospectively; moreover,this conventional criterion may be inaccurate due to contamination with other hematopoietic lineage cells. Human MSCs can be enriched by selection for LNGFR and THY-1,and this population may be analogous to murine PDGFR??+Sca-1+ cells,which are developmentally derived from neural crest cells (NCCs). Murine NCCs were labeled by fluorescence,which provided definitive proof of neural crest lineage,however,technical considerations prevent the use of a similar approach to determine the origin of human LNGFR+THY-1+ MSCs. To further clarify the origin of human MSCs,human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) were used in this study. Under culture conditions required for the induction of neural crest cells,human ESCs and iPSCs-derived cells highly expressed LNGFR and THY-1. These LNGFR+THY-1+ neural crest-like cells,designated as LT-NCLCs,showed a strong potential to differentiate into both mesenchymal and neural crest lineages. LT-NCLCs proliferated to form colonies and actively migrated in response to serum concentration. Furthermore,we transplanted LT-NCLCs into chick embryos,and traced their potential for survival,migration and differentiation in the host environment. These results suggest that LNGFR+THY-1+ cells identified following NCLC induction from ESCs/iPSCs shared similar potentials with multipotent MSCs. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells and human-induced pluripotent stem cells,are a renewable cell source for a wide range of applications in regenerative medicine and useful tools for human disease modeling and drug discovery. For these purposes,large numbers of high-quality cells are essential. Recently,we showed that a biological substrate,recombinant E8 fragments of laminin isoforms,sustains long-term self-renewal of hPSCs in defined,xeno-free medium with dissociated single-cell passaging. Here,we describe a modified culture system with similar performance to efficiently expand hPSCs under defined,xeno-free conditions using a non-biological synthetic substrate. View Publication

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood,aside from numerous congenital abnormalities. FA mouse models have been generated; however,they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero,a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology,we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential,disruption of the second allele causes a severe growth disadvantage. As a result,heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele,quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning,this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. ?? 2014. View Publication

Nature Genetics 47,469 (2015). doi:10.1038/ng.3258 View Publication