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

Insulin secretion is elaborately modulated in pancreatic ß cells within islets of three-dimensional (3D) structures. Using human pluripotent stem cells (hPSCs) to develop islet-like structures with insulin-producing ß cells for the treatment of diabetes is challenging. Here,we report that pancreatic islet-like clusters derived from hESCs are functionally capable of glucose-responsive insulin secretion as well as therapeutic effects. Pancreatic hormone-expressing endocrine cells (ECs) were differentiated from hESCs using a step-wise protocol. The hESC-derived ECs expressed pancreatic endocrine hormones,such as insulin,somatostatin,and pancreatic polypeptide. Notably,dissociated ECs autonomously aggregated to form islet-like,3D structures of consistent sizes (100-150 μm in diameter). These EC clusters (ECCs) enhanced insulin secretion in response to glucose stimulus and potassium channel inhibition in vitro. Furthermore,ß cell-deficient mice transplanted with ECCs survived for more than 40 d while retaining a normal blood glucose level to some extent. The expression of pancreatic endocrine hormones was observed in tissues transplanted with ECCs. In addition,ECCs could be generated from human induced pluripotent stem cells. These results suggest that hPSC-derived,islet-like clusters may be alternative therapeutic cell sources for treating diabetes. View Publication

PURPOSE: The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However,little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.backslashnbackslashnMETHODS: Limbal stromal cells were derived from corneoscleral rims. The SSEA-4(+) and SSEA-4(-) limbal stromal cells were sorted by fluorescence-activated cells sorting (FACS). Isolated cells were expanded and reanalyzed for their expression of SSEA-4. Expression of MSC and ESC markers on these cells were also analyzed by FACS. In addition,expression of limbal epithelial and corneal stromal proteins such as ATP-binding cassette sub-family G member 2 (ABCG2),tumour protein p63 (p63),paired box 6 (Pax6),cytokeratin 3 (AE5),cytokeratin 10,and keratocan sulfate were evaluated either by immunofluorecence staining or reverse transcription polymerase chain reaction. Appropriate induction medium was used to differentiate these cells into adipocytes,osteocytes,and chondrocytes.backslashnbackslashnRESULTS: Expanded limbal stromal cells expressed the majority of mesenchymal markers. These cells were negative for ABCG2,p63,Pax6,AE-5,and keratocan sulfate. After passaged,a subpopulation of these cells showed low expression of SSEA-4 but were negative for other important ESC surface markers such as Tra-1-60,Tra-1-81,and transcription factors like octamer-binding transcription factor 4 (Oct4),SRY(sex determining region Y)-box 2 (Sox2),and Nanog. Early passaged cells when induced were able to differentiate into adipocytes,osteocytes and chondrocytes.backslashnbackslashnCONCLUSIONS: The expanded limbal stromal cells showed features of multipotent MSC. Our study confirmed the expression of SSEA-4 by a subpopulation of cultured limbal stromal cells. However,despite the expression of SSEA-4,these cells did not express any other markers of ESC. Therefore,we conclude that the cells did not show properties of ESC. View Publication

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited,stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently,the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition,the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However,the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation,we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice,hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling,increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. View Publication

Human embryonic stem cells (hESCs) hold great value for future clinical applications. However,standard culture conditions maintain hESCs in a primed state,which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks,primed hESCs have been converted to a naive state,but this has restricted the efficiency of existing directed differentiation protocols. In mouse,WNT inhibition by inhibitor of WNT production-2,together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI),markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study,we show that DhiFI conditions similarly improved primed hESC traits,such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages,DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover,DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers,compared to primed-derived lines from the same PICMI. Overall,DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types. View Publication

Application of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems,we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas,high ALDH activity identifies the tumorigenic cell fraction,capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas,expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts. View Publication

The human brain is composed of a complex assembly of about 171 billion heterogeneous cellular units (86 billion neurons and 85 billion non-neuronal glia cells). A comprehensive description of brain cells is necessary to understand the nervous system in health and disease. Recently,advances in genomics have permitted the accurate analysis of the full transcriptome of single cells (scRNA-seq). We have built upon such technical progress to combine scRNA-seq with patch-clamping electrophysiological recording and morphological analysis of single human neurons in vitro. This new powerful method,referred to as Patch-seq,enables a thorough,multimodal profiling of neurons and permits us to expose the links between functional properties,morphology,and gene expression. Here,we present a detailed Patch-seq protocol for isolating single neurons from in vitro neuronal cultures. We have validated the Patch-seq whole-transcriptome profiling method with human neurons generated from embryonic and induced pluripotent stem cells (ESCs/iPSCs) derived from healthy subjects,but the procedure may be applied to any kind of cell type in vitro. Patch-seq may be used on neurons in vitro to profile cell types and states in depth to unravel the human molecular basis of neuronal diversity and investigate the cellular mechanisms underlying brain disorders. View Publication

Human natural killer (NK) cells express an abundant level of 2B4 and CD2 on their surface. Their counter-receptors,CD48 and CD58,are also expressed on the NK cell surface,raising a question about the functional consequences of potential 2B4/CD48 and CD2/CD58 interactions. Using blocking antibodies specific to each receptor,we demonstrated that both 2B4/CD48 and CD2/CD58 interactions were essential for the development of NK effector functions: cytotoxicity and cytokine secretion. However,only 2B4/CD48,but not CD2/CD58,interactions were shown to be critical for the optimal NK cell proliferation in response to interleukin (IL)-2. IL-2-activated NK cells cultured in the absence of 2B4/CD48 or CD2/CD58 interactions were severely impaired for their ability to induce intracellular calcium mobilization and subsequent ERK activation upon tumor target exposure,suggesting that the early signaling pathway of NK receptors leading to impaired cytolysis and interferon (IFN)-γ secretion was inhibited. Nevertheless,these defects did not fully account for the reduced proliferation of NK cells in the absence of 2B4/CD48 interactions,because anti-CD2 or anti-CD58 monoclonal antibody (mAb)-treated NK cells,showing defective signaling and effector functions,displayed normal proliferation upon IL-2 stimulation. These results propose the signaling divergence between pathways leading to cell proliferation and cytotoxicity/cytokine release,which can be differentially regulated by 2B4 and CD2 during IL-2-driven NK cell activation. Collectively,these results reveal the importance of homotypic NK-to-NK cell cross-talk through 2B4/CD48 and CD2/CD58 pairs and further present their differential and overlapping roles in human NK cells. View Publication

PURPOSE Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard,stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study,we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. METHODS AND MATERIALS A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer,170 keV/μm; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug,allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF,PlGF,SDF-1,and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling,a specific SCF/c-Kit inhibitor (ISCK03) was used. RESULTS Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control,20.71 ± 1.55; X-ray,36.44 ± 3.44; carbon ion,16.33 ± 1.03; number per microscopic field). Concordantly,irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray,36.44 ± 3.44; X-ray and ISCK03,4.33 ± 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. CONCLUSIONS The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells,a phenomenon that could not be observed with carbon ion irradiation. Thus,in this model system evaluating angiogenesis,carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models. View Publication

Ionizing radiation (IR) is a known mutagen that is widely employed for medical diagnostic and therapeutic purposes. To study the extent of genetic variations in DNA caused by IR,we used IR-sensitive human embryonic stem cells (hESCs). Four hESC cell lines,H1,H7,H9,and H14,were subjected to IR at 0.2 or 1 Gy dose and then maintained in culture for four days before being harvested for DNA isolation. Irradiation with 1 Gy dose resulted in significant cell death,ranging from 60% to 90% reduction in cell population. Since IR is often implicated as a risk for inducing cancer,a primer pool targeting genomic hotspot" regions that are frequently mutated in human cancer genes was used to generate libraries from irradiated and control samples. Using a semiconductor-based next-generation sequencing approach� View Publication

Engineered heart tissues made from human pluripotent stem cell-derived cardiomyocytes have been used for modeling cardiac pathologies,screening new therapeutics,and providing replacement cardiac tissue. Current methods measure the functional performance of engineered heart tissue by their twitch force and beating frequency,typically obtained by optical measurements. In this article,we describe a novel method for assessing twitch force and beating frequency of engineered heart tissue using magnetic field sensing,which enables multiple tissues to be measured simultaneously. The tissues are formed as thin structures suspended between two silicone posts,where one post is rigid and another is flexible and contains an embedded magnet. When the tissue contracts it causes the flexible post to bend in proportion to its twitch force. We measured the bending of the post using giant magnetoresistive (GMR) sensors located underneath a 24-well plate containing the tissues. We validated the accuracy of the readings from the GMR sensors against optical measurements. We demonstrated the utility and sensitivity of our approach by testing the effects of three concentrations of isoproterenol and verapamil on twitch force and beating frequency in real-time,parallel experiments. This system should be scalable beyond the 24-well format,enabling greater automation in assessing the contractile function of cardiomyocytes in a tissue-engineered environment. View Publication

Activin A belongs to the superfamily of transforming growth factor beta (TGF$\beta$) and is a critical regulatory cytokine in breast cancer and inflammation. However,the role of activin A in migration of breast cancer cells and immune cells was not well characterized. Here,a microfluidic device was used to examine the effect of activin A on the migration of human breast cancer cell line MDA-MB-231 cells and human blood neutrophils as well as their migratory interaction. We found that activin A promoted the basal migration but impaired epidermal growth factor (EGF)-induced migration of breast cancer cells. By contrast,activin A reduced neutrophil chemotaxis and transendothelial migration to N-Formyl-Met-Leu-Phe (fMLP). Finally,activin A promoted neutrophil chemotaxis to the supernatant from breast cancer cell culture. Collectively,our study revealed the different roles of activin A in regulating the migration of breast cancer cells and neutrophils and their migratory interaction. These findings suggested the potential of activin A as a therapeutic target for inflammation and breast cancers. View Publication

Human embryonic stem cells (hESCs) tend to lose genomic integrity during long periods of culture in vitro and to acquire a cancer-like phenotype. In this study,we aim at understanding the contribution of point mutations to the adaptation process and at providing a mechanistic explanation for their accumulation. We observed that,due to the absence of p21/Waf1/Cip1,cultured hESCs lack proper cell cycle checkpoints and are vulnerable to the kind of DNA damage usually repaired by the highly versatile nucleotide excision repair (NER) pathway. In response to UV-induced DNA damage,the majority of hESCs succumb to apoptosis; however,a subpopulation continues to proliferate,carrying damaged DNA and accumulating point mutations with a typical UV-induced signature. The UV-resistant cells retain their proliferative capacity and potential for pluripotent differentiation and are markedly less apoptotic to subsequent UV exposure. These findings demonstrate that,due to deficient DNA damage response,the modest NER activity in hESCs is insufficient to prevent increased mutagenesis. This provides for the appearance of genetically aberrant hESCs,paving the way for further major genetic changes. View Publication