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Myocilin is a secreted glycoprotein that is expressed in ocular and non-ocular tissues. Mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. Here we report that myocilin is expressed in bone marrow-derived mesenchymal stem cells (MSCs) and plays a role in their differentiation into osteoblasts in vitro and in osteogenesis in vivo. Expression of myocilin was detected in MSCs derived from mouse,rat,and human bone marrow,with human MSCs exhibiting the highest level of myocilin expression. Expression of myocilin rose during the course of human MSC differentiation into osteoblasts but not into adipocytes,and treatment with exogenous myocilin further enhanced osteogenesis. MSCs derived from Myoc-null mice had a reduced ability to differentiate into the osteoblastic lineage,which was partially rescued by exogenous extracellular myocilin treatment. Myocilin also stimulated osteogenic differentiation of wild-type MSCs,which was associated with activation of the p38,Erk1/2,and JNK MAP kinase signaling pathways as well as up-regulated expression of the osteogenic transcription factors Runx2 and Dlx5. Finally,cortical bone thickness and trabecular volume,as well as the expression level of osteopontin,a known factor of bone remodeling and osteoblast differentiation,were reduced dramatically in the femurs of Myoc-null mice compared with wild-type mice. These data suggest that myocilin should be considered as a target for improving the bone regenerative potential of MSCs and may identify a new role for myocilin in bone formation and/or maintenance in vivo. View Publication

Neurogenin3+ (Ngn3+) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin,glucagon,somatostatin,pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach,where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors,but the lineage and regulators of pancreatic gastrin+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin+ cells in the developing pancreas co-express glucagon,ghrelin or pancreatic polypeptide,but many gastrin+ cells do not express any other islet hormone. Pancreatic gastrin+ cells express the transcription factors Nkx6.1,Nkx2.2 and low levels of Pdx1,and derive from Ngn3+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3,Nkx2.2,NeuroD1 and Arx,but not Pax4 or Pax6. Finally,gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus,gastrin+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells. View Publication

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However,such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs),which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods,we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene,which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening. View Publication

INTRODUCTION The control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs. METHODS Expression of total YAP,its paralogue transcriptional co-activator with PDZ-binding motif (TAZ),and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis,C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally,YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry. RESULTS YAP,but not TAZ,was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants,however,was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1,Id2 and Id3 in response to BMP-2. In developing mouse limbs,Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage,suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo. CONCLUSIONS Our findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology. View Publication

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL),HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and,in turn,is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis,we found that in CLL cells,HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma,reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models,and prolongs survival in mice. Of interest,we found that in CLL cells,HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore,HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes,including CXCR4,thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis. View Publication

SummaryFocusing on the early stages of Alzheimer’s disease (AD) holds great promise. However,the specific events in neural cells preceding AD onset remain elusive. To address this,we utilized human-induced pluripotent stem cells carrying APPswe mutation to explore the initial changes associated with AD progression. We observed enhanced neural activity and early neuronal differentiation in APPswe cerebral organoids cultured for one month. This phenomenon was also evident when neural progenitor cells (NPCs) were differentiated into neurons. Furthermore,transcriptomic analyses of NPCs and neurons confirmed altered expression of neurogenesis-related genes in APPswe NPCs. We also found that the upregulation of reactive oxygen species (ROS) is crucial for early neuronal differentiation in these cells. In addition,APPswe neurons remained immature after initial differentiation with increased susceptibility to toxicity,providing valuable insights into the premature exit from the neural progenitor state and the increased vulnerability of neural cells in AD. Graphical abstract Highlights•APPswe organoids show increased neural activity and early differentiation•Enhanced ROS levels are necessary but insufficient to accelerate differentiation•Transcriptome analysis of APPswe NPCs shows gene expression shift to differentiation•Premature neural cells with APPswe exhibit increased vulnerability to toxicity Molecular biology; Neuroscience; Cell biology View Publication

The role of the bone marrow (BM) microenvironment in regulating the antitumor immune response in Waldenstrom macroglobulinemia (WM) remains poorly understood. Here we transcriptionally and phenotypically profiled non-malignant (CD19- CD138-) BM cells from WM patients with a focus on myeloid derived suppressive cells (MDSCs) to provide a deeper understanding of their role in WM. We found that HLA-DRlowCD11b+CD33+ MDSCs were significantly increased in WM patients as compared to normal controls,with an expansion of predominantly polymorphonuclear (PMN)-MDSCs. Single-cell immunogenomic profiling of WM MDSCs identified an immune-suppressive gene signature with upregulated inflammatory pathways associated with interferon and tumor necrosis factor (TNF) signaling. Gene signatures associated with an inflammatory and immune suppressive environment were predominately expressed in PMN-MDSCs. In vitro,WM PMN-MDSCs demonstrated robust T-cell suppression and their viability and expansion was notably enhanced by granulocyte colony stimulating factor (G-CSF) and TNFα. Furthermore,BM malignant B-cells attracted PMN-MDSCs to a greater degree than monocytic MDSCs. Collectively,these data suggest that malignant WM B cells actively recruit PMN-MDSCs which promote an immunosuppressive BM microenvironment through a direct T cell inhibition,while release of G-CSF/TNFα in the microenvironment further promotes PMN-MDSC expansion and in turn immune suppression. Targeting PMN-MDSCs may therefore represent a potential therapeutic strategy in patients with WM. View Publication

Aim of work was to locate a simple,reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited,namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size,pore orientation,porosity,and pore distribution were produced using two different techniques,such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity,seeding efficiency,and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method,as these conditions successfully improved the cellularization of polymeric patches. Furthermore,the investigation provided interesting information on patches' stability in physiological simulating experimental conditions. Considering the in vitro results,it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover,the protocol turned out to be simple,repeatable,and reproducible. View Publication

Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however,the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely,shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1,verteporfin,significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1(+). Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties,suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population. View Publication

Detection and characterization of rare circulating tumor cells (CTCs) in patients' blood is important for the diagnosis and monitoring of cancer. The traditional way of counting CTCs via fluorescent images requires a series of tedious experimental procedures and often impacts the viability of cells. Here we present a method for label-free detection of CTCs from patient blood samples,by taking advantage of data analysis of bright field microscopy images. The approach uses the convolutional neural network,a powerful image classification and machine learning algorithm to perform label-free classification of cells detected in microscopic images of patient blood samples containing white blood cells and CTCs. It requires minimal data pre-processing and has an easy experimental setup. Through our experiments,we show that our method can achieve high accuracy on the identification of rare CTCs without the need for advanced devices or expert users,thus providing a faster and simpler way for counting and identifying CTCs. With more data becoming available in the future,the machine learning model can be further improved and can serve as an accurate and easy-to-use tool for CTC analysis. View Publication