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

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening. View Publication

During an immune response,activated antigen (Ag)-specific T cells condition dendritic cells (DCs) to enhance DC function and survival within the inflamed draining lymph node (LN). It has been difficult to ascertain the role of the tumor necrosis factor (TNF) superfamily member lymphotoxin-alphabeta (LTalphabeta) in this process because signaling through the LTbeta-receptor (LTbetaR) controls multiple aspects of lymphoid tissue organization. To resolve this,we have used an in vivo system where the expression of TNF family ligands is manipulated only on the Ag-specific T cells that interact with and condition Ag-bearing DCs. We report that LTalphabeta is a critical participant required for optimal DC function,independent of its described role in maintaining lymphoid tissue organization. In the absence of LTalphabeta or CD40L on Ag-specific T cells,DC dysfunction could be rescued in vivo via CD40 or LTbetaR stimulation,respectively,suggesting that these two pathways cooperate for optimal DC conditioning. View Publication

Cancer stem cells (CSCs) play crucial roles in tumor metastasis,therapy resistance,and immune evasion. Identifying and understanding the factors that regulate the stemness of tumor cells presents promising opportunities for developing effective therapeutic strategies. In this study on oral squamous cell carcinoma (OSCC),we confirmed the key role of KLF7 in maintaining the stemness of OSCC. Using chromatin immunoprecipitation sequencing and dual-luciferase assays,we identified ITGA2,a membrane receptor,as a key downstream gene regulated by KLF7 in the maintenance of stemness. Tumor sphere formation assays,flow cytometry analyses,and in vivo limiting dilution tumorigenicity evaluations demonstrated that knocking down ITGA2 significantly impaired stemness. Upon binding to its extracellular matrix (ECM) ligand,type I collagen,ITGA2 activates stemness-associated signaling pathways,including PI3K-AKT,MAPK,and Hippo. TC-I 15,a small-molecule inhibitor of the ITGA2-collagen interaction,significantly sensitizes oral squamous cell carcinoma (OSCC) to cisplatin in xenograft models. In summary,we reveal that the KLF7/ITGA2 axis is a crucial modulator of stemness in OSCC. Our findings suggest that ITGA2 is a promising therapeutic target,offering a novel anti-CSC strategy. Subject terms: Cancer stem cells,Cancer therapeutic resistance View Publication

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages,necessary to understand the species specific pathogenic effects of HCMV,has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells,iPS-derived neural stem cells (NSCs),neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection,i.e.,they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate. View Publication

Casitas B-cell lymphoma (Cbl)-family E3 ubiquitin ligases are negative regulators of tyrosine kinase signaling. Recent work has revealed a critical role of Cbl in the maintenance of hematopoietic stem cell (HSC) homeostasis,and mutations in CBL have been identified in myeloid malignancies. Here we show that,in contrast to Cbl or Cbl-b single-deficient mice,concurrent loss of Cbl and Cbl-b in the HSC compartment leads to an early-onset lethal myeloproliferative disease in mice. Cbl,Cbl-b double-deficient bone marrow cells are hypersensitive to cytokines,and show altered biochemical response to thrombopoietin. Thus,Cbl and Cbl-b play redundant but essential roles in HSC regulation,whose breakdown leads to hematological abnormalities that phenocopy crucial aspects of mutant Cbl-driven human myeloid malignancies. View Publication

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here,we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the beta-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD,with 24.5 ± 7.6{\%} efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells,with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9{\%}. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that,by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification,the off-target effects including chromosomal rearrangements were significantly reduced. Taken together,our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs,a significant reduction in sickling of red blood cells,engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice. View Publication

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However,attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria. Using optimized isolation and culture conditions,in up to 63% of 59 low-volume UCB units,cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 x 10(8) mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition,osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However,in contrast to MSCs,MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than 15 hours,a net volume of more than 33 ml,and an MNC count of more than 1 x 10(8) MNCs. Because MSC-like cells can be isolated at high efficacy from full-term UCB donations,we regard UCB as an additional stem cell source for experimental and potentially clinical purposes. View Publication

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs),the molecular mechanism involved remains to be fully elucidated. Here,we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover,BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together,our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy,however,is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs,implying that activation of JNKs is essential for BMP9 osteoinductive activity. View Publication

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However,most of the studies used skin fibroblasts as the starting population for reprogramming,which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore,iPS cells can be readily derived from adult hASCs in a feeder-free condition,thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion,are easy to maintain in culture,and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells. View Publication

Motor neuron diseases (MNDs) are a group of neurological disorders that selectively affect motor neurons. There are currently no cures or efficacious treatments for these diseases. In recent years,significant developments in stem cell research have been applied to MNDs,particularly regarding neuroprotection and cell replacement. However,a consistent source of motor neurons for cell replacement is required. Human embryonic stem cells (hESCs) could provide an inexhaustible supply of differentiated cell types,including motor neurons that could be used for MND therapies. Recently,it has been demonstrated that induced pluripotent stem (iPS) cells may serve as an alternative source of motor neurons,since they share ES characteristics,self-renewal,and the potential to differentiate into any somatic cell type. In this review,we discuss several reproducible methods by which hESCs or iPS cells are efficiently isolated and differentiated into functional motor neurons,and possible clinical applications. View Publication

OBJECTIVE The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors,including PAX8 and NKX2-1,which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells,and TAZ interacts directly with both PAX8 and NKX2-1,leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes. METHODS The use of a small molecule,ethacridine,recently identified as a TAZ activator,in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First,endodermal cells were derived from hES cells using Activin A,followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH). RESULTS The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH,the thyroid-specific genes TG,TPO,TSHR,and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes,thyroid follicle formation and abundant TG protein expression were observed. Furthermore,such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation. CONCLUSION These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine,a TAZ activator,which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes,without any gene transfection or complex culture conditions,by directly manipulating the transcriptional machinery without interfering with intermediate signaling events. View Publication

Cardiovascular progenitor cells (CVPCs) derived from human embryonic stem cells and human induced pluripotent stem cells represent an invaluable potential source for the study of early embryonic cardiovascular development and stem cell-based therapies for congenital and acquired heart diseases. To fully realize their values,it is essential to establish an efficient and stable differentiation system for the induction of these pluripotent stem cells (PSCs) into the CVPCs and robustly expand them in culture,and then further differentiate these CVPCs into multiple cardiovascular cell types. Here we describe the protocols for efficient derivation,expansion,and differentiation of CVPCs from hPSCs in a chemically defined medium under feeder- and serum-free culture conditions. View Publication