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

Two groups recently reported the in vitro differentiation of human embryonic stem cells into insulin-secreting cells,achieving an elusive goal for regenerative medicine. Herein we provide a perspective regarding these developments,compare phenotypes of the insulin-containing cells to human $\beta$ cells,and discuss implications for type 1 diabetes research and clinical care. View Publication

The type III family of IFNs displays immunomodulatory and antiviral activity. Each member (IFN-lambda1,-2,and -3) signals through the same heterodimeric receptor complex,which consists of the binding and signaling subunit (IL-28Ralpha) plus the IL-10Rbeta chain. Although the receptor has a wide tissue distribution,the direct effects of IFN-lambda on various immune cell subsets have not been fully characterized. We have identified high levels of IL-28Ralpha mRNA in pDC from peripheral blood and hypothesized that IFN-lambda plays an important role in pDC maturation and development. We show that stimulation of pDC with HSV or Imiquimod causes an increase in IL-28Ralpha mRNA. In these cells,IFN-lambda1 alters expression of the costimulatory molecules CD80 and ICOS-L and synergizes with IFN-alpha to up-regulate CD83. In addition,IFN-lambda1 has a variable effect on the homing molecule expression of pDC and mDC. IFN-lambda1-treated pDC display a marked difference in their ability to stimulate production of the signature cytokines IL-13,IFN-gamma,and IL-10 in a MLR. This work characterizes the variable effects of IFN-lambda on DC surface molecule expression and identifies a role in pDC activation and immunostimulatory potential. View Publication

Although distinct human induced pluripotent stem cell (hiPSC) lines can display considerable epigenetic variation,it has been unclear whether such variability impacts their utility for disease modeling. Here,we show that although low-passage female hiPSCs retain the inactive X chromosome of the somatic cell they are derived from,over time in culture they undergo an erosion" of X chromosome inactivation (XCI). This erosion of XCI is characterized by loss of XIST expression and foci of H3-K27-trimethylation� View Publication

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG),affecting many cell types including those of the pancreas. Indeed,XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin,insulin,and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development,we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected),glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult $$-cells. Differentiated ARX knockout cells upregulated PAX4,NKX2.2,ISL1,HHEX,PCSK1,PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide,somatostatin,glucagon and insulin positive cells from hESCs. View Publication

An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry,expansion of the progenitor population,and delayed neuronal differentiation,all key features of the developing human cortex. In contrast,Pten deletion in mouse organoids does not lead to folding. Finally,we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex. View Publication

Functional cardiomyocytes can be efficiently derived from human pluripotent stem cells (hPSCs),which collectively include embryonic and induced pluripotent stem cells. This cellular platform presents exciting new opportunities for development of pharmacologically relevant in vitro screens to detect cardiotoxicity,validate novel drug candidates in preclinical trials and understand complex congenital cardiovascular disorders,to advance current clinical therapies. Here,we discuss the progress and impediments the field has faced in using hPSC-derived cardiomyocytes for these in vitro applications,and highlight that rigorous protocol optimisation and standardisation,scalability and automation are remaining obstacles for the generation of pure,mature and clinically relevant hPSC cardiomyocytes. View Publication

Under specific conditions,cultured human embryonic stem cells (hESCs) corresponding to primed post-implantation epiblasts can be converted back to a ‘naïve pluripotency’ state that resembles the pre-implantation epiblasts. The core pluripotency factor OCT4 is known to be crucial in regulating different states of pluripotency,but its potential regulatory role in human naïve pluripotency remains unexplored. In this study,we systematically mapped out phosphorylation sites in OCT4 protein that are differentially phosphorylated between two states of pluripotency,and further identified ATR as a key kinase that phosphorylated OCT4 in naïve but not primed hESCs. The kinase activity levels of ATR in naïve hESCs were higher than those in primed hESCs. Ablating cellular ATR activity significantly halted the induction of naïve hESCs from their primed counterparts,and increased early apoptotic death of naïve hESCs upon UV and CPT treatment. Thus,our work reveals the importance of ATR activity in safeguarding human naïve pluripotency,and implicates a potential association of OCT4 phosphorylation,DNA damage sensing and repairing system in regulating different states of pluripotency during early development. View Publication

Using live-cell microscopy,we find that loss of VPS13C in human neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal tethers,leading to impaired lysosomal motility and defective lysosomal function as well as a decreased phospho-Rab10-mediated lysosomal stress response. Loss-of-function mutations in VPS13C are linked to early-onset Parkinson’s disease (PD). While VPS13C has been previously studied in non-neuronal cells,the neuronal role of VPS13C in disease-relevant human dopaminergic neurons has not been elucidated. Using live-cell microscopy,we investigated the role of VPS13C in regulating lysosomal dynamics and function in human iPSC-derived dopaminergic neurons. Loss of VPS13C in dopaminergic neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal contacts,leading to impaired lysosomal motility and cellular distribution,as well as defective lysosomal hydrolytic activity and acidification. We identified Rab10 as a phospho-dependent interactor of VPS13C on lysosomes and observed a decreased phospho-Rab10-mediated lysosomal stress response upon loss of VPS13C. These findings highlight an important role of VPS13C in regulating lysosomal homeostasis in human dopaminergic neurons and suggest that disruptions in Rab10-mediated lysosomal stress response contribute to disease pathogenesis in VPS13C-linked PD. View Publication

Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study,we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures,including upregulation of inflammatory chemokines,lack of neuronal apoptosis,and axonal transmission of viruses in neuronal networks. In addition,we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies,which could lead to improved control methods. View Publication

Radiotherapy is a standard treatment of pediatric brain tumors. Though the survival rate has improved for many tumor types,most patients suffer long-term cognitive decline and there is currently no way of preventing radiation-induced damage to healthy brain tissue. Here,we used a human forebrain organoid model to investigate if the α2-adrenoceptor and I1-imidazoline receptor agonist clonidine could prevent radiotoxicity. We found that treatment of organoids with clonidine significantly reduced radiation-induced loss of neural progenitor cells,neurons,astrocytes,and oligodendrocyte lineage cells. Moreover,clonidine reduced overall DNA damage and signs of reactive gliosis in organoids. Our findings demonstrate that pharmacological rescue of radiation neurotoxicity is possible in a human brain organoid model and provides a rationale for future drug repurposing studies aiming to prevent radiation-induced brain injury in children treated with radiotherapy. View Publication