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

Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes,shRNAs,miRNAs,or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines. View Publication

Induced pluripotent stem (iPS) cells have great potential for regenerative medicine and gene therapy. Thus far,iPS cells have typically been generated using integrating viral vectors expressing various reprogramming transcription factors; nonintegrating methods have been less effective and efficient. Because there is a significant risk of malignant transformation and cancer involved with the use of iPS cells,careful evaluation of transplanted iPS cells will be necessary in small and large animal studies before clinical application. Here,we have generated and characterized nonhuman primate iPS cells with the goal of evaluating iPS cell transplantation in a clinically relevant large animal model. We developed stable Phoenix-RD114-based packaging cell lines that produce OCT4,SOX2,c-MYC,and KLF4 (OSCK) expressing gammaretroviral vectors. Using these vectors in combination with small molecules,we were able to efficiently and reproducibly generate nonhuman primate iPS cells from pigtailed macaques (Macaca nemestrina). The established nonhuman primate iPS cells exhibited pluripotency and extensive self-renewal capacity. The facile and reproducible generation of nonhuman primate iPS cells using defined producer cells as a source of individual reprogramming factors should provide an important resource to optimize and evaluate iPS cell technology for studies involving stem cell biology and regenerative medicine. View Publication

Human pluripotent stem cells (PSCs),encompassing embryonic stem cells and induced pluripotent stem cells,proliferate extensively and differentiate into virtually any desired cell type. PSCs endow regenerative medicine with an unlimited source of replacement cells suitable for human therapy. Several hurdles must be carefully addressed in PSC research before these theoretical possibilities are translated into clinical applications. These obstacles are: (1) cell proliferation; (2) cell differentiation; (3) genetic integrity; (4) allogenicity; and (5) ethical issues. We discuss these issues and underline the fact that the answers to these questions lie in a better understanding of the biology of PSCs. To contribute to this aim,we have developed a free online expression atlas,Amazonia!,that displays for each human gene a virtual northern blot for PSC samples and adult tissues (http://www.amazonia.transcriptome.eu). View Publication

Somatic cell nuclear transfer,cell fusion,or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors,Brn2 (also known as Pou3f2),Ascl1 and Myt1l,can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1,these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers,even after downregulation of the exogenous transcription factors. Importantly,the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells,as well as pluripotent stem cells,can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine. View Publication

Micro RNAs (miRNAs),small and labile ˜22 nucleotide-sized fragments of single stranded RNA,are important regulators of messenger (mRNA) complexity and in shaping the transcriptome of a cell. In this communication,we utilized amyloid beta 42 (Aβ42) peptides and interleukin-1beta (IL-1β) as a combinatorial,physiologically-relevant stress to induce miRNAs in human primary neural (HNG) cells (a co-culture of neurons and astroglia). Specific miRNA up-regulation was monitored using miRNA arrays,Northern micro-dot blots and RT-PCR. Selective NF-кB translocation and DNA binding inhibitors,including the chelator and anti-oxidant pyrollidine dithiocarbamate (PDTC) and the polyphenolic resveratrol analog CAY10512 (trans-3,5,4'-trihydroxystilbene),indicated the NF-кB sensitivity of several brain miRNAs,including miRNA-9,miRNA-125b and miRNA-146a. The inducible miRNA-125b and miRNA-146a,and their verified mRNA targets,including 15-lipoxygenase (15-LOX),synapsin-2 (SYN-2),complement factor H (CFH) and tetraspanin-12 (TSPAN12),suggests complex and highly interactive roles for NF-кB,miRNA-125b and miRNA-146a. These data further indicate that just two NF-кB-mediated miRNAs have tremendous potential to contribute to the regulation of neurotrophic support,synaptogenesis,neuroinflammation,innate immune signaling and amyloidogenesis in stressed primary neural cells of the human brain. View Publication

Regenerative medicine,relying on human embryonic stem cell (hESC) technology,opens promising new avenues for therapy of many severe diseases. However,this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress,growth rates,and heterogeneous cellular states under current culture conditions. In this study,we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states,precisely control growth rates,significantly increase cell production,and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple,robust,scalable,and suitable for high-throughput screening and drug discovery. View Publication

Glioblastoma growth is driven by cancer cells that have stem cell properties,but molecular determinants of their tumorigenic behavior are poorly defined. In cancer,altered activity of the epigenetic modifiers Polycomb and Trithorax complexes may contribute to the neoplastic phenotype. Here,we provide the first mechanistic insights into the role of the Trithorax protein mixed lineage leukemia (MLL) in maintaining cancer stem cell characteristics in human glioblastoma. We found that MLL directly activates the Homeobox gene HOXA10. In turn,HOXA10 activates a downstream Homeobox network and other genes previously characterized for their role in tumorigenesis. The MLL-Homeobox axis we identified significantly contributes to the tumorigenic potential of glioblastoma stem cells. Our studies suggest a role for MLL in contributing to the epigenetic heterogeneity between tumor-initiating and non-tumor-initiating cells in glioblastoma. View Publication

We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs),partially reprogrammed cells,somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over approximately 10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free,enriched to 95%-99% purity with textgreater80% survival,and had normal transcriptional profiles,differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells. View Publication

The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However,the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis,the non-canonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a two-day window showed significantly enhanced osteogenic yield. Mechanistically,rWNT5a supplementation up-regulated PKC,CamKII and JNK activity while antagonizing the key effector of canonical Wnt signaling: beta-catenin. Conversely,when recombinant WNT3a (rWNT3a) or other positive regulators of ?-catenin were employed during this same time-window there was a decrease in osteogenic marker expression. However,if rWNT3a was supplemented during a time-window following rWNT5a treatment,osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine. View Publication

There is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust,patient-specific tissue model systems for studying the pathogenesis of vascular disease,and for developing novel therapeutic interventions. Here,we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore,we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system,extendable to study other vascular proliferative diseases for drug screening. Thus,this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications. View Publication

The $\beta$-haemoglobinopathies,such as sickle cell disease and $\beta$-thalassaemia,are caused by mutations in the $\beta$-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure $\beta$-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably,we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90{\%} targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that,after differentiation into erythrocytes,express adult $\beta$-globin (HbA) messenger RNA,which confirms intact transcriptional regulation of edited HBB alleles. Collectively,these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for $\beta$-haemoglobinopathies. View Publication

The CD34 molecule belongs to the mucin membrane molecule family and is expressed on virtually all normal haematopoietic progenitor cells (HPC). Due to its heavy glycosylation,several different epitopes exist on the molecule. Based on the sensitivity of the glycosylated molecule to degradation with a glycoprotease from Pasteurella haemolytica and neuraminidase,three classes of epitopes have been identified. The class I and II epitopes are probably related to the glycosylated part of the molecule while class III epitopes are core protein related. It has been known for some time that CD34 class I epitopes are absent on CD34 molecules expressed on high endothelial venules. Here we review recent observations that expression of both class I and II epitopes,but not class III epitopes,is impaired on mature myeloid CD34-pos. HPC while no diverse class epitope expression was observed on immature HPC. In addition,cells from patients with CD34-pos. acute myeloid leukaemia of FAB classification M4-M5,i.e.,leukaemic blast cells of relatively mature morphologic phenotype,also express less class I and II epitopes than class III epitopes. It therefore seems that HPC maturation and class I and II epitope deprivation are concomitant events and that CD34 class I and II epitopes are lost prior to downregulation of the CD34 molecule per se. The biological significance of this observation is discussed as well as the need to carefully select CD34-specific monoclonal antibodies for research and clinical purposes. View Publication