技术资料

Primitive erythroid cells,the first red blood cells produced in the mammalian embryo,are necessary for embryonic survival. Erythropoietin and its receptor EpoR,are absolutely required for survival of late-stage definitive erythroid progenitors in the fetal liver and adult bone marrow. Epo- and Epor-null mice die at E13.5 with a lack of definitive erythrocytes. However,the persistence of circulating primitive erythroblasts raises questions about the role of erythropoietin/EpoR in primitive erythropoiesis. Using Epor-null mice and a novel primitive erythroid 2-step culture we found that erythropoietin is not necessary for specification of primitive erythroid progenitors. However,Epor-null embryos develop a progressive,profound anemia by E12.5 as primitive erythroblasts mature as a synchronous cohort. This anemia results from reduced primitive erythroblast proliferation associated with increased p27 expression,from advanced cellular maturation,and from markedly elevated rates of apoptosis associated with an imbalance in pro- and anti-apoptotic gene expression. Both mouse and human primitive erythroblasts cultured without erythropoietin also undergo accelerated maturation and apoptosis at later stages of maturation. We conclude that erythropoietin plays an evolutionarily conserved role in promoting the proliferation,survival,and appropriate timing of terminal maturation of primitive erythroid precursors. View Publication

Realization of the full potential of human induced pluripotent stem cells (hiPSC) in clinical applications requires the development of well-defined culture conditions for their long-term growth and directed differentiation. This paper describes a novel fully defined synthetic peptide-decorated substrate that supports self-renewal of hiPSC in commercially available xeno-free,chemically defined medium. The Au surface was deposited by a poly(OEGMA-co-HEMA) film,using the surface-initiated polymerization method (SIP) with the further step of carboxylation. The hiPSC generated from umbilical cord mesenchymal cells were successfully cultured for 10 passages on the peptide-tethered poly(OEGMA-co-HEMA) brushes for the first time. Cells maintained their characteristic morphology,proliferation and expressed high levels of markers of pluripotency,similar to the cells cultured on Matrigel???. Moreover,the cell adhesion could be tuned by the pattern and peptide concentration on the substrate. This well-defined,xeno-free and safe substrate,which supports long-term proliferation and self-renewal of hiPSC,will not only help to accelerate the translational perspectives of hiPSC,but also provide a platform to elucidate the underlying molecular mechanisms that regulate stem cell proliferation and differentiation via SIP technology. ?? 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. View Publication

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

INTRODUCTION: The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming,however,represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA),an excisable lentiviral reprogramming vector,provides,in our hands,the most consistent reprogramming approach that addresses this safety concern. Nevertheless,most viral integrations occur in genes,and exactly how the integration,epigenetic reprogramming,and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. METHODS: In this study,we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry,karyotyping and teratoma formation,and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. RESULTS: We found that a STEMCCA-derived iPSC line that contains a single integration,found to be located in an intronic location in an actively transcribed gene,PRPF39,displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs,differentiated them into multiple clinically relevant cell types (including oligodendrocytes,hepatocytes,and cardiomyocytes),and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. CONCLUSION: For the first time,these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and,in light of the limited availability of current good manufacturing practice cellular manufacturing facilities,highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics. View Publication

Human pluripotent stem cells (hPSCs) have been differentiated to astroglia,but the utilization of hPSC-derived astroglia as cell therapy for neurological diseases has not been well studied. Astroglia are heterogeneous,and not all astroglia are equivalent in promoting neural repair. A prerequisite for cell therapy is to derive defined cell populations with superior therapeutic effects. Here we use an Olig2-GFP human embryonic stem cell (hESC) reporter to demonstrate that hESC-derived Olig2(+) progenitors generate a subtype of previously uncharacterized astroglia (Olig2PC-Astros). These Olig2PC-Astros differ substantially from astroglia differentiated from Olig2-negative hESC-derived neural progenitor cells (NPC-Astros),particularly in their neuroprotective properties. When grafted into brains subjected to global ischaemia,Olig2PC-Astros exhibit superior neuroprotective effects and improved behavioural outcome compared to NPC-Astros. Thus,this new paradigm of human astroglial differentiation is useful for studying the heterogeneity of human astroglia,and the unique Olig2PC-Astros may constitute a new cell therapy for treating cerebral ischaemia and other neurological diseases. View Publication

Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling,which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs,suggesting that they may influence self-renewal. To test this hypothesis,we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation,whereas SPRY4 KD enhanced survival. In both cases,after KD the cells were able to differentiate into cells of the three germ layers,although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage,explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs. View Publication

AIMS: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however,resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells. METHODS: We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9,PC9GR,H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry. RESULTS: In the present study,we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity,whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels. CONCLUSION: Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated. View Publication

Hepatocytes play a central and crucial role in cholesterol and lipid homeostasis,and their proper function is of key importance for cardiovascular health. In particular,hepatocytes (especially periportal hepatocytes) endogenously synthesize large amounts of cholesterol and secrete it into circulating blood via apolipoprotein particles. Cholesterol-secreting hepatocytes are also the clinically-relevant cells targeted by statin treatment in vivo. The study of cholesterol homeostasis is largely restricted to the use of animal models and immortalized cell lines that do not recapitulate those key aspects of normal human hepatocyte function that result from genetic variation of individuals within a population. Hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells can provide a cell culture model for the study of cholesterol homeostasis,dyslipidemias,the action of statins and other pharmaceuticals important for cardiovascular health. We have analyzed expression of core components for cholesterol homeostasis in untreated human iPS cells and in response to pravastatin. Here we show the production of differentiated cells resembling periportal hepatocytes from human pluripotent stem cells. These cells express a broad range of apolipoproteins required for secretion and elimination of serum cholesterol,actively secrete cholesterol into the medium,and respond functionally to statin treatment by reduced cholesterol secretion. Our research shows that HLCs derived from human pluripotent cells provide a robust cell culture system for the investigation of the hepatic contribution to human cholesterol homeostasis at both cellular and molecular levels. Importantly,it permits for the first time to also functionally assess the impact of genetic polymorphisms on cholesterol homeostasis. Finally,the system will also be useful for mechanistic studies of heritable dyslipidemias,drug discovery,and investigation of modes of action of cholesterol-modulatory drugs. View Publication

Renal lineages including kidney are derived from intermediate mesoderm,which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined,serum-free,adherent,monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm,odd-skipped related 1 (Osr1) and Wilm’s Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist,but not by retinoid X receptor (RXR) agonists. Furthermore,the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney. View Publication

Cell fate can be reprogrammed by modifying intrinsic and extrinsic cues. Here we show that two small molecules (forskolin and dorsomorphin) enable the transcription factor Neurogenin 2 (NGN2) to convert human fetal lung fibroblasts into cholinergic neurons with high purity (textgreater90%) and efficiency (up to 99% of NGN2-expressing cells). The conversion is direct without passing through a proliferative progenitor state. These human induced cholinergic neurons (hiCN) show mature electrophysiological properties and exhibit motor neuron-like features,including morphology,gene expression and the formation of functional neuromuscular junctions. Inclusion of an additional transcription factor,SOX11,also efficiently converts postnatal and adult skin fibroblasts from healthy and diseased human patients to cholinergic neurons. Taken together,this study identifies a simple and highly efficient strategy for reprogramming human fibroblasts to subtype-specific neurons. These findings offer a unique venue for investigating the molecular mechanisms underlying cellular plasticity and human neurodegenerative diseases. View Publication

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here,we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates textgreater700 guide RNA variants and SpCas9-induced indel mutation levels at textgreater100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner,sensitive to the number,position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications,we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. View Publication

Pluripotent stem cells can be induced from somatic cells,providing an unlimited cell resource,with potential for studying disease and use in regenerative medicine. However,genetic manipulation and technically challenging strategies such as nuclear transfer used in reprogramming limit their clinical applications. Here,we show that pluripotent stem cells can be generated from mouse somatic cells at a frequency up to 0.2% using a combination of seven small-molecule compounds. The chemically induced pluripotent stem cells resemble embryonic stem cells in terms of their gene expression profiles,epigenetic status,and potential for differentiation and germline transmission. By using small molecules,exogenous master genes" are dispensable for cell fate reprogramming. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications." View Publication