技术资料

Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle,two orthogonal strategies for reducing off-target cleavage,truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs),could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally,we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing. View Publication

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1$$ (HIF-1$$) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1$$ in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study,we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1$$ and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1$$. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats,DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone,leading to bone regeneration. Collectively,the results indicate that DMOG,via activation of the PI3K/Akt pathway,promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration. View Publication

Infection with a recombinant murine-feline gammaretrovirus,MoFe2,or with the parent virus,Moloney murine leukemia virus,caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective,in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state. View Publication

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases,including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells,direct cell reprogramming driven by lineagedetermining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter,we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover,they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination,but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling,drug screening,and cell therapy of retinal degeneration. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia,haploid spermatocytes,or spermatids. Here,we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages,including postmeiotic,spermatid-like cells,in vitro without genetic manipulation. Furthermore,our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-,PLZF-,and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin,transition protein 1,and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro View Publication

The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human nonmobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4,SOX2,KLF4,and c-MYC. The PB- and BM-derived iPSCs were quite similar to human embryonic stem cells with regard to morphology,expression of surface antigens and pluripotency-associated transcription factors,global gene expression profiles,and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines,although transduction efficiencies were not high. We found that 5 × 10(5) PB MNCs,which corresponds to less than 1 mL of PB,was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of nonmobilized PB,with its relative efficiency and ease of harvesting,could enable the therapeutic use of patient-specific pluripotent stem cells. View Publication

Human engineered heart tissues have potential to revolutionize cardiac development research,drug-testing,and treatment of heart disease; however,implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment,we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward,ontomimetic approach,imitating the process of development,requires only a single cell-handling step,provides reproducible results for a range of tested geometries and size scales,and overcomes inherent limitations in cell maintenance and maturation,while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation,mimicking heart development,and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue. View Publication

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here,we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore,in vitro,iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice,human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together,our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons. View Publication

Advancing pluripotent stem cell technologies for modelling haematopoietic stem cell development and blood therapies requires identifying key regulators of haematopoietic commitment from human pluripotent stem cells (hPSCs). Here,by screening the effect of 27 candidate factors,we reveal two groups of transcriptional regulators capable of inducing distinct haematopoietic programs from hPSCs: pan-myeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1). In both cases,these transcription factors directly convert hPSCs to endothelium,which subsequently transform into blood cells with pan-myeloid or erythro-megakaryocytic potential. These data demonstrate that two distinct genetic programs regulate the haematopoietic development from hPSCs and that both of these programs specify hPSCs directly to haemogenic endothelial cells. In addition,this study provides a novel method for the efficient induction of blood and endothelial cells from hPSCs via the overexpression of modified mRNA for the selected transcription factors. View Publication

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined,low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements,express neural precursor markers,generate neurons and glia in vitro,and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment. View Publication

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4,MEF2C,and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However,GMT alone appears insufficient in human fibroblasts,at least in vitro. Here,we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells,fetal heart,and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming,including sarcomere formation,calcium transients,and action potentials,although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore,we found that transforming growth factor β signaling was important for,and improved the efficiency of,human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage,and lay the foundation for future refinements in vitro and in vivo. textcopyright 2013 The Authors. View Publication

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors,useful for both basic research and clinical applications. Besides their characterization in colony assays,protocols exist for the cultivation of lymphoid,myeloid,and erythroid cells. With the possible exception of mast cells,however,long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here,we describe for the first time an efficient yet easy strategy to generate mass cultures of pure,immature erythroid progenitors from mouse ES cells (ES-EPs),using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived,adult,definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions,ES-EPs differentiated into mature,enucleated erythrocytes. Importantly,ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition,the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells. View Publication