技术资料

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is a crucial step in cell therapy. In the present study,we demonstrate that functional CD34(+) progenitor cells can be efficiently produced from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) by combined modulation of 2 signaling pathways. A higher proportion of CD34(+) cells (∼ 20%) could be derived from hPSCs by inhibition of mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling and activation of bone morphogenic protein-4 (BMP4) signaling. hPSC-derived CD34(+) progenitor cells further developed to endothelial and smooth muscle cells with functionality. Moreover,they contributed directly to neovasculogenesis in ischemic mouse hind limbs,thereby resulting in improved blood perfusion and limb salvage. Our results suggest that combined modulation of signaling pathways may be an efficient means of differentiating hPSCs into functional CD34(+) progenitor cells. View Publication

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine granulocyte colony-stimulating factor (G-CSF) has become the preferred source of HSPCs for stem cell transplants. However,G-CSF fails to mobilize sufficient numbers of stem cells in up to 10% of donors,precluding autologous transplantation in those donors or substantially delaying transplant recovery time. Consequently,new regimens are needed to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in mice,we mapped the gene encoding the epidermal growth factor receptor (Egfr) to a genetic region modifying G-CSF-mediated HSPC mobilization. Amounts of EGFR in HSPCs inversely correlated with the cells' ability to be mobilized by G-CSF,implying a negative role for EGFR signaling in mobilization. In combination with G-CSF treatment,genetic reduction of EGFR activity in HSPCs (in waved-2 mutant mice) or treatment with the EGFR inhibitor erlotinib increased mobilization. Increased mobilization due to suppression of EGFR activity correlated with reduced activity of cell division control protein-42 (Cdc42),and genetic Cdc42 deficiency in vivo also enhanced G-CSF-induced mobilization. Our findings reveal a previously unknown signaling pathway regulating stem cell mobilization and provide a new pharmacological approach for improving HSPC mobilization and thereby transplantation outcomes. View Publication

Chemical compounds have emerged as powerful tools for modulating ESC functions and deriving induced pluripotent stem cells (iPSCs),but documentation of compound-induced efficient directed differentiation in human ESCs (hESCs) and human iPSC (hiPSCs) is limited. By screening a collection of chemical compounds,we identified compound C (also denoted as dorsomorphin),a protein kinase inhibitor,as a potent regulator of hESC and hiPSC fate decisions. Compound C suppresses mesoderm,endoderm,and trophoectoderm differentiation and induces rapid and high-efficiency neural conversion in both hESCs and hiPSCs,88.7% and 70.4%,respectively. Interestingly,compound C is ineffective in inducing neural conversion in mouse ESCs (mESCs). Large-scale kinase assay revealed that compound C targets at least seven transforming growth factor beta (TGF-β) superfamily receptors,including both type I and type II receptors,and thereby blocks both the Activin and bone morphogenesis protein (BMP) signaling pathways in hESCs. Dual inhibition of Activin and BMP signaling accounts for the effects of compound C on hESC differentiation and neural conversion. We also identified muscle segment homeobox gene 2 (MSX2) as a downstream target gene of compound C and a key signaling intermediate of the BMP pathway in hESCs. Our findings provide a single-step cost-effective method for efficient derivation of neural progenitor cells in adherent culture from human pluripotent stem cells. Therefore,it will be uniquely suitable for the production of neural progenitor cells in large scale and should facilitate the use of stem cells in drug screening and regenerative medicine and study of early human neural development. View Publication

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells,including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate,as well as reversing those decisions during induced pluripotency (iPS),have focused largely on transcriptomic controls. Here,we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore,p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However,expression of constitutively active p70 S6K,but not wild-type p70 S6K,induces differentiation. Additionally,hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation. 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

The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter,we describe protocols for culture,maintenance,and cardiac differentiation of hiPSCs. Then,we provide a basic procedure to transduce hiPSC-CMs. View Publication

The gene of ATP-binding cassette subfamily C member 8 (Abcc8) is cytogenetically located at 11p15.1 and encodes the sulfonylurea receptor (SUR1). SUR1 is a subunit of ATP-sensitive potassium channel (KAPT) in the β-cell regulating insulin secretion. Mutations of ABCC8 are responsible for congenital hyperinsulinism (CHI). Here we reported that an Abcc8 heterozygous mutant cell line was generated by CRISPR/Cas9 technique with 1bp insertion resulting in abnormal splicing on human embryonic stem cell line H1. The phenotypic characteristics of this cell line reveal defective KATP channel and diazoxide-responsive that provides ideal model for molecular pathology research and drug screening for CHI. View Publication

Human embryonic stem cell (hESC) line chHES-480 was derived from abnormal blastocyst diagnosed with adrenoleukodystrophy (ALD) after preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that chHES-480 cell line carried a hemizygous missense mutation c.1825GtextgreaterA(p.Glu609Lys) of ABCD1 gene. Characteristic tests proved that the chHES-480 cell line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. View Publication

Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However,marker-free genome editing using standard protocols remains inefficient,yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing,so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA),donor DNA and piggyBac transposase resulted in efficient,targeted genome editing and concurrent scarless transgene excision. Using this approach,in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%. View Publication

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular,this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells),hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here,we described a new platform which enables,rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular,we devised a protocol that,combining the expression of the CRISPR components with neurogenic factors,generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy,fast and that does not require the generation of stable isogenic clones,practice that is time consuming and for some genes not feasible. View Publication

Human induced pluripotent stem cell (hiPSC) utility is limited by variations in the ability of these cells to undergo lineage-specific differentiation. We have undertaken a transcriptional comparison of human embryonic stem cell (hESC) lines and hiPSC lines and have shown that hiPSCs are inferior in their ability to undergo neuroectodermal differentiation. Among the differentially expressed candidates between hESCs and hiPSCs,we identified a mitochondrial protein,CHCHD2,whose expression seems to correlate with neuroectodermal differentiation potential of pluripotent stem cells. We provide evidence that hiPSC variability with respect to CHCHD2 expression and differentiation potential is caused by clonal variation during the reprogramming process and that CHCHD2 primes neuroectodermal differentiation of hESCs and hiPSCs by binding and sequestering SMAD4 to the mitochondria,resulting in suppression of the activity of the TGFβ signaling pathway. Using CHCHD2 as a marker for assessing and comparing the hiPSC clonal and/or line differentiation potential provides a tool for large scale differentiation and hiPSC banking studies. View Publication

Induced pluripotent stem cells (iPSCs) are being pursued as a source of cells for autologous therapies,many of which will be aimed at aged patients. To explore the impact of age on iPSC quality,we produced iPSCs from blood cells of 16 donors aged 21-100. We find that iPSCs from older donors retain an epigenetic signature of age,which can be reduced through passaging. Clonal expansion via reprogramming also enables the discovery of somatic mutations present in individual donor cells,which are missed by bulk sequencing methods. We show that exomic mutations in iPSCs increase linearly with age,and all iPSC lines analyzed carry at least one gene-disrupting mutation,several of which have been associated with cancer or dysfunction. Unexpectedly,elderly donors (textgreater90 yrs) harbor fewer mutations than predicted,likely due to a contracted blood progenitor pool. These studies establish that donor age is associated with an increased risk of abnormalities in iPSCs and will inform clinical development of reprogramming technology. View Publication