技术资料

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs,the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA)--induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8),which are expressed in simple epithelial cells,while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4- simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent cells have the potential to provide an unlimited source of tissues for regenerative medicine. For this purpose,development of defined/xeno-free culture systems under feeder-free conditions is essential for the expansion of hESCs. Most defined/xeno-free media for the culture of hESCs contain basic fibroblast growth factor (bFGF). Therefore,bFGF is thought to have an almost essential role for the expansion of hESCs in an undifferentiated state. Here,we report identification of small molecules,some of which were neurotransmitter antagonists (trimipramine and ethopropazine),which promote long-term hESC self-renewal without bFGF in the medium. The hESCs maintained high expression levels of pluripotency markers,had a normal karyotype after 20 passages,and could differentiate into all three germ layers. ?? 2013 Elsevier Inc. View Publication

The mammalian central nervous system (CNS) undergoes significant expansion postnatally,producing astrocytes,oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy,a condition commonly treated with valproate/valproic acid (VPA),a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs,however,may be essential for the differentiation of distinct subpopulations of neurons and glia. Here,we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7-P14,combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG,rostral migratory stream (RMS),and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed,however,that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA,OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity,we found that short term treatment with VPA in vivo reduced neurosphere numbers and size,a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor,Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively,these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered,especially in individuals whose brains are actively undergoing key postnatal time windows of development. View Publication

Background: The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). Method: Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4,KChIP2 and Kv4.3 were quantified from BCs. Results: BCs exhibited spontaneous beating (60.5??2.6bpm) and maximum-diastolic-potential (MDP) of 67.8??0.8mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7??1.9 pA/pF at +40mV; n=14). Recovery of Ito from inactivation (at -80mV) showed slow kinetics (??1=200??110ms (12%) and ??2=2380??240ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. Conclusion: The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype. ?? 2013 Elsevier Ltd. View Publication

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2),synthesised by PIKfyve,regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex,leading to even a mild reduction in PtdIns(3,5)P 2,results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity,using YM-201636,significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly,YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II,an effect that was potentiated by inhibition of lysosomal proteases,suggesting that alterations in autophagy could be a contributing factor to neuronal cell death. View Publication

ALDH1(+)CD44(+) cells are putative tumor-initiating cells (TIC) in head and neck squamous cell carcinomas (HNC). miR-145 regulates tumorigenicity in various cancers but the breadth of its mechanistic contributions and potential therapeutic applications are not completely known. Here,we report that ALDH1(+)CD44(+)-HNC cells express reduced levels of miR145. SPONGE-mediated inhibition of miR-145 (Spg-miR145) was sufficient to drive tumor-initiating characteristics in non-TICs/ALDH1(-)CD44-negative HNC cells. Mechanistic analyses identified SOX9 and ADAM17 as two novel miR145 targets relevant to this process. miR-145 expression repressed TICs in HNC in a manner associated with SOX9 interaction with the ADAM17 promoter,thereby activating ADAM17 expression. Notably,the SOX9/ADAM17 axis dominated the TIC-inducing activity of miR-145. Either miR-145 suppression or ADAM17 overexpression in non-TICs/ALDH1(-)CD44(-)-HNC cells increased expression and secretion of interleukin (IL)-6 and soluble-IL-6 receptor (sIL-6R). Conversely,conditioned medium from Spg-miR145-transfected non-TICs/ALDH1(-)CD44(-)-HNC cells was sufficient to confer tumor-initiating properties in non-TICs/ALDH1(-)CD44(-)-HNC and this effect could be abrogated by an IL-6-neutralizing antibody. We found that curcumin administration increased miR-145 promoter activity,thereby decreasing SOX9/ADAM17 expression and eliminating TICs in HNC cell populations. Delivery of lentivral-miR145 or orally administered curcumin blocked tumor progression in HNC-TICs in murine xenotransplant assays. Finally,immunohistochemical analyses of patient specimens confirmed that an miR-145(low)/SOX9(high)/ADAM17(high) phenotype correlated with poor survival. Collectively,our results show how miR-145 targets the SOX9/ADAM17 axis to regulate TIC properties in HNC,and how altering this pathway may partly explain the anticancer effects of curcumin. By inhibiting IL-6 and sIL-6R as downstream effector cytokines in this pathway,miR-145 seems to suppress a paracrine signaling pathway in the tumor microenvironment that is vital to maintain TICs in HNC. View Publication

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study,we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4,hSox2,hKlf4,and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci,a hallmark of DM1,were detected in DM1 iPSCs,neural stem cells (NSCs),and terminally differentiated neurons and astrocytes. In conclusion,we have successfully established disease-specific human DM1 iPSC lines,NSCs,and neuronal lineages with pathognomonic intranuclear RNA foci,which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development. View Publication

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA),known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as MELAS demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multi-lineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts,the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA,resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. Upon comparative differentiation of iPSC clones,improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared to isogenic clones with high heteroplasmy. Thus,mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny,and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. View Publication

Human pluripotent stem cells (hPSCs) have an unparalleled potential to generate limitless quantities of any somatic cell type. However,current methods for producing populations of various somatic cell types from hPSCs are generally not standardized and typically incorporate undefined cell culture components often resulting in variable differentiation efficiencies and poor reproducibility. To address this,we have developed a defined approach for generating epithelial progenitor and epidermal cells from hPSCs. In doing so,we have identified an optimal starting cell density to maximize yield and maintain high purity of K18+/p63+ simple epithelial progenitors. In addition,we have shown that the use of synthetic,defined substrates in lieu of Matrigel and gelatin can successfully facilitate efficient epithelial differentiation,maintaining a high (backslashtextgreater75%) purity of K14+/p63+ keratinocyte progenitor cells and at a two to threefold higher yield than a previously reported undefined differentiation method. These K14+/p63+ cells also exhibited a higher expansion potential compared to cells generated using an undefined differentiation protocol and were able to terminally differentiate and recapitulate an epidermal tissue architecture in vitro. In summary,we have demonstrated the production of populations of functional epithelial and epidermal cells from multiple hPSC lines using a new,completely defined differentiation strategy. 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

Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However,the low efficiency of iPSC derived from somatic cells (0.01-0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal,proliferation,and maintenance of embryonic stem cells (ESCs),but the contribution of this pathway or its well-known negative regulator,phosphatase,and tensin homolog deleted on chromosome ten (Pten),to somatic cell reprogramming remains largely unknown. Here,we show that activation of the PI3K pathway by the Pten inhibitor,dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate,improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs. View Publication

Monocytic lineage cells (monocytes,macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established,these methods depend on the use of xenogeneic materials and,therefore,have a relatively poor-reproducibility. Here,we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6) ± 0.3 × 10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine,disease-specific iPSC studies and drug discovery. View Publication