技术资料

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

Under stress conditions such as infection or inflammation the body rapidly needs to generate new blood cells that are adapted to the challenge. Haematopoietic cytokines are known to increase output of specific mature cells by affecting survival,expansion and differentiation of lineage-committed progenitors,but it has been debated whether long-term haematopoietic stem cells (HSCs) are susceptible to direct lineage-specifying effects of cytokines. Although genetic changes in transcription factor balance can sensitize HSCs to cytokine instruction,the initiation of HSC commitment is generally thought to be triggered by stochastic fluctuation in cell-intrinsic regulators such as lineage-specific transcription factors,leaving cytokines to ensure survival and proliferation of the progeny cells. Here we show that macrophage colony-stimulating factor (M-CSF,also called CSF1),a myeloid cytokine released during infection and inflammation,can directly induce the myeloid master regulator PU.1 and instruct myeloid cell-fate change in mouse HSCs,independently of selective survival or proliferation. Video imaging and single-cell gene expression analysis revealed that stimulation of highly purified HSCs with M-CSF in culture resulted in activation of the PU.1 promoter and an increased number of PU.1(+) cells with myeloid gene signature and differentiation potential. In vivo,high systemic levels of M-CSF directly stimulated M-CSF-receptor-dependent activation of endogenous PU.1 protein in single HSCs and induced a PU.1-dependent myeloid differentiation preference. Our data demonstrate that lineage-specific cytokines can act directly on HSCs in vitro and in vivo to instruct a change of cell identity. This fundamentally changes the current view of how HSCs respond to environmental challenge and implicates stress-induced cytokines as direct instructors of HSC fate. View Publication

Induced pluripotent stem cells (iPSCs) hold promise for the treatment of motoneuron diseases because of their distinct features including pluripotency,self-derivation and potential ability to differentiate into motoneurons. However,it is still unknown whether human iPSC-derived motoneurons can functionally innervate target muscles in vivo,which is the definitive sign of successful cell therapy for motoneuron diseases. In the present study,we demonstrated that human iPSCs derived from mesenchymal cells of the umbilical cord possessed a high yield in neural differentiation. Using a chemically-defined in vitro system,human iPSCs efficiently differentiated into motoneurons which displayed typical morphology,expressed specific molecules,and generated repetitive trains of action potentials. When transplanted into the injured musculocutaneous nerve of rats,they survived robustly,extended axons along the nerve,and formed functional connections with the target muscle (biceps brachii),thereby protecting the muscle from atrophy. Our study provides evidence for the first time that human iPSC-derived motoneurons are truly functional not only in vitro but also in vivo,and they have potential for stem cell-based therapies for motoneuron diseases. textcopyright 2013 Elsevier B.V. View Publication

The epithelial-mesenchymal transition (EMT) is activated in cancer cells by ZEB1,a member of the zinc finger/homeodomain family of transcriptional repressors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in human carcinoma cells. The present studies in breast cancer cells demonstrate that the oncogenic MUC1-C subunit induces expression of ZEB1 by a NF-$$B (nuclear factor kappa B) p65-dependent mechanism. MUC1-C occupies the ZEB1 promoter with NF-$$B p65 and thereby promotes ZEB1 transcription. In turn,ZEB1 associates with MUC1-C and the ZEB1/MUC1-C complex contributes to the transcriptional suppression of miR-200c,an inducer of epithelial differentiation. The co-ordinate upregulation of ZEB1 and suppression of miR-200c has been linked to the induction of EMT. In concert with the effects of MUC1-C on ZEB1 and miR-200c,we show that MUC1-C induces EMT and cellular invasion by a ZEB1-mediated mechanism. These findings indicate that (i) MUC1-C activates ZEB1 and suppresses miR-200c with the induction of EMT and (ii) targeting MUC1-C could be an effective approach for the treatment of breast and possibly other types of cancers that develop EMT properties. View Publication