技术资料

Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of dextran (DEX) onto polyethylene glycol (PEG)-covered cells though a glass capillary needle connected to a pneumatic pump. This technique is applied to purify colonies of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblast (MEF) feeder cultures and inefficiently induced iPSC colonies by selectively dissociating the iPSCs with proteases. View Publication

The family of calcium activated potassium channels of low and intermediate conductance,known as SK channels,consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes,such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date,the role of SK channels in developmental processes has been merely a marginal focus of investigation,although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently,several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein,we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells,neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly,we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis. ?? 2013 Elsevier GmbH. View Publication

Human somatic cells can be reprogrammed to the pluripotent state to become human-induced pluripotent stem cells (hiPSC). This reprogramming is achieved by activating signaling pathways that are expressed during early development. These pathways can be induced by ectopic expression of four transcription factors—Oct4,Sox2,Klf4,and c-Myc. Although there are many ways to deliver these transcription factors into the somatic cells,this chapter will provide protocols that can be used to generate hiPSC from lentiviruses. View Publication

Induced pluripotent stem cells (iPSC) have successfully been derived from somatic fibroblasts through transfection of synthetic modified mRNA encoding transcription factors. This technique obviates the use of recombinant DNA and viral vectors in cellular reprogramming. The present study derived iPSC from adipose-derived mesenchymal stem cells (of a 50-year-old female patient) by utilizing a similar technique,but with defined culture medium without feeder cells,during both reprogramming and propagation. Clonal selection was performed to yield 12 putative iPSC lines from individual colonies of nascent reprogrammed cells,starting from 150,000 cells. However,only seven lines maintained their undifferentiated state after 10 continuous serial passages. These seven lines were then subjected to a rigorous battery of analyses to confirm their identity as iPSC. These tests included immunostaining,flow cytometry,qRT-PCR,in vitro differentiation assay,and teratoma formation assay within SCID mice. Positive results were consistently observed in all analyses,thus verifying the cells as fully reprogrammed iPSC. While all 7 iPSC lines displayed normal karyogram up to passage 13,chromosomal anomalies occurred in 4 of 7 lines with extended in vitro culture beyond 24 serial passages. Only three lines retained normal karyotype of 46,XX. The remaining four lines displayed mosaicism of normal and abnormal karyotypes. Hence,this study successfully derived iPSC from abundant and easily accessible adipose tissues of a middle-aged patient; utilizing a mRNA-based integration-free technique under feeder-free conditions. This is a step forward in translating iPSC into personalized regenerative medicine within the clinic. ?? 2013 Elsevier Inc. View Publication

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however,their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC),human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC),and compared their in-vitro tube formation,migration and cytokine expression profiles,and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless,BM-EC,hESC-EC and hiPSC-EC exhibited typical cobblestone morphology,had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein,and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (Ptextgreater0.05). While increased expression of major angiogenic factors including epidermal growth factor,hepatocyte growth factor,vascular endothelial growth factor,placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (Ptextless0.05),the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (Ptextless0.05). Compared with medium,transplanting BM-EC (n = 6),HUVEC (n = 6),hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion,functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases,and hESC-EC or iPSC-EC are readily available as off-the-shelf" format for the treatment of tissue ischemia." View Publication

Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors,histone-modification enzymes,and mRNA processing proteins. Recent evidence suggests that nucleoporins,well known components that control nucleo-cytoplasmic trafficking,have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation,which initially has been described in fungi and flies,also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition,we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively,genes that are highly induced can interact with NUP98 in the nuclear interior,away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation,revealing a role of a nuclear pore protein in regulating developmental gene expression programs. View Publication

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets,the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study,we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation,cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating,even after cell sheets were detached from culture dishes. Furthermore,extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue. View Publication

Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease,and offer potentialbackslashrbackslashnfor patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function,and to gain insight into cardiac growth and disease,tissue engineers must quantify the contractile forces of these single cells. Currently,axial contractile forces of isolated adult heart cells can only be measuredbackslashrbackslashnby two-point methods such as carbon fiber techniques,which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificialbackslashrbackslashnsupport layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput,is functionally analogous to current gold-standard axial force assays for adult heart cells,and prescribes elongated cell shapes without protein patterning. Finally,we calibrate these force posts withbackslashrbackslashnpiezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far betterbackslashrbackslashnthan that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culturebackslashrbackslashnplatforms will enable improved cell placement and the complex suspension of cells across 3D constructs. View Publication

Cell Research 23,122 (2013). doi:10.1038/cr.2012.119 View Publication

The Hippo pathway is crucial in organ size control,and its dysregulation contributes to tumorigenesis. However,upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here,we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2,thereby activating YAP and TAZ transcription coactivators,which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression,cell migration,and proliferation. In contrast,stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity,thereby inhibiting YAP function. Thus,GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR. View Publication

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally,NPCs are produced in 2D cultures,in low yields,using a laborious process that includes generation of embryonic bodies,plating,and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs,we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC,using iPS (IMR90) as a model cell line. In the expansion stage,a process of cell propagation in serum-free MC culture was developed first in static culture,which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×10�?� cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1-60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×10�?� cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage,NPCs were generated with 78%-85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally,the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into βIII-tubulin�?� neurons,GFAP�?� astrocytes,and O4�?� oligodendrocytes,showing that cells maintained their multilineage differentiation potential. View Publication

Human embryonic stem cells (hESCs) tend to lose genomic integrity during long periods of culture in vitro and to acquire a cancer-like phenotype. In this study,we aim at understanding the contribution of point mutations to the adaptation process and at providing a mechanistic explanation for their accumulation. We observed that,due to the absence of p21/Waf1/Cip1,cultured hESCs lack proper cell cycle checkpoints and are vulnerable to the kind of DNA damage usually repaired by the highly versatile nucleotide excision repair (NER) pathway. In response to UV-induced DNA damage,the majority of hESCs succumb to apoptosis; however,a subpopulation continues to proliferate,carrying damaged DNA and accumulating point mutations with a typical UV-induced signature. The UV-resistant cells retain their proliferative capacity and potential for pluripotent differentiation and are markedly less apoptotic to subsequent UV exposure. These findings demonstrate that,due to deficient DNA damage response,the modest NER activity in hESCs is insufficient to prevent increased mutagenesis. This provides for the appearance of genetically aberrant hESCs,paving the way for further major genetic changes. View Publication