技术资料

Current protocols for human pluripotent stem cell (hPSC) expansion require feeder cells or matrices from animal sources that have been the major obstacle to obtain clinical grade hPSCs due to safety issues,difficulty in quality control,and high expense. Thus,feeder-free,chemically defined synthetic platforms have been developed,but are mostly confined to typical polystyrene culture plates. Here,we report a chemically defined,material-independent,bio-inspired surface coating allowing for feeder-free expansion and maintenance of self-renewal and pluripotency of hPSCs on various polymer substrates and devices. Polydopamine (pDA)-mediated immobilization of vitronectin (VN) peptides results in surface functionalization of VN-dimer/pDA conjugates. The engineered surfaces facilitate adhesion,proliferation,and colony formation of hPSCs via enhanced focal adhesion,cell-cell interaction,and biophysical signals,providing a chemically defined,xeno-free culture system for clonal expansion and long-term maintenance of hPSCs. This surface engineering enables the application of clinically-relevant hPSCs to a variety of biomedical systems such as tissue-engineering scaffolds and medical devices. View Publication

Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell-cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here,we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions,resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions,which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity. View Publication

The development of xeno-free,chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard,various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal,i.e.,proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly,when surface chemistry of the substrates was uniformly controlled by collagen conjugation,the stiffness of substrate was inversely related to the sphericity,a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture,implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall,we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal. View Publication

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD,little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD,we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially,no significant phenotypic differences were observed between iPSCs derived from the different family members. However,upon directed neural differentiation,we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity,including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3,a known regulator of WNT signaling,was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together,these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention. View Publication

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However,modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator,VP64-p65-Rta (VPR),fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes,targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs). View Publication

Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However,these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation,thereby limiting their range of applications. In this protocol,we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA). View Publication

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy,drug development,and studies of cellular differentiation and development. However,the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures,a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging,and feeder-free vs. mouse embryonic fibroblast feeder substrate,on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages,we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability,higher rates of cell proliferation,and persistence of OCT4/POU5F1-positive cells in teratomas,with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers,we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53,which was associated with decreased mRNA expression of TP53,as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures,we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies. View Publication

The use of human pluripotent cell progeny for cardiac disease modeling,drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success,recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR,which in turn induces the Activin-A and Bmp pathways,is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis,and to ultimately promote myocyte maturation,we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq,induction with CHIR during Day 1 (Days 0-1) was followed by immediate expression of Nodal ligands and receptors,followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50-100 ng/ml) during Day 0-1 efficiently induced definitive endoderm,whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency,even when CHIR alone was ineffective. Moreover,co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis,as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast,co-induction with CHIR plus low levels (3-10 ng/ml) of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings,which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression,are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages,while Bmp levels regulate subsequent allocation into mesodermal cell types. View Publication

Reprogramming somatic cells into a pluripotent state involves the overexpression of transcription factors leading to a series of changes that end in the formation of induced pluripotent stem cells (iPSCs). These iPSCs have a wide range of potential uses from drug testing and in vitro disease modelling to personalized cell therapies for patients. While viral methods for reprogramming factor delivery have been traditionally preferred due to their high efficiency,it is now possible to generate iPSCs using nonviral methods at similar efficiencies. We developed a robust reprogramming strategy that combines episomal plasmids and the use of commercially available animal free reagents that can be easily adapted for the GMP manufacture of clinical grade cells. View Publication

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro,increase our understanding of human embryonic development,and provide clinically relevant cell types for transplantation,drug testing,and toxicology studies. Since their discovery,numerous advances have been made in order to eliminate issues such as vector integration into the host genome,low reprogramming efficiency,incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally,we illustrate methods by which to validate pluripotency of the resulting stem cell population. View Publication

UNLABELLED In the classical form of $\$1-antitrypsin deficiency (ATD),aberrant intracellular accumulation of misfolded mutant $\$1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function,proteotoxic" mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation View Publication

Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS),the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility,gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors,important regulators of cell/tissue functions in vivo,influence the survival and growth of human embryonic stem cells. Thus,this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening. View Publication