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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

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

Recent studies have suggested that REX1 (reduced expression 1) plays an important role in pluripotency,proliferation,and differentiation. However,the molecular mechanisms involved in REX1-dependent regulation of diverse cellular processes remain unclear. To elucidate the regulatory functions of REX1 in human embryonic stem cells (hESCs),comparative proteomic analysis was performed on REX1 RNAi specifically silenced hESCs. Analysis of the proteome via nano-LC-MS/MS identified 140 differentially expressed proteins (DEPs) displaying a textgreater2-fold difference in expression level between control and REX1 knockdown (KD) hESCs,which were then compared with transcriptome data and validated by quantitative real-time RT-PCR and Western blotting. These DEPs were analyzed by GO,pathway,and functional clustering analyses to determine the molecular functions of the proteins and pathways regulated by REX1. The REX1 KD-mediated DEPs mapped to major biological processes involved in the regulation of ribosome-mediated translation and mitochondrial function. Functional network analysis revealed a highly interconnected network among these DEPs and indicated that these interconnected proteins are predominantly involved in translation and the regulation of mitochondrial organization. These findings regarding REX1-mediated regulatory network have revealed the contributions of REX1 to maintaining the status of hESCs and have improved our understanding of the molecular events that underlie the fundamental properties of hESCs. View Publication

BACKGROUND Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs),but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches,but the global proteome has not been examined. Furthermore,most hESC-CM studies focus on pathways important for cardiac differentiation,rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs,hESC-derived VCMs,human fetal and human adult ventricular and atrial CMs. METHODS AND RESULTS Using two-dimensional-differential-in-gel electrophoresis,121 differentially expressed (textgreater1.5-fold; Ptextless0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator-activated receptor $\$ in cardiac maturation. Consistently,WY-14643,a peroxisome proliferator-activated receptor $\$,increased fatty oxidative enzyme level,hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line,treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold,signifying their maturation. CONCLUSIONS We conclude that the peroxisome proliferator-activated receptor $\$ thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs. View Publication

Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes,shRNAs,miRNAs,or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines. View Publication

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders,some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here,we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program,TALENSeek,(2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol,and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify,construct,and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity. View Publication

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling,drug development,screening,and the potential for patient-matched" cellular therapies in neurodegenerative diseases. In this study� View Publication

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) $$$$ and $$$$ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells. View Publication

The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously,we identified a human pluripotent stem-cell-specific lectin probe,called rBC2LCN,by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A,termed rBC2LCN-PE23,which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells,followed by its internalization,allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus,rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies. View Publication

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Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases,both to study them and to explore therapies. However,a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study,we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV),adenoviruses and lentiviral vectors in hESC,hiPSC,and the derived cardiomyocytes. In undifferentiated cells,adenoviral and lentiviral vectors were superior,whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes,1,2,6,and 9,of which 2 and 6 were superior in their transduction efficiency. Interestingly,we observed that AAVs severely diminished the viability of undifferentiated cells,an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore,we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally,adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion,adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines,whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells. View Publication

Reprogramming of patient cells to human induced pluripotent stem cells (hiPSC) has facilitated in vitro disease modeling studies aiming at deciphering the molecular and cellular mechanisms that contribute to disease pathogenesis and progression. To fully exploit the potential of hiPSC for biomedical applications,technologies that enable the standardized generation and expansion of hiPSC from large numbers of donors are required. Paralleled automated processes for the expansion of hiPSC could provide an opportunity to maximize the generation of hiPSC collections from patient cohorts while minimizing hands-on time and costs. In order to develop a simple method for the parallel expansion of human pluripotent stem cells (hPSC) we established a protocol for their cultivation as undifferentiated aggregates in a bench-top bioreactor system (BioLevitator™). We show that long-term expansion (10 passages) of hPSCs either in mTeSR or E8 medium preserved a normal karyotype,three-germ-layer differentiation potential and high expression of pluripotency-associated markers. The system enables the expansion from low inoculation densities (0.3 × 10(5) cells/mL) and provides a simplified,cost-efficient and time-saving method for the provision of hiPSC at midi-scale. Implementation of this protocol in cell production schemes has the potential to advance cell manufacturing in many areas of hiPSC-based medical research. View Publication