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Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells,the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease - dyslipidemia,insulin resistance,hypoglycemia,lipodystrophy,motor-neuron death,and hepatitis C infection. We found little evidence of TALEN off-target effects,but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines,we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease. textcopyright 2013 Elsevier Inc. View Publication

Realization of the full potential of human induced pluripotent stem cells (hiPSCs) in clinical applications requires development of well-defined conditions for their growth and differentiation. A novel fully defined polyvinyl alcohol/hyaluronan (PVA/HA) polysaccharide nanofiber was developed for hiPSCs culture in commercially available xeno-free,chemically defined medium. Vitronectin peptide (VP) was immobilized to PVA/HA nanofibers through NHS/EDC chemistry. The hiPSCs successfully grew and proliferated on the VP-decorated PVA/HA nanofibers,similar to those on MatrigelTM. Such well-defined,xeno-free and safe nanofiber substrate that supports culture of hiPSCs will not only help to accelerate the translational perspectives of hiPSCs,but also provide a platform to investigate the cell-nanofiber interaction mechanisms that regulate stem cell proliferation and differentiation. ?? 2013 Elsevier Ltd. All rights reserved. View Publication

Traditionally,human embryonic stem cells (hESCs) are cultured on inactivated live feeder cells. For clinical application using hESCs,there is a requirement to minimize the risk of contamination with animal components. Extracellular matrix (ECM) derived from feeder cells is the most natural way to provide xeno-free substrates for hESC growth. In this study,we optimized the step-by-step procedure for ECM processing to develop a xeno-free ECM that supports the growth of undifferentiated hESCs. In addition,this newly developed xeno-free substrate can be stored at 4°C and is ready to use upon request,which serves as an easier way to amplify hESCs for clinical applications. View Publication

A large membrane proteinase 3 (mPR3)-positive neutrophil subset (mPR3high) is a risk for Wegener's granulomatosis (WG). The relationship between mPR3 expression and clinical manifestations was investigated in 81 WG patients and mPR3 expression was studied in CD34+ stem cell-derived human neutrophils. The mPR3high neutrophil percentage correlated with renal function,anemia,and albumin at the time of presentation. The mPR3high neutrophil percentage and renal failure severity correlated directly after 5 yr. For elucidating mechanisms that govern mPR3 expression,studies were conducted to determine whether the genetic information that governs mPR3 expression resides within the neutrophils,even without stimuli possibly related to disease. CD34+ hematopoietic stem cells were differentiated to neutrophils,and their mPR3 expression was determined. A two-step amplification/differentiation protocol was used to differentiate human CD34+ hematopoietic stem cells into neutrophils with G-CSF. The cells progressively expressed the neutrophil surface markers CD66b,CD35,and CD11b. The ferricytochrome C assay demonstrated a strong respiratory burst at day 14 in response to PMA but none at day 0. Intracellular PR3 was detectable from day 4 by Western blotting. An increasing percentage of a mPR3-positive neutrophil subset became detectable by flow cytometry,whereas a second subset remained negative,consistent with a bimodal expression. Finally,human PR3-anti-neutrophil cytoplasmic autoantibodies induced a stronger respiratory burst,compared with human control IgG in stem cell-derived neutrophils. Taken together,these studies underscore the clinical importance of the WG mPR3 phenotype. The surface mPR3 on resting cells is probably genetically determined rather than being dictated by external factors. View Publication

Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated,millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo,in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling,this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis,this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies,as well as providing a source of cells for tissue engineering and cell therapy approaches. View Publication

Neural cells derived from induced pluripotent stem cells (iPSCs) have the potential for autologous cell therapies in treating patients with severe neurological disorders or injury. However,further study of efficacy and safety are needed in large animal preclinical models that have similar neural anatomy and physiology to humans such as the pig. The pig model for pluripotent stem cell therapy has been made possible for the first time with the development of pig iPSCs (piPSCs) capable of in vitro and in vivo differentiation into tissues of all three germ layers. Still,the question remains if piPSCs are capable of undergoing robust neural differentiation using a system similar to those being used with human iPSCs. In this study,we generated a new line of piPSCs from fibroblast cells that expressed pluripotency markers and were capable of embryoid body differentiation into all three germ layers. piPSCs demonstrated robust neural differentiation forming βIII-TUB/MAP2+ neurons,GFAP+ astrocytes,and O4+ oligodendrocytes and demonstrated strong upregulation of neural cell genes representative of all three major neural lineages of the central nervous system. In the presence of motor neuron signaling factors,piPSC-derived neurons showed expression of transcription factors associated with motor neuron differentiation (HB9 and ISLET1). Our findings demonstrate that SSEA4 expression is required for piPSCs to differentiate into neurons,astrocytes,and oligodendrocytes and furthermore develop specific neuronal subtypes. This indicates that the pigs can fill the need for a powerful model to study autologous neural iPSC therapies in a system similar to humans. View Publication

Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here,we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G(0) residency) of murine and human hematopoietic cells. In human cord blood,quiescent fractions (CD34(+)CD38(-)Hoechst(lo)Pyronin Y(lo)) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells,reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF,but enforcing MEF expression does not prevent GATA-2-conferred quiescence,suggesting additional regulators are involved. Although known quiescence regulators p21(CIP1) and p27(KIP1) do not appear to be responsible,enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3,CDK4,and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2(hi)) failed to contribute to hematopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice,whereas GATA-2(lo) cells contributed with delayed kinetics and low efficiency,with reduced expression of Ki-67. Thus,GATA-2 activity inhibits cell cycle in vitro and in vivo,highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells. View Publication

The potential for human disease treatment using human pluripotent stem cells,including embryonic stem cells and induced pluripotent stem cells (iPSCs),also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies,which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study,a comparison of DNA repair pathways in pluripotent cells,as compared to those in non-pluripotent cells,demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair,we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells,while differentiated cells lacked response to this stimulus,and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition,the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype,but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together,these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines,in order to characterize their genomic stability,prior to their pre-clinical and clinical use. View Publication

Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However,currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability,painful and invasive cell-harvesting procedures,and tumorigenesis. Previously,we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan,fibromodulin (FMOD),circumventing gene transduction. In this study,we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect,contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence,engraftment,and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together,we have provided an extended potency,safety,and molecular profile of FReP cell-based bone regeneration. Therefore,FReP cells present a high potential for cellular and gene therapy products for bone regeneration. View Publication

Stem cells hold great promise for therapies aimed at regenerating damaged tissue,drug screening and studying in vitro models of human disease. However,many challenges remain before these applications can become a reality. One such challenge is developing chemically defined and scalable culture conditions for derivation and expansion of clinically viable human pluripotent stem cells,as well as controlling their differentiation with high specificity. Interaction of stem cells with their extracellular microenvironment plays an important role in determining their differentiation commitment and functions. Regenerative medicine approaches integrating cell-matrix and cell-cell interactions,and soluble factors could lead to development of robust microenvironments to control various cellular responses. Indeed,several of these recent developments have provided significant insight into the design of microenvironments that can elicit the targeted cellular response. In this article,we will focus on some of these developments with an emphasis on matrix-mediated expansion of human pluripotent stem cells while maintaining their pluripotency. We will also discuss the role of matrix-based cues and cell-cell interactions in the form of soluble signals in directing stem cell differentiation into musculoskeletal lineages. View Publication

Despite significant advances in commercially available media and kits and the differentiation approaches for human neural stem cell (NSC) generation,NSC production from the differentiation of human pluripotent stem cell (hPSC) is complicated by its time-consuming procedure,complex medium composition,and purification step. In this study,we developed a convenient and simplified NSC production protocol to meet the demand of NSC production. We demonstrated that NSCs can be generated efficiently without requirement of specific small molecules or embryoid body formation stage. Our experimental results suggest that a short suspension culture period may facilitate ectoderm lineage specification rather than endoderm or mesoderm lineage specification from hPSCs. The method developed in this study shortens the turnaround time of NSC production from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) differentiation. It provides a straightforward and useful strategy for generating NSCs that can benefit a wide range of research applications for human brain research. View Publication

To search for new indicators of self-renewing hematopoietic stem cells (HSCs),highly purified populations were isolated from adult mouse marrow,micromanipulated into a specially designed microscopic array,and cultured for 4 days in 300 ng/ml Steel factor,20 ng/ml IL-11,and 1 ng/ml flt3-ligand. During this period,each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (textgreater1% contribution to lymphoid and myeloid lineages). In a first experiment,6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity,demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions. View Publication