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Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study,we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4,nanog,and sox2,formed embryoid bodies in vitro,and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast,redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts,with 605 genes differing by more than twofold. After redifferentiation,112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation,only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly,of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts,293 genes were changed toward control after redifferentiation. In conclusion,functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs. View Publication

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC,these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further,the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial,ventricular (V),and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2,H7,and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4,Rho kinase inhibitor,activin-A,and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16,up to 95% of cells were cTnT(+). Of which,93%,94%,100%,92%,and 92% of cardiac derivatives from HES2,H7,H9,and two iPSC lines,respectively,were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to $\$-adrenergic stimulation. Our simple,cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol,the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors. View Publication

Schizophrenia has been considered a devastating clinical syndrome rather than a single disease. Nevertheless,the mechanisms behind the onset of schizophrenia have been only partially elucidated. Several studies propose that levels of trace elements are abnormal in schizophrenia; however,conflicting data generated from different biological sources prevent conclusions being drawn. In this work,we used synchrotron radiation X-ray microfluorescence spectroscopy to compare trace element levels in neural progenitor cells (NPCs) derived from two clones of induced pluripotent stem cell lines of a clozapine-resistant schizophrenic patient and two controls. Our data reveal the presence of elevated levels of potassium and zinc in schizophrenic NPCs. Neural cells treated with valproate,an adjunctive medication for schizophrenia,brought potassium and zinc content back to control levels. These results expand the understanding of atomic element imbalance related to schizophrenia and may provide novel insights for the screening of drugs to treat mental disorders. ?? 2014 Elsevier B.V. View Publication

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development,modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (˜70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (textgreater250%) MN production in chemically defined adherent cultures. Furthermore,we show that Islet-1 is essential for formation of mature and functional human MNs,but,unlike its mouse counterpart,does not regulate cell survival or suppress the V2a interneuron fate. Together,our discoveries improve the strategy for MN derivation,advance our understanding of human neural specification and MN development,and provide invaluable tools for human developmental studies,drug discovery and regenerative medicine. View Publication

Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases,and for ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities,the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are,thus,of interest,as the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However,characterization of such MSC-like cells is currently inadequate,especially with regard to the question of whether these cells are equivalent or identical to MSCs. In this study,we have derived MSC-like cells [induced PSC-derived MSC-like progenitor cells (iMPCs)] using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells (BMSCs),and compared the iMPCs directly with the originating parental BMSCs. The iMPCs exhibited typical MSC/fibroblastic morphology and MSC-typical surface marker profile,and they were capable of differentiation in vitro along the osteogenic,chondrogenic,and adipogenic lineages. However,compared with the parental BMSCs,iMPCs displayed a unique expression pattern of mesenchymal and pluripotency genes and were less responsive to traditional BMSC differentiation protocols. We,therefore,conclude that iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells which exhibit MSC-like characteristics. View Publication

GABAergic interneurons regulate cortical neural networks by providing inhibitory inputs,and their malfunction,resulting in failure to intricately regulate neural circuit balance,is implicated in brain diseases such as Schizophrenia,Autism,and Epilepsy. During early development,GABAergic interneuron progenitors arise from the ventral telencephalic area such as medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) by the actions of secreted signaling molecules from nearby organizers,and migrate to their target sites where they form local synaptic connections. In this study,using combinatorial and temporal modulation of developmentally relevant dorsoventral and rostrocaudal signaling pathways (SHH,Wnt,and FGF8),we efficiently generated MGE cells from multiple human pluripotent stem cells. Most importantly,modulation of FGF8/FGF19 signaling efficiently directed MGE versus CGE differentiation. Human MGE cells spontaneously differentiated into Lhx6-expressing GABAergic interneurons and showed migratory properties. These human MGE-derived neurons generated GABA,fired action potentials,and displayed robust GABAergic postsynaptic activity. Transplantation into rodent brains results in well-contained neural grafts enriched with GABAergic interneurons that migrate in the host and mature to express somatostatin or parvalbumin. Thus,we propose that signaling modulation recapitulating normal developmental patterns efficiently generate human GABAergic interneurons. This strategy represents a novel tool in regenerative medicine,developmental studies,disease modeling,bioassay,and drug screening. View Publication

The purpose of this study was to examine the in vitro and in vivo osteogenic potential of human induced pluripotent stem cells (hiPSCs) against that of human bone marrow mesenchymal stem cells (hBMMSCs). Embryoid bodies (EBs),which were formed from undifferentiated hiPSCs,were dissociated into single cells and underwent osteogenic differentiation using the same medium as hBMMSCs for 14 days. Osteoinduced hiPSCs were implanted on the critical-size calvarial defects and long bone segmental defects in rats. The healing of defects was evaluated after 8 weeks and 12 weeks of implantation,respectively. Osteoinduced hiPSCs showed relatively lower and delayed in vitro expressions of the osteogenic marker COL1A1 and bone sialoprotein,as well as a weaker osteogenic differentiation through alkaline phosphatase staining and mineralization through Alizarin red staining compared with hBMMSCs. Calvarial defects treated with osteoinduced hiPSCs had comparable quality of new bone formation,including full restoration of bone width and robust formation of trabeculae,to those treated with hBMMSCs. Both osteoinduced hiPSCs and hBMMSCs persisted in regenerated bone after 8 weeks of implantation. In critical-size long bone segmental defects,osteoinduced hiPSC treatment also led to healing of segmental defects comparable to osteoinduced hBMMSC treatment after 12 weeks. In conclusion,despite delayed in vitro osteogenesis,hiPSCs have an in vivo osteogenic potential as good as hBMMSCs. View Publication

PURPOSE Demonstrate a novel manufacturing method to generate extracellular matrix scaffolds from cardiac fibroblasts (CF-ECM) as a therapeutic mesenchymal stem cell-transfer device. MATERIALS AND METHODS Rat CF were cultured at high-density (˜1.6×10(5)/cm(2)) for 10-14 days. Cell sheets were removed from the culture dish by incubation with EDTA and decellularized with water and peracetic acid. CF-ECM was characterized by mass spectrometry,immunofluorescence and scanning electron microscopy. CF-ECM seeded with human embryonic stem cell derived mesenchymal stromal cells (hEMSCs) were transferred into a mouse myocardial infarction model. 48 hours later,mouse hearts were excised and examined for CF-ECM scaffold retention and cell transfer. RESULTS CF-ECM scaffolds are composed of fibronectin (82%),collagens type I (13%),type III (3.4%),type V (0.2%),type II (0.1%) elastin (1.3%) and 18 non-structural bioactive molecules. Scaffolds remained intact on the mouse heart for 48 hours without the use of sutures or glue. Identified hEMSCs were distributed from the epicardium to the endocardium. CONCLUSIONS High density cardiac fibroblast culture can be used to generate CF-ECM scaffolds. CF-ECM scaffolds seeded with hEMSCs can be maintained on the heart without suture or glue. hEMSC are successfully delivered throughout the myocardium. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells and human-induced pluripotent stem cells,are a renewable cell source for a wide range of applications in regenerative medicine and useful tools for human disease modeling and drug discovery. For these purposes,large numbers of high-quality cells are essential. Recently,we showed that a biological substrate,recombinant E8 fragments of laminin isoforms,sustains long-term self-renewal of hPSCs in defined,xeno-free medium with dissociated single-cell passaging. Here,we describe a modified culture system with similar performance to efficiently expand hPSCs under defined,xeno-free conditions using a non-biological synthetic substrate. View Publication

Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T),cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18),and they displayed the ability to generate NP-like tissue in vitro,which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs,and eventually towards regenerative therapies for disc degeneration. View Publication

The generation of human induced pluripotent stem cells (hiPSC) from somatic cells has enabled the possibility to provide patient-specific hiPSC for cell-based therapy,drug discovery,and other translational applications. Two major obstacles in using hiPSC for clinical application reside in the risk of genomic modification when they are derived with viral transgenes and risk of teratoma formation if undifferentiated cells are engrafted. In this study,we report the generation of footprint-free" hiPSC-derived astrocytes. These are efficiently generated� View Publication

Spinal and bulbar muscular atrophy (SBMA,Kennedy's disease) is a motor neuron disease caused by polyglutamine repeat expansion in the androgen receptor. Although degeneration occurs in the spinal cord and muscle,the exact mechanism is not clear. Induced pluripotent stem cells from spinal and bulbar muscular atrophy patients provide a useful model for understanding the disease mechanism and designing effective therapy. Stem cells were generated from six patients and compared to control lines from three healthy individuals. Motor neurons from four patients were differentiated from stem cells and characterized to understand disease-relevant phenotypes. Stem cells created from patient fibroblasts express less androgen receptor than control cells,but show androgen-dependent stabilization and nuclear translocation. The expanded repeat in several stem cell clones was unstable,with either expansion or contraction. Patient stem cell clones produced a similar number of motor neurons compared to controls,with or without androgen treatment. The stem cell-derived motor neurons had immunoreactivity for HB9,Isl1,ChAT,and SMI-32,and those with the largest repeat expansions were found to have increased acetylated ??-tubulin and reduced HDAC6. Reduced HDAC6 was also found in motor neuron cultures from two other patients with shorter repeats. Evaluation of stably transfected mouse cells and SBMA spinal cord showed similar changes in acetylated ??-tubulin and HDAC6. Perinuclear lysosomal enrichment,an HDAC6 dependent process,was disrupted in motor neurons from two patients with the longest repeats. SBMA stem cells present new insights into the disease,and the observations of reduced androgen receptor levels,repeat instability,and reduced HDAC6 provide avenues for further investigation of the disease mechanism and development of effective therapy. ?? 2014. View Publication