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UNLABELLED: Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated,there has been limited progress in iPSC-based drug screening/discovery for liver diseases,and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency,for which there is currently no drug or gene therapy available,we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay,based on our hepatic differentiation protocol,was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients,we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening,five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition,using the recently developed transcription activator-like effector nuclease technology,we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications.backslashnbackslashnCONCLUSIONS: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs,both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells,but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (textgreater60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. View Publication

Pluripotent stem cells hold great promise for regenerative medicine,due to their unlimited self-renewal potential and the ability to differentiate into all somatic cell types. Differences between the rodent disease models and the situation in humans can be narrowed down with non-human primate models. The common marmoset monkey (Callithrix jacchus) is an interesting model for biomedical research because these animals are easy to breed,get relatively old (≤ 13 years),are small in size,are relatively cost-effective and have a high genetic proximity to the human. In particular,diseases of the liver and pancreas are interesting for cell replacement therapies but the in vitro differentiation of ESCs into the definitive endoderm germ layer is still a demanding task. Membrane-permeable,chemically defined small molecules can possibly replace recombinant growth factors used in most directed differentiation protocols. However,the potent small molecules IDE-1 and IDE-2 were not able to induce definitive endoderm-like cells when ESCs from the common marmoset were treated with these compounds,whereas the recombinant growth factor Activin A could force the differentiation into this lineage. Our results indicate that ESCs from the common marmoset are less sensitive or even insensitive to these small molecules. Thus,differences between the species of human ESCs and ESCs of this non-human primate might be a useful model to further evaluate the exact mode of action of these compounds. View Publication

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes,mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture,hES cells expressing cell-surface NGFR protein (CD271,p75NTR) were isolated by immunoaffinity adsorption,and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression,examined by quantitative RT-PCR,found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR,SNAI1,NTRK3,SOX9,and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer,mRNAs typifying adult stromal stem cells were detected,including BMI1,KIT,NES,NOTCH1,and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1,B3GNT7,PTDGS,and ALDH3A1 were upregulated. mRNA for keratocan (KERA),a cornea-specific proteoglycan,was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate,a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells,therefore,may provide a renewable source of material for development of treatment of corneal stromal opacities. View Publication

Human pluripotent stem cells (hPSCs) have an unparalleled potential to generate limitless quantities of any somatic cell type. However,current methods for producing populations of various somatic cell types from hPSCs are generally not standardized and typically incorporate undefined cell culture components often resulting in variable differentiation efficiencies and poor reproducibility. To address this,we have developed a defined approach for generating epithelial progenitor and epidermal cells from hPSCs. In doing so,we have identified an optimal starting cell density to maximize yield and maintain high purity of K18+/p63+ simple epithelial progenitors. In addition,we have shown that the use of synthetic,defined substrates in lieu of Matrigel and gelatin can successfully facilitate efficient epithelial differentiation,maintaining a high (backslashtextgreater75%) purity of K14+/p63+ keratinocyte progenitor cells and at a two to threefold higher yield than a previously reported undefined differentiation method. These K14+/p63+ cells also exhibited a higher expansion potential compared to cells generated using an undefined differentiation protocol and were able to terminally differentiate and recapitulate an epidermal tissue architecture in vitro. In summary,we have demonstrated the production of populations of functional epithelial and epidermal cells from multiple hPSC lines using a new,completely defined differentiation strategy. View Publication

Monocytic lineage cells (monocytes,macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established,these methods depend on the use of xenogeneic materials and,therefore,have a relatively poor-reproducibility. Here,we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6) ± 0.3 × 10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine,disease-specific iPSC studies and drug discovery. View Publication

Induced pluripotent stem cells (iPSCs) hold promise for the treatment of motoneuron diseases because of their distinct features including pluripotency,self-derivation and potential ability to differentiate into motoneurons. However,it is still unknown whether human iPSC-derived motoneurons can functionally innervate target muscles in vivo,which is the definitive sign of successful cell therapy for motoneuron diseases. In the present study,we demonstrated that human iPSCs derived from mesenchymal cells of the umbilical cord possessed a high yield in neural differentiation. Using a chemically-defined in vitro system,human iPSCs efficiently differentiated into motoneurons which displayed typical morphology,expressed specific molecules,and generated repetitive trains of action potentials. When transplanted into the injured musculocutaneous nerve of rats,they survived robustly,extended axons along the nerve,and formed functional connections with the target muscle (biceps brachii),thereby protecting the muscle from atrophy. Our study provides evidence for the first time that human iPSC-derived motoneurons are truly functional not only in vitro but also in vivo,and they have potential for stem cell-based therapies for motoneuron diseases. textcopyright 2013 Elsevier B.V. View Publication

AbstractIntroduction
Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited,there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast,and express HLA-G,a marker of EVT. The goals of the present study were to further characterize the HLA-G+ cells derived from BMP4-treated hESC,and determine their suitability as a model.
Methods
HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G+ and HLA-G−/TACSTD2+ populations with immunomagnetic beads and expression profiles analyzed by microarray.
Results
There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent,stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7,and the majority were also HLA-G+. Gene expression profiles revealed that HLA-G+,BMP4-treated hESC were similar to,but distinct from,HLA-G+ cells isolated from first trimester placentas. Whereas HLA-G+ and HLA-G− cells from first trimester placentas had highly divergent gene expression profiles,HLA-G+ and HLA-G− cells from BMP4-treated hESC had somewhat similar profiles,and both expressed genes characteristic of early trophoblast development.
Conclusions
We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage. View Publication

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue,but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus,fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies,but that of BOECs was lower. In terms of invasiveness of biopsy sampling,biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs,but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative. View Publication

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited,stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently,the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition,the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However,the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation,we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice,hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling,increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. View Publication

Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells,the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here,we attempted to reprogram E-PZ cells by forced expression of Oct4,Sox2,c-Myc,and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81,SSEA-3,Nanog,Sox2,and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5,CK14,and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal,mesodermal,and endodermal cells in vitro,although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly,E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44,p63,MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR),and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA),a hallmark of secretory cells,suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally,when injected into mice,E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme,E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore,provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. View Publication