Scientific Resources

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here,we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore,in vitro,iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice,human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together,our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons. View Publication

Human embryonic stem cells (hESCs) can differentiate into all somatic lineages including stratified squamous epithelia. Thus,efficient methods are required to direct hESC differentiation to obtain a pure subpopulation for tissue engineering. The study aimed to assess the effects of retinoic acid (RA),bone morphogenetic protein-4 (BMP4),and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro. The first media contained AA and BMP4; the second contained RA,AA,and BMP4; the third was commercial-defined keratinocyte serum-free medium,which was used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors. Real-time RT-PCR,immunofluorescence,and flow-cytometry were used to characterize the differentiated cells. Cells induced by AA + BMP4 + RA showed the typical epithelial morphology,while cells induced by AA + BMP4 showed multiple appearances. CK14 and p63 messenger RNA (mRNA) expressions in the AA + BMP4 + RA-treated cells were higher than those of the AA + BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18 mRNA expressions at 14 d of differentiation and keratinocyte marker CK14 and transcription factor p63 mRNA expressions at 35 d of differentiation were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18 10.51 ± 3.26 vs. 6.67 ± 1.28; CK14 9.27 ± 3.61 vs. 5.32 ± 1.86; p63 0.73 ± 0.06 vs. 0.44 ± 0.12,all P textless 0.05) After hESC induction by AA+BMP4+RA,CK14 mRNA expression was upregulated after day 21,peaking by 35 d of differentiation. Combined RA,BMP4,and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering. View Publication

PURPOSE Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however,the mechanisms leading to enhanced RPE proliferation,migration,and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease. METHODS ARPE-19 cells,primary cultures of porcine RPE,and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGF$$2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor,trichostatin A (TSA; 0.1 $$M),were assessed for contraction and migration through collagen contraction and scratch assays,respectively. Western blotting and immunofluorescence analysis were performed to assess $$-smooth muscle actin ($$-SMA) and $$-catenin expression after TGF$$2 treatment alone or in combination with TSA. RESULTS TGF$$2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 $$M). In agreement with these data,immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased $$-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGF$$2-mediated iPS-RPE cell migration. CONCLUSIONS Our findings indicate a role of acetylation in RPE activation. Specifically,the HDAC inhibitor TSA decreased RPE cell proliferation and TGF$$2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR. View Publication

ALS is a rapidly progressive,devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression,and molecular insights into pathogenesis and progression are sorely needed. In that context,we used high-depth,next generation RNA sequencing (RNAseq,Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned textgreater50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2,DEseq2,EdgeR) for identification of differentially expressed genes (DEG's). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples,with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNF$$-induced protein 2 (TNFAIP2) as a major network hub" gene (WGCNA). Using the oPOSSUM algorithm� View Publication

Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications,there is a need to develop scalable cell culture platforms to propagate hESCs. In this study,we encapsulated hESCs in calcium alginate microfibers as single cells,for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (textgreater10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM),which did not affect hESCs viability,was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical,as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line,HUES7,for up to five passages while maintaining pluripotency. Immunohistochemistry,polymerase chain reaction,and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells,possessed normal karyotype,and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively,our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions. View Publication

Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin-specific gene expression that is regulated by a differentially methylated region. Gene mutations or failures in the imprinting process lead to the development of imprinting disorders,such as Angelman syndrome. The symptoms of Angelman syndrome are caused by the absence of functional UBE3A protein in neurons of the brain. To create a human neuronal model for Angelman syndrome,we reprogrammed dermal fibroblasts of a patient carrying a defined three-base pair deletion in UBE3A into induced pluripotent stem cells (iPSCs). In these iPSCs,both parental alleles are present,distinguishable by the mutation,and express UBE3A. Detailed characterization of these iPSCs demonstrated their pluripotency and exceptional stability of the differentially methylated region regulating imprinted UBE3A expression. We observed strong induction of SNHG14 and silencing of paternal UBE3A expression only late during neuronal differentiation,in vitro. This new Angelman syndrome iPSC line allows to study imprinted gene regulation on both parental alleles and to dissect molecular pathways affected by the absence of UBE3A protein. View Publication

Next-generation mass spectrometric (MS) techniques such as SWATH-MS have substantially increased the throughput and reproducibility of proteomic analysis,but ensuring consistent quantification of thousands of peptide analytes across multiple liquid chromatography-tandem MS (LC-MS/MS) runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion,we developed TRIC (http://proteomics.ethz.ch/tric/),a software tool that utilizes fragment-ion data to perform cross-run alignment,consistent peak-picking and quantification for high-throughput targeted proteomics. TRIC reduced the identification error compared to a state-of-the-art SWATH-MS analysis without alignment by more than threefold at constant recall while correcting for highly nonlinear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem cells,TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups. Thus,TRIC fills a gap in the pipeline for automated analysis of massively parallel targeted proteomics data sets. View Publication

Recent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here,we demonstrate that both glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using 13C/2H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth,chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration,altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development. Zhang et al. apply metabolic flux analysis to comprehensively characterize the metabolism of human pluripotent stem cells cultured in different media. Cells maintained in chemically defined media significantly upregulate lipid biosynthesis and redox pathways to compensate for medium lipid deficiency while downregulating oxidative mitochondrial metabolism. View Publication

Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated,primed,pluripotent states established by specific culture conditions. Increased intracellular α-ketoglutarate (αKG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of αKG or αKG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased αKG or αKG-to-succinate ratios accelerate,and elevated succinate levels delay,primed PSC differentiation. αKG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede αKG-accelerated primed PSC differentiation. Instead,αKG induced,and succinate impaired,global histone and DNA demethylation in primed PSCs. The data support αKG promotion of self-renewal or differentiation depending on the pluripotent state. View Publication

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH,however,lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore,other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs),characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming,and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation,the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients. View Publication

CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical,iPSC generation must be rapid and efficient. Therefore,we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector,rCLAE-R6,that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently,the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% ($$(A)/[$$(S)+$$(A)]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected,patient-specific iPSCs for therapeutic applications. View Publication

The epigenetic background of pluripotent stem cells can influence transcriptional and functional behavior. Most of these data have been obtained in standard monolayer cell culture systems. In this study,we used exome sequencing,array comparative genomic hybridization (CGH),miRNA array,DNA methylation array,three-dimensional (3D) tissue engineering,and immunostaining to conduct a comparative analysis of two induced pluripotent stem cell (iPSC) lines used in engineering of 3D human epidermal equivalent (HEE),which more closely approximates epidermis. Exome sequencing and array CGH suggested that their genome was stable following 3 months of feeder-free culture. While the miRNAome was also not affected,≈7% of CpG sites were differently methylated between the two lines. Analysis of the epidermal differentiation complex,a region on chromosome 1 that contains multiple genes involved in skin barrier maturation (including trichohyalin,TCHH),found that in one of the iPSC clones (iKCL004),TCHH retained a DNA methylation signature characteristic of the original somatic cells,whereas in other iPSC line (iKCL011),the TCHH methylation signature matched that of the human embryonic stem cell line KCL034. The difference between the two iPSC clones in TCHH methylation did not have an obvious effect on its expression in 3D HEE,suggesting that differentiation and tissue formation may mitigate variations in the iPSC methylome. View Publication