Scientific Resources

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope,since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but,in contrast to over-expression systems,cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8,in agreement with the observed reduction of VAPB in sporadic ALS. View Publication

By providing access to affected neurons,human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease,defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system,causing relentless progression toward severe mental retardation. Partially digested proteoglycans,which affect fibroblast growth factor signaling,accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages,patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures,undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions,whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together,these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues,which possibly affect Golgi organization,cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development,once HS proteoglycan accumulation becomes prominent in the affected child brain. View Publication

A practical synthesis of the Rho-Kinase inhibitor Y-27632 and two new fluoro derivatives was achieved in seven steps and with a good overall yield of 45% starting from commercially available (R)-1-phenylethylamine. Compared to Y-27632 the new fluoro derivatives showed reduced or no effect on hPSC vitality and expansion after dissociation in human pluripotent stem cells. View Publication

The mitochondrial contribution to the maintenance of human embryonic stem cell (hESC) pluripotency and culture homeostasis remains poorly understood. Here,we sought to determine whether hESC adaptation to different feeder-free culture conditions is linked to a mitochondrial adaptation. The expression of ESC pluripotency factors and parameters of mitochondrial contribution including mitochondrial membrane potential,mtDNA content,and the expression of master mitochondrial genes implicated in replication,transcription,and biogenesis were determined in 8 hESC lines maintained in 2 distinct human feeders-conditioned media (CM): human foreskin fibroblast-CM (HFF-CM) and mesenchymal stem cell-CM (MSC-CM). We show a robust parallel trend between the expression of ESC pluripotency factors and the mitochondrial contribution depending on the culture conditions employed to maintain the hESCs,with those in MSC-CM consistently displaying increased levels of pluripotency markers associated to an enhanced mitochondrial contribution. The differences in the mitochondrial status between hESCs maintained in MSC-CM versus HFF-CM respond to coordinated changes in mitochondrial gene expression and biogenesis. Importantly,the culture conditions determine the mitochondrial distribution within the stage-specific embryonic antigen 3 positive (SSEA3(+)) and negative (SSEA3(-)) isolated cell subsets. hESC colonies in MSC-CM display an intrinsic" high mitochondrial status which may suffice to support undifferentiated growth� View Publication

Pluripotent stem cells,both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC),can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However,the tumorigenic potential of these cells remains a great concern,as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice,most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal,cardiac,or endothelial cells prior to human transplantation,drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study,we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells,and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer,whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall,our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation,and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy. View Publication

Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors,Oct4,Sox2,Klf4 and c-Myc,also known as the Yamanaka factors. In practice,initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology,but often may go on to fail subsequent assays,such as the alkaline phosphate (AP) assay. In this study,we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly,we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system,and then transferred to a feeder layer for expansion. Furthermore,colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies,45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies,while colonies screened by the feeder-free system were 84% AP+ colonies,16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers,OCT4,SOX2,NANOG,TRA-1-60,TRA-1-81,SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study,we report a simplistic,one-step method for selection of AP+ AF-iPS cells via feeder-free screening. View Publication

Forced expression of selected transcription factors can transform somatic cells into embryonic stem cell (ESC)-like cells,termed induced pluripotent stem cells (iPSCs). There is no consensus regarding the preferred tissue from which to harvest donor cells for reprogramming into iPSCs,and some donor cell types may be more prone than others to accumulation of epigenetic imprints and somatic cell mutations. Here,we present a simple,reproducible,noninvasive method for generating human iPSCs from renal tubular cells present in urine. This procedure eliminates many problems associated with other protocols,and the resulting iPSCs display an excellent ability to differentiate. These data suggest that urine may be a preferred source for generating iPSCs. View Publication

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical,environmental,and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part,previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation,which are highly nonphysiologic,or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair,we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs,compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus,the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny. View Publication

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings,however,requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study,we show that once specified to neuro-epithelial lineage,hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However,our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes,namely,migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes,a new potential source for therapeutic use for brain tumors and other neurological diseases. View Publication

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells,free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death,which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore,we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly,three compounds currently used as clinically approved drugs,nortriptyline,benzethonium chloride and methylbenzethonium chloride,induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs,whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline,a tricyclic antidepressant,has also been described as a potent inhibitor of mitochondrial permeability transition,one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline,although with a different behavior. Our results indicate differential sensitivity of ESCs,hiPSCs and fibroblasts to certain chemical compounds,which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases. View Publication

Somatic cell nuclear transfer,cell fusion,or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors,Brn2 (also known as Pou3f2),Ascl1 and Myt1l,can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1,these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers,even after downregulation of the exogenous transcription factors. Importantly,the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells,as well as pluripotent stem cells,can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine. View Publication

RATIONALE: Cardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis,the key process underlying excitation-contraction coupling. OBJECTIVE: We investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs). METHODS AND RESULTS: We differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes,spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore,in contrast with hESC-derived cardiomyocytes,ryanodine did not reduce the amplitudes,maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition,spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2),sacroplasmic recticulum calcium-ATPase (SERCA),junction (Jun) and triadin (TRDN),were significantly lower in hiPSC than in hESCs. CONCLUSIONS: The results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts. View Publication