Derivation of induced pluripotent stem cells from orangutan skin fibroblasts. Ramaswamy, K; Yik, WY; Wang, XM; Oliphant, EN; Lu, W; Shibata, D; Ryder, OA; Hacia, JG BMC research notes
8
577
2015
显示摘要
Orangutans are an endangered species whose natural habitats are restricted to the Southeast Asian islands of Borneo and Sumatra. Along with the African great apes, orangutans are among the closest living relatives to humans. For potential species conservation and functional genomics studies, we derived induced pluripotent stem cells (iPSCs) from cryopreserved somatic cells obtained from captive orangutans.Primary skin fibroblasts from two Sumatran orangutans were transduced with retroviral vectors expressing the human OCT4, SOX2, KLF4, and c-MYC factors. Candidate orangutan iPSCs were characterized by global gene expression and DNA copy number analysis. All were consistent with pluripotency and provided no evidence of large genomic insertions or deletions. In addition, orangutan iPSCs were capable of producing cells derived from all three germ layers in vitro through embryoid body differentiation assays and in vivo through teratoma formation in immune-compromised mice.We demonstrate that orangutan skin fibroblasts are capable of being reprogrammed into iPSCs with hallmark molecular signatures and differentiation potential. We suggest that reprogramming orangutan somatic cells in genome resource banks could provide new opportunities for advancing assisted reproductive technologies relevant for species conservation efforts. Furthermore, orangutan iPSCs could have applications for investigating the phenotypic relevance of genomic changes that occurred in the human, African great ape, and/or orangutan lineages. This provides opportunities for orangutan cell culture models that would otherwise be impossible to develop from living donors due to the invasive nature of the procedures required for obtaining primary cells. | | | 26475477
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Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines. Cerbini, T; Funahashi, R; Luo, Y; Liu, C; Park, K; Rao, M; Malik, N; Zou, J PloS one
10
e0116032
2015
显示摘要
Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells. | | | 25587899
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A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach. Chou, BK; Gu, H; Gao, Y; Dowey, SN; Wang, Y; Shi, J; Li, Y; Ye, Z; Cheng, T; Cheng, L Stem cells translational medicine
4
320-32
2015
显示摘要
Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. | | | 25742692
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Impaired respiratory function in MELAS-induced pluripotent stem cells with high heteroplasmy levels. Kodaira, M; Hatakeyama, H; Yuasa, S; Seki, T; Egashira, T; Tohyama, S; Kuroda, Y; Tanaka, A; Okata, S; Hashimoto, H; Kusumoto, D; Kunitomi, A; Takei, M; Kashimura, S; Suzuki, T; Yozu, G; Shimojima, M; Motoda, C; Hayashiji, N; Saito, Y; Goto, Y; Fukuda, K FEBS open bio
5
219-25
2015
显示摘要
Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS-specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS-iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243Agreater than G heteroplasmy. MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243Agreater than G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity. Our data indicate that MELAS-iPSCs can be models for MELAS but we should carefully select MELAS-iPSCs with appropriate heteroplasmy levels and respiratory functions for mitochondrial disease modeling. | | | 25853038
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A method for human teratogen detection by geometrically confined cell differentiation and migration. Xing, J; Toh, YC; Xu, S; Yu, H Scientific reports
5
10038
2015
显示摘要
Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time, cost and high inter-species variability. Here, we developed a human-relevant in vitro model, which recapitulated two cellular events characteristic of embryogenesis, to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation, epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds' teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human. | | | 25966467
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An effective freezing/thawing method for human pluripotent stem cells cultured in chemically-defined and feeder-free conditions. Nishishita, N; Muramatsu, M; Kawamata, S American journal of stem cells
4
38-49
2015
显示摘要
Culturing human Pluripotent Stem Cells (hPSC)s in chemically defined medium and feeder-free condition can facilitate metabolome and proteome analysis of culturing cells and medium, and reduce regulatory concerns for clinical application of cells. And in addition, if hPSC are passaged and cryopreserved in single cells it also facilitates quality control of cells at single cell level. Here we report a robust single cell freezing and thawing method of hPSCs cultured in chemically-defined medium TeSR(TM)-E8(TM) and on cost-effective recombinant human Vitronectin-N (rhVTN-N)-coated dish. Cells are dissociated into single cells with recombinant TrypLE(TM) Select and 0.5 mM EDTA/PBS (3:1 solution) in the presence of Rock inhibitor and cryopreserved with chemically defined CryoStem(TM). Approximately 60% of cells were viable after dissociation. Aggrewell(TM) 400 was used to form cell clumps of 500 cells after thaw in the presence of Rock inhibitor and cells were cultured for two days with TeSR-E8. Cells clumps were then seeded on rhVTN-N-coated dish and cultured with TeSR-E8 for two days prior to the first passage after thawing. Number of viable cells at the first passage increased around 10 times of that just before freezing. This robust single cell freezing method for hPSCs cultured in chemically defined medium will facilitate quality control of cultured cells at single cell level before cryopreservation and consequently assure the quality of cells in frozen vials for further manipulation after thawing. | | | 25973330
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Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations. Turner, J; Quek, LE; Titmarsh, D; Krömer, JO; Kao, LP; Nielsen, L; Wolvetang, E; Cooper-White, J PloS one
9
e112757
2014
显示摘要
As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion. | Western Blotting | | 25412279
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Synaptic dysregulation in a human iPS cell model of mental disorders. Wen, Z; Nguyen, HN; Guo, Z; Lalli, MA; Wang, X; Su, Y; Kim, NS; Yoon, KJ; Shin, J; Zhang, C; Makri, G; Nauen, D; Yu, H; Guzman, E; Chiang, CH; Yoritomo, N; Kaibuchi, K; Zou, J; Christian, KM; Cheng, L; Ross, CA; Margolis, RL; Chen, G; Kosik, KS; Song, H; Ming, GL Nature
2014
显示摘要
Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders, and 'a disease of synapses' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies, little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare, multiply affected, large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and, furthermore, dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders. | | | 25132547
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Pathological roles of the VEGF/SphK pathway in Niemann-Pick type C neurons. Lee, H; Lee, JK; Park, MH; Hong, YR; Marti, HH; Kim, H; Okada, Y; Otsu, M; Seo, EJ; Park, JH; Bae, JH; Okino, N; He, X; Schuchman, EH; Bae, JS; Jin, HK Nature communications
5
5514
2014
显示摘要
Sphingosine is a major storage compound in Niemann-Pick type C disease (NP-C), although the pathological role(s) of this accumulation have not been fully characterized. Here we found that sphingosine kinase (SphK) activity is reduced in NP-C patient fibroblasts and NP-C mouse Purkinje neurons (PNs) due to defective vascular endothelial growth factor (VEGF) levels. Sphingosine accumulation due to inactivation of VEGF/SphK pathway led to PNs loss via inhibition of autophagosome-lysosome fusion in NP-C mice. VEGF activates SphK by binding to VEGFR2, resulting in decreased sphingosine storage as well as improved PNs survival and clinical outcomes in NP-C cells and mice. We also show that induced pluripotent stem cell (iPSC)-derived human NP-C neurons are generated and the abnormalities caused by VEGF/SphK inactivity in these cells are corrected by replenishment of VEGF. Overall, these results reveal a pathogenic mechanism in NP-C neurons where defective SphK activity is due to impaired VEGF levels. | Immunofluorescence | | 25417698
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Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs. Liu, GH; Suzuki, K; Li, M; Qu, J; Montserrat, N; Tarantino, C; Gu, Y; Yi, F; Xu, X; Zhang, W; Ruiz, S; Plongthongkum, N; Zhang, K; Masuda, S; Nivet, E; Tsunekawa, Y; Soligalla, RD; Goebl, A; Aizawa, E; Kim, NY; Kim, J; Dubova, I; Li, Y; Ren, R; Benner, C; del Sol, A; Bueren, J; Trujillo, JP; Surralles, J; Cappelli, E; Dufour, C; Esteban, CR; Izpisua Belmonte, JC Nature communications
5
4330
2014
显示摘要
Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells. | | | 24999918
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