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Introduction: Fuchs endothelial corneal dystrophy (FECD) is the leading cause of corneal blindness in developed countries. Corneal endothelial cells in FECD are susceptive to oxidative stress, leading to mitochondrial dysfunction and cell death. Oxidative stress causes many forms of cell death including parthanatos, which is characterized by translocation of apoptosis-inducing factor (AIF) to the nucleus with upregulation of poly (ADP-ribose) polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). Although cell death is an important aspect of FECD, previous reports have often analyzed immortalized cell lines, making the evaluation of cell death difficult. Therefore, we established a new in vitro FECD model to evaluate the pathophysiology of FECD. Methods: Corneal endothelial cells were derived from disease-specific induced pluripotent stem cells (iPSCs). Hydrogen peroxide (H2O2) was used as a source for oxidative stress to mimic the pathophysiology of FECD. We investigated the responses to oxidative stress and the involvement of parthanatos in FECD-corneal endothelial cells. Results: Cell death ratio and oxidative stress level were upregulated in FECD with H2O2 treatment compared with non-FECD control, indicating the vulnerability of oxidative stress in FECD. We also found that intracellular PAR, as well as PARP-1 and AIF in the nucleus were upregulated in FECD. Furthermore, PARP inhibition, but not pan-caspase inhibition, rescued cell death, DNA double-strand breaks, mitochondrial membrane potential depolarization and energy depletion, suggesting that cell death was mainly due to parthanatos. Conclusions: We report that parthanatos may be involved in the pathophysiology of FECD and targeting this cell death pathway may be a potential therapeutic approach for FECD.
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GM1 gangliosidosis is a lysosomal storage disease caused by loss of lysosomal ß-galactosidase activity and characterized by progressive neurodegeneration due to massive accumulation of GM1 ganglioside in the brain. Here, we generated induced pluripotent stem cells (iPSCs) derived from patients with GM1 gangliosidosis, and the resultant neurons showed impaired neurotransmitter release as a presynaptic function and accumulation of GM1 ganglioside. Treatment of normal neurons with GM1 ganglioside also disturbed presynaptic function. A high-content drug-screening system was then established and identified two compounds as drug candidates for GM1 gangliosidosis. Treatment of the patient-derived neurons with the candidate agents activated autophagy pathways, reducing GM1 ganglioside accumulation in vitro and in vivo, and restoring the presynaptic dysfunction. Our findings thus demonstrated the potential value of patient-derived iPSC lines as cellular models of GM1 gangliosidosis and revealed two potential therapeutic agents for future clinical application.
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Autofagia , Gangliosídeo G(M1)/metabolismo , Gangliosidose GM1/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Células Cultivadas , Desenvolvimento de Medicamentos/métodos , Gangliosidose GM1/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.
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Células-Tronco Pluripotentes Induzidas , Linfócitos do Interstício Tumoral , Melanoma/patologia , Transferência Adotiva , Humanos , Imunoterapia AdotivaRESUMO
Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy.
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UNLABELLED: Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. SIGNIFICANCE: Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature ß cell phenotype would lead to safe islet replacement therapy for diabetes.
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Diabetes Mellitus Tipo 1/cirurgia , Diabetes Mellitus Tipo 2/cirurgia , Células-Tronco Pluripotentes Induzidas/transplante , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/cirurgia , Queratinócitos/transplante , Regeneração , Teratocarcinoma/prevenção & controle , Adulto , Idoso , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Reprogramação Celular , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Xenoenxertos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Queratinócitos/metabolismo , Queratinócitos/patologia , Lentivirus/genética , Masculino , Camundongos SCID , Fenótipo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Teratocarcinoma/patologia , TransfecçãoRESUMO
Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier.
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Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Células-Tronco Neurais/citologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Regulação para Cima , Biópsia , Diferenciação Celular , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Inativação Gênica , Vetores Genéticos , Genótipo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Cariotipagem , Linfócitos/citologia , Masculino , Microscopia Confocal , Mutação , Linhagem , Fenótipo , RNA Interferente Pequeno/metabolismo , Pele/patologiaRESUMO
Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.
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Anemia Hemolítica Congênita não Esferocítica/genética , Anemia Hemolítica Congênita não Esferocítica/terapia , Células Eritroides/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Piruvato Quinase/deficiência , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/genética , Erros Inatos do Metabolismo dos Piruvatos/terapia , Alelos , Sequência de Bases , Contagem de Células , DNA Complementar/genética , Células Eritroides/metabolismo , Marcação de Genes , Terapia Genética , Humanos , Leucócitos Mononucleares/metabolismo , Recombinação GenéticaRESUMO
Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21.
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Envelhecimento/fisiologia , Diferenciação Celular/fisiologia , Síndrome de Down/genética , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Animais , Células Cultivadas , Fibroblastos/citologia , Humanos , Mitocôndrias/genéticaRESUMO
Niemann-Pick disease type C (NPC) is a lysosomal storage disease characterized by abnormal accumulation of free cholesterol and glycolipids. Here, we established induced pluripotent stem cell (iPSC) lines from NPC patients. Hepatocyte-like cells (HLCs) and neural progenitors derived from the iPSC lines accumulated cholesterol and displayed impaired autophagy and ATP production. A molecular signature related to lipid metabolism was also impaired in the NPC-iPSC-derived HLCs. These findings indicate that iPSC-derived cells can phenocopy human NPC. We also newly found that 2-hydroxypropyl-γ-cyclodextrin (HPGCD) could reduce the cholesterol accumulation and restore the functional and molecular abnormalities in the NPC patient-derived cells, and do so more effectively than 2-hydroxypropyl-ß-cyclodextrin treatment. In addition, NPC model mice showed an improved liver status and prolonged survival with HPGCDs. Thus, iPSC lines derived from patient cells are powerful tools to study cellular models of NPC, and HPGCD is a potential new drug candidate for future treatment of this disease.
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Células-Tronco Pluripotentes Induzidas/patologia , Doença de Niemann-Pick Tipo C/patologia , beta-Ciclodextrinas/farmacologia , gama-Ciclodextrinas/farmacologia , 2-Hidroxipropil-beta-Ciclodextrina , Animais , Técnicas de Cocultura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/metabolismo , Resultado do Tratamento , beta-Ciclodextrinas/uso terapêutico , gama-Ciclodextrinas/uso terapêuticoRESUMO
Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.
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Diferenciação Celular/genética , Vetores Genéticos , Vírus Sendai/genética , Transdução Genética , Animais , Linhagem Celular , Reprogramação Celular/genética , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Pan troglodytes , TransgenesRESUMO
Human dental pulp cells (hDPCs) are a promising resource for regenerative medicine and tissue engineering and can be used for derivation of induced pluripotent stem cells (iPSCs). However, current protocols use reagents of animal origin (mainly fetal bovine serum, FBS) that carry the potential risk of infectious diseases and unwanted immunogenicity. Here, we report a chemically defined protocol to isolate and maintain the growth and differentiation potential of hDPCs. hDPCs cultured under these conditions showed significantly less primary colony formation than those with FBS. Cell culture under stringently defined conditions revealed a donor-dependent growth capacity; however, once established, the differentiation capabilities of the hDPCs were comparable to those observed with FBS. DNA array analyses indicated that the culture conditions robustly altered hDPC gene expression patterns but, more importantly, had little effect on neither pluripotent gene expression nor the efficiency of iPSC induction. The chemically defined culture conditions described herein are not perfect serum replacements, but can be used for the safe establishment of iPSCs and will find utility in applications for cell-based regenerative medicine.
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Polpa Dentária/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Adolescente , Diferenciação Celular , Células Cultivadas , Humanos , Cultura Primária de Células/métodosRESUMO
Familial amyloidotic polyneuropathy (FAP) is a hereditary amyloidosis induced by amyloidogenic transthyretin (ATTR). Because most transthyretin (TTR) in serum is synthesized by the liver, liver transplantation (LT) is today the only treatment available to halt the progression of FAP, even though LT is associated with several problems. Despite the urgent need to develop alternatives to LT, the detailed pathogenesis of FAP is still unknown; also, no model fully represents the relevant processes in patients with FAP. The induction of induced pluripotent stem (iPS) cells has allowed development of pluripotent cells specific for patients and has led to useful models of human diseases. Because of the need for a tool to elucidate the molecular pathogenesis of FAP, in this study we sought to establish heterozygous ATTR mutant iPS cells, and were successful, by using a Sendai virus vector mixture containing four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to reprogram dermal fibroblasts derived from FAP patients. Moreover, FAP-specific iPS cells had the potential to differentiate into hepatocyte-like cells and indeed expressed ATTR. FAP-specific iPS cells demonstrated the possibility of serving as a pathological tool that will contribute to understanding the pathogenesis of FAP and development of FAP treatments.
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Neuropatias Amiloides Familiares/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Pré-Albumina/metabolismo , Neuropatias Amiloides Familiares/genética , Animais , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , CamundongosRESUMO
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.
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Envelhecimento/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Reprogramação Celular , Senescência Celular , Criança , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/transplante , Neurônios Dopaminérgicos/ultraestrutura , Fibroblastos/metabolismo , Humanos , Lamina Tipo A , Mesencéfalo/patologia , Camundongos , Pessoa de Meia-Idade , Doença de Parkinson/patologia , Fenótipo , Doadores de TecidosRESUMO
Studies of rare genetic bone disorders are often limited due to unavailability of tissue specimens and the lack of animal models fully replicating phenotypic features. Craniometaphyseal dysplasia (CMD) is a rare monogenic disorder characterized by hyperostosis of craniofacial bones concurrent with abnormal shape of long bones. Mutations for autosomal dominant CMD have been identified in the ANK gene (ANKH). Here we describe a simple and efficient method to reprogram adherent cells cultured from peripheral blood to human induced pluripotent stem cells (hiPSCs) from eight CMD patients and five healthy controls. Peripheral blood mononuclear cells (PBMCs) were separated from 5-7 mL of whole blood by Ficoll gradient, expanded in the presence of cytokines and transduced with Sendai virus (SeV) vectors encoding OCT3/4, SOX2, KLF4, and c-MYC. SeV vector, a cytoplasmic RNA vector, is lost from host cells after propagation for 10-13 passages. These hiPSCs express stem cell markers, have normal karyotypes, and are capable of forming embryoid bodies in vitro as well as teratomas in vivo. Further differentiation of these patient-specific iPSCs into osteoblasts and osteoclasts can provide a useful tool to study the effects CMD mutations on bone, and this approach can be applied for disease modeling of other rare genetic musculoskeletal disorders.
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Doenças do Desenvolvimento Ósseo/sangue , Reprogramação Celular , Anormalidades Craniofaciais/sangue , Vetores Genéticos , Hiperostose/sangue , Hipertelorismo/sangue , Células-Tronco Pluripotentes Induzidas/citologia , Vírus Sendai/genética , Adulto , Sequência de Bases , Estudos de Casos e Controles , Criança , Primers do DNA , Feminino , Rearranjo Gênico do Linfócito T , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Cancer is initiated by the transformation of stem cells or progenitor cells via a dedifferentiation process that leads to cancer stem cells; however, the process involves the activation of growth-promoting oncogenes and the inactivation of growth-constraining tumor suppressor genes. The introduction of defined factors, such as those encoded by c-Myc, Sox2, Oct3/4 and Klf4, in normal somatic cells results in their dedifferentiation into induced pluripotent stem (iPS) cells. We previously reported that these defined factors induced the development of induced multipotent cancer (iPC) cells from gastrointestinal cancer cells by reducing tumor aggressiveness. Previous studies indicated that although reprogramming may be facilitated by p53 inhibition, gain-of-function oncogenic mutations in p53 and oncogenic mutations in Kras-stimulated tumorigenic activity, and their roles in vivo are imperfectly understood. Hence, in the present study, the effect of direct injection of a Sendai virus (SeV) vector encoding four defined factors in vivo was studied using various backgrounds of transgenic and knockout mice, and was compared with that of direct injection of microRNAs (miRNAs) diluted with cationic lipid. The in vivo imaging data revealed transformation hot spots for p53 deficiency or conditional activation of mutant Kras, and the sizes were concordant with those in immuno-deficient NOD/SCID and uPA-NOG mice, as well as larger compared with those in the control mice. Overall, the present data on in vivo reprogramming indicated that Kras activation may facilitate the effect of cellular reprogramming in normal liver cells, and the effect of Kras activation is more apparent than that of tumor suppressor p53 deficiency. The results also revealed that immunodeficiency may increase the effect of reprogramming, presumably by blocking the immunosurveillance of transformed cells. These findings provide a rationale for further studies to develop a therapeutic approach involving direct in vivo reprogramming.
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The discovery of induced pluripotent stem cells (iPSCs) holds great promise for regenerative medicine since it is possible to produce patient-specific pluripotent stem cells from affected individuals for potential autologous treatment. Using nonintegrating cytoplasmic Sendai viral vectors, we generated iPSCs efficiently from adult mobilized CD34⺠and peripheral blood mononuclear cells. After 5-8 passages, the Sendai viral genome could not be detected by real-time quantitative reverse transcription-polymerase chain reaction. Using the spin embryoid body method, we showed that these blood cell-derived iPSCs could efficiently be differentiated into hematopoietic stem and progenitor cells without the need of coculture with either mouse or human stromal cells. We obtained up to 40% CD34⺠of which ~25% were CD34âº/CD43⺠hematopoietic precursors that could readily be differentiated into mature blood cells. Our study demonstrated a reproducible protocol for reprogramming blood cells into transgene-free iPSCs by the Sendai viral vector method. Maintenance of the genomic integrity of iPSCs without integration of exogenous DNA should allow the development of therapeutic-grade stem cells for regenerative medicine.
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Células Sanguíneas/citologia , Reprogramação Celular , Vetores Genéticos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Vírus Sendai/genética , Fatores de Transcrição/metabolismo , Adulto , Animais , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Reprogramação Celular/genética , Feminino , Perfilação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Transgenes/genéticaRESUMO
The induced pluripotent stem cell (iPSC) technology enables derivation of patient-specific pluripotent stem cells from adult somatic cells without using an embryonic cell source. Redifferentiation of iPSCs from diabetic patients into pancreatic islets will allow patient-specific disease modeling and autologous cell replacement therapy for failing islets. To date, diabetes-specific iPSCs have been generated from patients with type 1 diabetes using integrating retroviral vectors. However, vector integration into the host genome could compromise the biosafety and differentiation propensities of derived iPSCs. Although various integration-free reprogramming systems have been described, their utility to reprogram somatic cells from patients remains largely undetermined. Here, we used nonintegrating Sendai viral vectors to reprogram cells from patients with type 1 and type 2 diabetes (T2D). Sendai vector infection led to reproducible generation of genomic modification-free iPSCs (SV-iPSCs) from patients with diabetes, including an 85-year-old individual with T2D. SV-iPSCs lost the Sendai viral genome and antigens within 8-12 passages while maintaining pluripotency. Genome-wide transcriptome analysis of SV-iPSCs revealed induction of endogenous pluripotency genes and downregulation of genes involved in the oxidative stress response and the INK4/ARF pathways, including p16(INK4a), p15(INK4b), and p21(CIP1). SV-iPSCs and iPSCs made with integrating lentiviral vectors demonstrated remarkable similarities in global gene expression profiles. Thus, the Sendai vector system facilitates reliable reprogramming of patient cells into transgene-free iPSCs, providing a pluripotent platform for personalized diagnostic and therapeutic approaches for diabetes and diabetes-associated complications.
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Diabetes Mellitus Tipo 1/terapia , Diabetes Mellitus Tipo 2/terapia , Células-Tronco Pluripotentes Induzidas/metabolismo , Transgenes , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Regulação da Expressão Gênica , Genes p16 , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Genoma Viral , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Queratinócitos/citologia , Queratinócitos/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Estresse Oxidativo , Vírus Sendai/genética , Vírus Sendai/metabolismo , Transdução de Sinais , TranscriptomaRESUMO
Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by progressive ossification of soft tissues. FOP is caused by mutations in activin receptor-like kinase 2 (ALK2) that cause its constitutive activation and result in dysregulation of BMP signaling. Here, we show that generation of induced pluripotent stem cells (iPSCs) from FOP-derived skin fibroblasts is repressed because of incomplete reprogramming and inhibition of iPSC maintenance. This repression was mostly overcome by specific suppression of ALK2 expression and treatment with an ALK2 inhibitor, indicating that the inhibition of iPSC generation and maintenance observed in FOP-derived skin fibroblasts results from constitutive activation of ALK2. Using this system, we identified an ALK2 inhibitor as a potential candidate for future drug development. This study highlights the potential of the inhibited production and maintenance of iPSCs seen in diseases as a useful phenotype not only for studying the molecular mechanisms underlying iPS reprogramming but also for identifying drug candidates for future therapies.
Assuntos
Receptores de Ativinas Tipo I/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Miosite Ossificante/patologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Mutação de Sentido Incorreto , Miosite Ossificante/genética , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Pele/patologia , TranscriptomaRESUMO
CD34+ cord blood cells can be reprogrammed effectively on dishes coated with a synthetic RGD motif polymer (PronectinF®) using a temperature sensitive Sendai virus vector (SeV TS7) carrying reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC. Dish-shaped human ES cell-like colonies emerged in serum-free primate ES cell medium (supplemented with bFGF) in 20% O2 culture conditions. The copy numbers of SeV TS7 vectors in the cytoplasm were drastically reduced by a temperature shift at 38°C for three days. Then, single cells from colonies were seeded on PronectinF®-coated 96-well plates and cultured under naïve culture conditions (N2B27-based medium supplemented with LIF, forskolin, a MAPK inhibitor, and a GSK inhibitor in 5% O2) for cloning purpose. Dome-shaped mouse ES cell-like colonies from single cells emerged on PronectinF®-coated dishes. These cells were collected and cultured again in primate ES cell medium supplemented with bFGF in 20% O2 and maintained on PronectinF®-coated dishes. Cells were assessed for reprogramming, including the absence of residual SeV and their potential for three germ layer differentiation. Generation of virus-free induced pluripotent stem cell (iPSC) clones from single cells under feeder-free conditions will solve some of the safety concerns related to use of xeno- or allogeneic-material in culture, and contribute to the characterization and the standardization of iPS cells intended for use in a clinical setting.
Assuntos
Células Clonais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Células Cultivadas , Células Clonais/imunologia , Meios de Cultura , Vetores Genéticos , Humanos , Cariotipagem , Fator 4 Semelhante a Kruppel , Camundongos , Células-Tronco Pluripotentes/imunologia , Primatas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai/genéticaRESUMO
To identify therapeutic molecular targets for glioma, we performed modified serological identification of antigens by recombinant complementary DNA (cDNA) expression cloning using sera from a mouse glioma model. Two clones, kinesin family member 23 (Kif23) and structural maintenance of chromosomes 4 (Smc4), were identified as antigens through immunological reaction with sera from mice harboring synergic GL261 mouse glioma and intratumoral inoculation with a mutant herpes simplex virus. The human Kif23 homolog KIF23 is a nuclear protein that localizes to the interzone of mitotic spindles, acting as a plus-end-directed motor enzyme that moves antiparallel microtubules in vitro. Expression analysis revealed a higher level of KIF23 expression in glioma tissues than in normal brain tissue. The introduction of small interfering RNA (siRNA) targeting KIF23 into two different glioma cell lines, U87MG and SF126, downregulated KIF23 expression, which significantly suppressed glioma cell proliferation in vitro. KIF23 siRNA-treated glioma cells exhibited larger cell bodies with two or more nuclei compared with control cells. In vivo analysis using mouse xenograft showed that KIF23 siRNA/DNA chimera-treated tumors were significantly smaller than tumors treated with control siRNA/DNA chimera. Taken together, our results indicate that downregulation of KIF23 decreases proliferation of glioma cells and that KIF23 may be a novel therapeutic target in malignant glioma.