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1.
Stem Cell Res Ther ; 15(1): 73, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38475911

RESUMO

BACKGROUND: Cell- or tissue-based regenerative therapy is an attractive approach to treat heart failure. A tissue patch that can safely and effectively repair damaged heart muscle would greatly improve outcomes for patients with heart failure. In this study, we conducted a preclinical proof-of-concept analysis of the efficacy and safety of clinical-grade human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches. METHODS: A clinical-grade hiPSC line was established using peripheral blood mononuclear cells from a healthy volunteer that was homozygous for human leukocyte antigens. The hiPSCs were differentiated into cardiomyocytes. The obtained hiPSC-CMs were cultured on temperature-responsive culture dishes for patch fabrication. The cellular characteristics, safety, and efficacy of hiPSCs, hiPSC-CMs, and hiPSC-CM patches were analyzed. RESULTS: The hiPSC-CMs expressed cardiomyocyte-specific genes and proteins, and electrophysiological analyses revealed that hiPSC-CMs exhibit similar properties to human primary myocardial cells. In vitro and in vivo safety studies indicated that tumorigenic cells were absent. Moreover, whole-genome and exome sequencing revealed no genomic mutations. General toxicity tests also showed no adverse events posttransplantation. A porcine model of myocardial infarction demonstrated significantly improved cardiac function and angiogenesis in response to cytokine secretion from hiPSC-CM patches. No lethal arrhythmias were observed. CONCLUSIONS: hiPSC-CM patches are promising for future translational research and may have clinical application potential for the treatment of heart failure.


Assuntos
Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Suínos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares , Miocárdio , Insuficiência Cardíaca/terapia
2.
Stem Cell Reports ; 13(4): 761-774, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31564644

RESUMO

The first-in-human trial of induced pluripotent stem cell (iPSC)-based autologous transplantation was successfully performed on a female patient with age-related macular degeneration. Here we delineated the base-resolution methylome of the iPSC-derived retinal pigment epithelium (iRPE) used in this trial. The methylome of iRPE closely resembled that of native RPE (nRPE), although partially methylated domains (PMDs) emerged in iRPE but not nRPE. Most differentially methylated regions between iRPE and nRPE appeared to originate from (de)methylation errors during differentiation, whereas errors at reprogramming resulted in aberrant genomic imprinting and X chromosome reactivation. Moreover, non-CpG methylation was prominent in nRPE but not iRPE. Intriguingly, xenotransplantation to mouse remodeled the iRPE methylome to demethylate a subset of suppressed genes and accumulate non-CpG methylation, but failed to resolve PMDs and hypermethylated CpG islands. Although the impacts of these alterations remain elusive, our findings should provide a useful guide for methylome analyses of other iPSC-derived cells.


Assuntos
Epigenoma , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Epitélio Pigmentado da Retina/citologia , Transplante de Células-Tronco , Reprogramação Celular , Biologia Computacional/métodos , Ilhas de CpG , Metilação de DNA , Humanos , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/terapia , Transplante Autólogo , Sequenciamento Completo do Genoma
3.
Cell Stem Cell ; 24(4): 566-578.e7, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30853558

RESUMO

Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world's population, greatly facilitating iPSC-based regenerative medicine applications.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Antígenos HLA/genética , Histocompatibilidade/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Linhagem Celular , Feminino , Antígenos HLA/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD
4.
Cell Rep ; 23(2): 361-375, 2018 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-29641997

RESUMO

Here, we report that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC.


Assuntos
Reprogramação Celular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/metabolismo , Antígenos de Superfície/metabolismo , Linhagem Celular , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fosforilação , Proteoglicanas/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
5.
N Engl J Med ; 376(11): 1038-1046, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28296613

RESUMO

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/citologia , Idoso , Técnicas de Cultura de Células , Diferenciação Celular , Estudos de Viabilidade , Feminino , Fibroblastos , Humanos , Masculino , Epitélio Pigmentado da Retina/transplante , Transplante Autólogo
6.
Sci Rep ; 6: 30013, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27418197

RESUMO

Cardiovascular complications are the leading cause of death in autosomal dominant polycystic kidney disease (ADPKD), and intracranial aneurysm (ICA) causing subarachnoid hemorrhage is among the most serious complications. The diagnostic and therapeutic strategies for ICAs in ADPKD have not been fully established. We here generated induced pluripotent stem cells (iPSCs) from seven ADPKD patients, including four with ICAs. The vascular cells differentiated from ADPKD-iPSCs showed altered Ca(2+) entry and gene expression profiles compared with those of iPSCs from non-ADPKD subjects. We found that the expression level of a metalloenzyme gene, matrix metalloproteinase (MMP) 1, was specifically elevated in iPSC-derived endothelia from ADPKD patients with ICAs. Furthermore, we confirmed the correlation between the serum MMP1 levels and the development of ICAs in 354 ADPKD patients, indicating that high serum MMP1 levels may be a novel risk factor. These results suggest that cellular disease models with ADPKD-specific iPSCs can be used to study the disease mechanisms and to identify novel disease-related molecules or risk factors.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Aneurisma Intracraniano/patologia , Metaloproteinase 1 da Matriz/sangue , Rim Policístico Autossômico Dominante/patologia , Hemorragia Subaracnóidea/patologia , Idoso , Animais , Biomarcadores/sangue , Diferenciação Celular , Células Cultivadas , Metilação de DNA/genética , Feminino , Humanos , Aneurisma Intracraniano/sangue , Masculino , Metaloproteinase 1 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/mortalidade , Fatores de Risco , Canais de Cátion TRPP/genética
7.
Biol Pharm Bull ; 32(4): 665-70, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19336902

RESUMO

The stems of Akebia plants, Akebiae Caulis, have long been used in traditional Chinese and Japanese medicines, and are mainly produced in western Japan. Three Akebia plants, Akebia quinata (AQ), A. trifoliata (AT), and A. pentaphylla (AP) grow wild in Japan. With the aim of carrying out molecular biological identification of Akebia plant species and discriminating Akebiae Caulis from other related crude drugs originating from non-Akebia plants, sequencing analysis of Akebia plants collected from various parts of Japan and the southern Korean Peninsula was performed. Specimens identified morphologically as AQ and AT had their respective common internal transcribed spacer one (ITS1) sequences, which could be distinguished. Cloning experiments of AP specimens showed that their ITS1 contained both common sequences of AQ and AT as well as their chimera. These chimeric sequences were not identical between AP specimens, suggesting that AP is not a species with uniform DNA sequences but a group of individuals with hybrid genomes of AQ and AT. Based on the sequences of Akebia species found here, we propose polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) methods to discriminate Akebiae Caulis from the related crude drugs and to distinguish three Akebia plants. Comparison of triterpene-rich fractions of extracts from Akebia plants by TLC showed that AP had an intermediate profile of AQ and AT.


Assuntos
Medicina Tradicional do Leste Asiático , Preparações de Plantas/química , Ranunculaceae/química , Ranunculaceae/genética , Cromatografia em Camada Fina , DNA de Plantas/biossíntese , DNA de Plantas/genética , Japão , Coreia (Geográfico) , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
J Nat Med ; 63(3): 368-74, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19277830

RESUMO

The stem of the Akebia plant, "Mokutsu", is a crude diuretic and antiphlogistic drug. Japanese products prepared from wild Akebia plants cover most of the Mokutsu market. Two Akebia plants, Akebia quinata Decaisne (Aq) and A. trifoliata Koidzumi (At) of Lardizabalaceae, are standardized as Mokutsu in Japanese pharmacopoeia. These two Akebia plants along with A. x pentaphylla Makino (Ap), which is considered a hybrid with the morphology of Aq and At, can be distinguished by DNA sequence analysis of internal transcribed spacers 1 and 2 (ITS) of nuclear ribosome DNA. Here, we report the results of molecular genetic analysis of Akebia plants grown in various wild habitats in Japan. We found that each of three Akebia plants could be distinguished in terms of their locality according to their nucleotide sequence in ITS, specifically at positions 91, 128, 133, 134, and 221. Plants with a comparable habitat had similar nucleotide sequences at these five points. We also found Aq with ITS and nucleotide deletion at position 86 that was distributed only around Awajishima in Shikoku (A), Harimanada (B), and Kinki (C), including the chief production center of Akebia Caulis. The results of these ITS sequences enabled discrimination of plants originating from Akebia Caulis.


Assuntos
DNA Espaçador Ribossômico/genética , Variação Genética , Magnoliopsida/genética , Geografia , Japão , Magnoliopsida/classificação , Reação em Cadeia da Polimerase
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