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1.
Stem Cell Res ; 54: 102438, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34214898

RESUMO

Friedreich's ataxia (FRDA) is a rare neurodegenerative disorder which is caused by triplet repeat expansion (GAA) in the first intron of FXN gene. In this present study, we generated induced pluripotent stem cells (iPSC) lines from fibroblasts of three unrelated FRDA patients using integration-free episomal vectors. All iPSC lines express the pluripotency markers such as OCT4 and SSEA4, display normal karyotypes and can differentiate into all three germ layers via in vivo teratoma formation assay.


Assuntos
Ataxia de Friedreich , Células-Tronco Pluripotentes Induzidas , Proteínas de Ligação ao Ferro , Ataxia de Friedreich/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons/genética , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Expansão das Repetições de Trinucleotídeos , Frataxina
2.
Epigenetics Chromatin ; 14(1): 32, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215314

RESUMO

BACKGROUND: The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. RESULTS: We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. CONCLUSIONS: Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.


Assuntos
Reprogramação Celular , Histona-Lisina N-Metiltransferase , Fatores de Transcrição , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Proteômica , Fatores de Transcrição/metabolismo
3.
Cell Death Dis ; 11(8): 658, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814763

RESUMO

Complete hydatidiform mole (HM) is a gestational trophoblastic disease resulting in hyperproliferation of trophoblast cells and absence of embryo development. Mutations in the maternal-effect gene NLRP7 are the major cause of familial recurrent complete HM. Here, we established an in vitro model of HM using patient-specific induced pluripotent stem cells (iPSCs) derived trophoblasts harboring NLRP7 mutations. Using whole transcriptome profiling during trophoblast differentiation, we showed that impaired NLRP7 expression results in precocious downregulation of pluripotency factors, activation of trophoblast lineage markers, and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. Interestingly, we found that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition corrected the excessive trophoblast differentiation of patient-derived iPSCs. Our human iPSC model of a genetic placental disease recapitulates aspects of trophoblast biology, highlights the broad utility of iPSC-derived trophoblasts for modeling human placental diseases and identifies NLRP7 as an essential modulator of key developmental cell fate regulators.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Trofoblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteína Morfogenética Óssea 4/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Mola Hidatiforme/genética , Mola Hidatiforme/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Biológicos , Placenta/metabolismo , Gravidez , Transdução de Sinais/fisiologia , Transcriptoma/genética
4.
Stem Cell Reports ; 13(4): 627-641, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31522975

RESUMO

Organoid technologies have become a powerful emerging tool to model liver diseases, for drug screening, and for personalized treatments. These applications are, however, limited in their capacity to generate functional hepatocytes in a reproducible and efficient manner. Here, we generated and characterized the hepatic organoid (eHEPO) culture system using human induced pluripotent stem cell (iPSC)-derived EpCAM-positive endodermal cells as an intermediate. eHEPOs can be produced within 2 weeks and expanded long term (>16 months) without any loss of differentiation capacity to mature hepatocytes. Starting from patient-specific iPSCs, we modeled citrullinemia type 1, a urea cycle disorder caused by mutations in the argininosuccinate synthetase (ASS1) enzyme. The disease-related ammonia accumulation phenotype in eHEPOs could be reversed by the overexpression of the wild-type ASS1 gene, which also indicated that this model is amenable to genetic manipulation. Thus, eHEPOs are excellent unlimited cell sources to generate functional hepatic organoids in a fast and efficient manner.


Assuntos
Diferenciação Celular , Suscetibilidade a Doenças , Endoderma/citologia , Hepatócitos/citologia , Fígado/citologia , Fígado/embriologia , Organogênese , Organoides/citologia , Biomarcadores , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Técnicas de Cultura de Tecidos
5.
In Vitro Cell Dev Biol Anim ; 55(7): 473-481, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31214928

RESUMO

Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.


Assuntos
Blastocisto/citologia , Proliferação de Células/efeitos dos fármacos , Leptina/farmacologia , Células-Tronco Embrionárias Murinas/citologia , Teratoma/metabolismo , Animais , Fator de Transcrição CDX2/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária , Corpos Embrioides/citologia , Antígenos CD15/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína Homeobox Nanog/biossíntese , Fator 3 de Transcrição de Octâmero/biossíntese , Teratoma/induzido quimicamente
6.
Methods Mol Biol ; 1353: 215-31, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26126451

RESUMO

Induced pluripotent stem cells (iPSCs) offer great promise as tools for basic biomedical research, disease modeling, and drug screening. In this chapter, we describe the generation of patient-specific, transgene-free iPSCs from skin biopsies and peripheral blood mononuclear cells through electroporation of episomal vectors and growth under two different culture conditions. The resulting iPSC lines are characterized with respect to pluripotency marker expression through immunostaining, tested for transgene integration by PCR, and assayed for differentiation capacity via teratoma formation.


Assuntos
Técnicas de Cultura de Células/métodos , Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Leucócitos Mononucleares/citologia , Amidas/farmacologia , Animais , Biomarcadores/metabolismo , Biópsia , Diferenciação Celular/efeitos dos fármacos , Colágeno/química , Criopreservação , Combinação de Medicamentos , Eletroporação , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Laminina/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Plasmídeos/genética , Plasmídeos/metabolismo , Cultura Primária de Células , Proteoglicanas/química , Piridinas/farmacologia , Pele/citologia , Pele/metabolismo , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Transgenes
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