RESUMEN
We describe a protocol for efficient generation of human-induced pluripotent stem cells (hiPSCs) from urine-derived cells (UDCs) obtained from adult donors using self-replicative RNA containing the reprogramming factors OCT3/4, SOX2, KLF4, GLIS1, and c-MYC (ReproRNA-OKSGM). After electroporation, transfection efficiency is quantified by measuring OCT3/4-expressing UDCs using flow cytometry and should be ≥0.1%. hiPSC colonies emerge within 3 weeks after transfection and express multiple pluripotency markers. Moreover, the UDC-derived hiPSCs are able to differentiate into cells of all three germ layers and display normal karyotypes. ReproRNA-OKSGM is available commercially and only requires a single transfection step so that the protocol is readily accessible, as well as straightforward. In addition to a detailed step-by-step description for generating clonal hiPSCs from UDCs using ReproRNA-OKSGM, we provide guidance for basic pluripotency characterization of the hiPSC lines. © 2020 The Authors. Basic Protocol: Reprogramming of urine-derived cells using ReproRNA-OKSGM Support Protocol 1: Determination of the pluripotency status of hiPSCs by flow cytometry Support Protocol 2: Characterization of functional pluripotency of hiPSCs.
Asunto(s)
Técnicas de Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Orina/citología , Células Cultivadas , Electroporación , Humanos , Factor 4 Similar a Kruppel , ARN/metabolismoRESUMEN
Fibroblasts from a patient carrying a heterozygous 18bp deletion in exon 8 of the ACVRL1 gene (c.1120del18) were reprogrammed using episomal vectors. The in-frame deletion in ACVRL1 causes the loss of 6 amino acids of the protein, which is associated with Hereditary Hemorrhagic Telangiectasia (HHT) type 2 (Letteboer et al., 2005). CRISPR-Cas9 editing was used to genetically correct the mutation in the induced pluripotent stem cells (iPSCs). The top5-predicted off-target sites were not altered. Patient and isogenic iPSCs showed high pluripotent marker expression, in vitro differentiation capacity into all three germ layers and displayed a normal karyotype. The obtained isogenic pairs will enable proper in vitro disease modelling of HHT (Roman and Hinck, 2017).
Asunto(s)
Células Madre Pluripotentes Inducidas , Telangiectasia Hemorrágica Hereditaria , Receptores de Activinas Tipo II/genética , Células Clonales , Heterocigoto , Humanos , Mutación/genética , Telangiectasia Hemorrágica Hereditaria/genéticaRESUMEN
Lymphoblast cells from four individuals of a family of two genetically unrelated parents and their monozygotic twins were used to generate integration-free human induced pluripotent stem cells (hiPSCs). Reprogramming factors were delivered by co-electroporation of three episomal-based plasmids expressing OCT3/4, SOX2, KLF4, L-MYC and LIN28. The hiPSCs showed a normal karyotype, expressed pluripotency-associated markers, displayed the capacity for in vitro differentiation into the three germ layers and were Epstein Barr virus-free. These hiPSC lines offer the possibility to compare genetically unrelated and genetically identical tissues from different individuals and to study genotype-specific effects, which are particularly relevant for toxicology testing.
Asunto(s)
Línea Celular/citología , Células Madre Pluripotentes Inducidas/patología , Linfocitos/patología , Padres , Gemelos Monocigóticos/genética , Adulto , Anciano , Femenino , Humanos , Factor 4 Similar a Kruppel , Masculino , Reproducibilidad de los ResultadosRESUMEN
Spinocerebellar ataxia type 1 (SCA1) is a hereditary neurodegenerative disease caused by a CAG repeat expansion in exon 8 of the ATXN1 gene. We generated induced pluripotent stem cells (hiPSCs) from a SCA1 patient and his non-affected sister by using non-integrating Sendai Viruses (SeV). The resulting hiPSCs are SeVfree, express pluripotency markers, display a normal karyotype, retain the mutation (length of the CAG repeat expansion in the ATXN1 gene) and are able to differentiate into the three germ layers in vitro.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Ataxias Espinocerebelosas/metabolismo , Adulto , Diferenciación Celular , Línea Celular , Femenino , Humanos , Masculino , HermanosRESUMEN
The ability to form teratomas in vivo containing multiple somatic cell types is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal dependent, laborious, and only qualitative, the PluriTest and the hPSC ScoreCard assay have been developed as in vitro alternatives. Here we compared normal hPSCs, induced hPSCs (hiPSCs) with reactivated reprogramming transgenes, and human embryonal carcinoma cells (hECs) in these assays. While normal hPSCs gave rise to typical teratomas, the xenografts of the hECs and the hiPSCs with reactivated reprogramming transgenes were largely undifferentiated and malignant. The hPSC ScoreCard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analyzed by the PluriTest, only hECs were identified as abnormal whereas all other cell lines were indistinguishable and resembled normal hPSCs. Our results indicate that pluripotency assays are best selected on the basis of intended downstream applications.