RESUMO
Runt-Related Transcription Factor 1c (RUNX1c) plays an important role in regulating the development of hematopoietic stem cells (HSC). Using CRISPR/Cas9 gene editing technology, we established a RUNX1c-eGFP reporter cell line from the MUSIi012-A cell line. The MUSIi012-A-4 cell line has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro and in vivo. This cell line serves as a valuable model to observe the expression of RUNX1c via eGFP tracking during human hematopoietic development.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Edição de Genes , Diferenciação CelularRESUMO
Natural killer (NK) cells were isolated from human umbilical cord blood from a healthy newborn and reprogrammed by episomal vectors carrying reprograming factors L-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1, and shRNA against p53 delivered using nucleofection. The obtained MUSIi013-A human induced pluripotent stem cell (iPSC) line highly expressed pluripotency markers, had the capacity to differentiate into derivatives of the three germ layers, while retained a normal karyotype. This cell line may be a useful tool to study epigenic memory that may predispose hiPSCs to enhanced NK differentiation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Sangue Fetal , Humanos , Células Matadoras Naturais , Fator 4 Semelhante a KruppelRESUMO
Yes-associated protein (YAP) is an important transcriptional coactivator in the Hippo signaling pathway. Using CRISPR/Cas9 technology, we established a stable YAP-knockdown (YAP-KD) induced pluripotent stem cell (iPSC) from the MUSIi012-A cell line. The YAP-KD iPSC MUSIi012-A-2 maintained the pluripotent phenotype, the ability to differentiate into all three embryonic germ layers, and it maintained the normal karyotype.
Assuntos
Proteínas de Ciclo Celular/genética , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Feminino , HumanosRESUMO
MUSIe001-A cell line was derived from a Southeast Asian (SEA) type deletion α0-thalassemia embryo. The SEA deletion embryo was donated for research with informed consent. This cell line shows normal hESC morphology, expresses all pluripotent markers, and has the potential to differentiate into all three germ layers in vitro and in vivo. The MUSIe001-A line has normal karyotype and is free from mycoplasma contamination. PCR analysis confirmed the MUSIe001-A cell line to be a SEA type deletion. MUSIe001-A is a valuable proof of principle model for gene therapy that will facilitate the development of new treatments for affected foetuses.
Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Talassemia alfa/genética , Animais , Linhagem Celular , Deleção de Genes , Células-Tronco Embrionárias Humanas/citologia , Humanos , Camundongos , Camundongos NusRESUMO
WWTR1 or TAZ (WWTR1/TAZ) is a transcriptional coactivator that acts as a downstream regulatory target in the Hippo signaling pathway, which plays a pivotal role in regulating cell proliferation and anti-apoptosis. It has been shown in other cell types that WWTR1/TAZ plays a redundant role to its homolog YAP1. Using CRISPR/Cas9 gene editing, we established the WWTR1/TAZ-KO cell line, which features homozygous deletion of WWTR1 gene from human iPSCs. The established WWTR1/YAZ-KO cell line maintained the pluripotent phenotype, the ability to differentiate into all three embryonic germ layers, and normal karyotype.
Assuntos
Sistemas CRISPR-Cas/genética , Linhagem Celular/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mutação/genética , Transativadores/genética , Sequência de Bases , Feminino , Humanos , Reprodutibilidade dos Testes , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
CD34+ cells were isolated from mobilized peripheral blood of a healthy donor and reprogrammed by nucleofection with episomal plasmids carrying l-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1, and shRNA against p53. The obtained MUSIi012-A cell line maintained the pluripotent phenotype, the ability to differentiate into all three germ layers, and a normal karyotype.
Assuntos
Diferenciação Celular , Reprogramação Celular , Células-Tronco de Sangue Periférico/citologia , Teratoma/patologia , Células Cultivadas , Feminino , Humanos , Fator 4 Semelhante a Kruppel , PlasmídeosRESUMO
Human induced pluripotent stem cells (hiPSCs) derived from dermal fibroblasts having wild type (WT) SCN5A were engineered by CRISPR/Cas9-mediated genome editing to harbor a specific point mutation (C2204>T) in SCN5A, which results in a substitution of the WT alanine by valine at codon 735 (A735V). The established MUSli009-A-1 hiPSC line has a homozygous C2204>T mutation on exon 14 of SCN5A that was confirmed by DNA sequencing analysis. The cells exhibited normal karyotype, expressed pluripotent markers and retained its capability to differentiate into three germ layers. The cardiomyocytes derived from this line would be a useful model for investigating cardiac channelopathy.
Assuntos
Síndrome de Brugada/genética , Sistemas CRISPR-Cas/genética , Técnicas de Cultura de Células/métodos , Linhagem Celular/patologia , Edição de Genes , Células-Tronco Pluripotentes Induzidas/patologia , Mutação/genética , Sequência de Bases , Humanos , MasculinoRESUMO
BACKGROUND: Due to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells (hESCs) have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy. The first hESC line was generated in 1998 by culturing inner cell mass (ICM) cells isolated from human blastocysts using an immunosurgery technique. Since then, many techniques including mechanical ICM isolation, laser dissection, and whole embryo culture have been used to derive hESC lines. However, the hESC derivation efficiency remains low, usually less than 50%, and it requires a large number of human embryos to derive a significant number of hESC lines. Due to a shortage of and restricted access to human embryos, a novel approach with better hESC derivation efficiency is badly needed to decrease the number of embryos used. METHODS: We hypothesized that the low hESC derivation efficiency might be due to extensive proliferation of trophoblast (TE) cells which could interfere with ICM proliferation. We therefore developed a methodology to minimize TE cell proliferation by culturing ICM in a feeder-free system for 3 days before transferring them onto feeder cells. RESULTS: This minimized trophoblast cell proliferation (MTP) technique could be successfully used to derive hESCs from normal, abnormal, and frozen-thawed embryos with better derivation efficiency of more than 50% (range 50-100%; median 70%). CONCLUSIONS: We successfully developed a better hESC derivation methodology using the "MTP" culture system. This methodology can be effectively used to derive hESCs from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies. With this methodology, large-scale production of clinical-grade hESCs is feasible.