RESUMEN
E-cadherin, a transmembrane protein, is essential for maintaining the integrity and structure of human pluripotent stem cells (hPSCs) by facilitating strong cell-cell adhesion and communication, which is crucial for their colony formation and pluripotency. Here, we used the CRISPR/Cas9 system to introduce the enhanced green fluorescent protein (EGFP)-tagged CDH1 into the AAVS1 locus, a safe harbour site, of human induced pluripotent stem cells (hiPSCs). The engineered cell line, KSCBi002-A-3, expressed functional CDH1-EGFP fusion protein, exhibited normal cell morphology, maintained a normal karyotype, and retained pluripotent state.
RESUMEN
GATA6 is expressed during early embryogenesis and localizes to endoderm- and mesoderm-derived tissues during later embryogenesis. Here, we established a human induced pluripotent stem cell (hiPSC) line expressing EGFP under GATA6 gene. EGFP coding sequence was introduced into the C-terminus of GATA6 in KSCBi017-A hiPSCs through homologous recombination using CRISPR/Cas9 system. The successfully edited line, KSCBi017-A-1, was selected and confirmed by sequencing. The line had a normal karyotype and exhibited potential to differentiate into three germ layers while it expressed EGFP upon endoderm induction. KSCBi017-A-1 cells can be used to monitor the expression of GATA6 during differentiation. This cell line is available from Korea National Stem Cell Bank.
Asunto(s)
Sistemas CRISPR-Cas , Factor de Transcripción GATA6 , Proteínas Fluorescentes Verdes , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Humanos , Factor de Transcripción GATA6/metabolismo , Factor de Transcripción GATA6/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Línea Celular , Diferenciación CelularRESUMEN
Human induced pluripotent stem cells (hiPSCs) have potential use in regerenrative medicine for disease modeling and drug screening studies. The AAVS1 locus has been validated as a stable transgene expression and safe genomic location. Therefore, we inserted the enhanced green fluorescent protein (EGFP) gene into the AAVS1 locus of hiPSCs, using CRISPR/Cas9 genome editing. The results showed that the hiPSCs stably expressed EGFP in pluripotency and differentiated into three germ lineages. Our results strongly indicate that the EGFP-tagged cell line has potential for use in in vivo and in vitro experiments for monitoring cell location and type.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular , Proteínas Fluorescentes Verdes/metabolismoRESUMEN
Cultured human pluripotent stem cells (hPSCs) grow as colonies that require breakdown into small clumps for further propagation. Although cell death mechanism by single-cell dissociation of hPSCs has been well defined, how hPSCs respond to the deadly stimulus and recover the original status remains unclear. Here we show that dissociation of hPSCs immediately activates ERK, which subsequently activates RSK and induces DUSP6, an ERK-specific phosphatase. Although the activation is transient, DUSP6 expression persists days after passaging. DUSP6 depletion using the CRISPR/Cas9 system reveals that DUSP6 suppresses the ERK activity over the long term. Elevated ERK activity by DUSP6 depletion increases both viability of hPSCs after single-cell dissociation and differentiation propensity towards mesoderm and endoderm lineages. These findings provide new insights into how hPSCs respond to dissociation in order to maintain pluripotency.
Asunto(s)
Células Madre Pluripotentes , Transducción de Señal , Humanos , Retroalimentación , Diferenciación Celular , Muerte Celular , Fosfatasa 6 de Especificidad Dual/genética , Fosfatasa 6 de Especificidad Dual/metabolismoRESUMEN
A human induced pluripotent cell (hiPSC) line, KSCBi012-A, was generated from a 40-year-old male individual using non-integrating episomal vectors expressing reprogramming factors. The generated hiPSCs were integration-free, expressed pluripotency markers, exhibited the potential for differentiation into three germ layers in vivo, and maintained the normal karyotype. This cell line can be used as a control for a disease model and is available from Korea National Stem Cell Bank.
Asunto(s)
Células Madre Pluripotentes Inducidas , Adulto , Diferenciación Celular , Línea Celular , Reprogramación Celular , Células Epiteliales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , PlásmidosRESUMEN
Assessment of differentiation potential is a basic requirement to obtain qualified human pluripotent stem cells (hPSCs). Here, we report a simple differentiation method using fetal bovine serum (FBS) to estimate differentiation potential and propensity of hPSCs. PluriTest using RNA-sequencing showed that cells differentiated after treatment with 5% FBS. Expression patterns of three germ layer markers revealed that cells cultured in Knockout Serum Replacement-containing medium (KSR) with mouse feeder cells had higher differentiation potential than cells cultured in a chemically defined medium (E8) with recombinant matrix proteins, especially into the mesoderm and endoderm lineages. Analysis of differentially expressed genes between KSR and E8 identified DUSP6 as a marker for where cells had been cultured. Expression of DUSP6 correlated with FGF-ERK signaling activity. Fine-tuning of FGF-ERK signaling activity to a range that can shut down DUSP6 transcription but sustain NANOG transcription partially increased the differentiation potential. Our data suggest that differentiation with 5% FBS is good to estimate differentiation potential and propensity at the early stage, and that DUSP6 is an excellent marker to monitor ERK signaling activity.
Asunto(s)
Diferenciación Celular , Fosfatasa 6 de Especificidad Dual/análisis , Sistema de Señalización de MAP Quinasas , Células Madre Pluripotentes/metabolismo , Suero , Animales , Biomarcadores/análisis , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/farmacología , Células Nutrientes , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Ratones , Células Madre Pluripotentes/citologíaRESUMEN
We generated a human induced pluripotent stem cell (hiPSC) line, KSCBi003-A, from adipose tissue-derived mesenchymal stem cells (Ad-MSCs) using a Sendai virus-based gene delivery system. We confirmed that the KSCBi003-A has a normal karyotype and short tandem repeat (STR)-based identities that match the parent cells. We also confirmed that the cell line expresses pluripotent stem cell markers such as Nanog, OCT4, SSEA-4, TRA-1-60, and TRA-1-81. We also analyzed that the KSCBi003-A has an ability to differentiate three germ layers (ectoderm, mesoderm, endoderm). This cell line is registered and available at the National Stem Cell Bank, Korea National Institute of Health.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Diferenciación Celular , HumanosRESUMEN
We generated human induced pluripotent stem cells (KSCBi002-B and KSCBi002-B-1) from the dermal fibroblasts of a donor using a modified RNA-based gene delivery method. According to GTG-banding analysis, the generated KSCBi002-B line has a cytogenetic abnormality (46,XY, t(1;4)(q21;q25)) that is distinct from that of the donor, whereas KSCBi002-B-1 has a normal karyotype (46,XY). These cell lines can be useful as a model for characterizing the hiPSCs generated by a non-viral and non-integrative system, or as a chromosomal balanced translocation model. These two cell lines are registered and available from the National Stem Cell Bank, Korea National Institute of Health.
Asunto(s)
Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , ARN/metabolismo , Línea Celular , Humanos , MasculinoRESUMEN
We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts using a Sendai virus (SeV)-based gene delivery method. The generated hiPSC line, KSCBi002-A, has a normal karyotype (46,XY). The pluripotency and differentiation capacity were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from the National Stem Cell Bank, Korea National Institute of Health.
Asunto(s)
Dermis/metabolismo , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Virus Sendai , Transducción Genética , Línea Celular , Dermis/citología , Fibroblastos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , MasculinoRESUMEN
Urinary cells can be an ideal source for generating hiPSCs and progenitors, as they are easily accessible, non-invasive, and universally available. We generated human induced pluripotent stem cells (hiPSCs) from the urinary cells of a healthy donor using a Sendai virus-based gene delivery method. The generated hiPSC line, KSCBi001-A, has a normal karyotype (46,XY). The pluripotency and capacity of multilineage differentiation were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from National Stem Cell Bank, Korea National Institute of Health.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Virus Sendai , Transducción Genética , Orina/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , MasculinoRESUMEN
Anti cancer agent 5-FU (Fluoro Uracil) is a prodrug that can be metabolized and then activated to interfere with RNA and DNA homeostasis. However, the majority of administered 5-FU is known to be catabolized in vivo in the liver where Dihydropyrimidine dehydrogenase (DPD) is abundantly expressed to degrade 5-FU. The biological factors that correlate with the response to 5-FU-based chemotherapy have been proposed to include uridine phosphorylase (UPP), thymidine phosphorylase (TPP), p53 and microsatellite instability. Among these, the expression of UPP is known to be controlled by cytokines such as TNF-alpha, IL1 and IFN-gamma. Our preliminary study using a DNA microarray technique showed that basic fibroblast growth factor (bFGF) markedly induced the expression of UPP1 at the transcription level. In the present study, we investigated whether bFGF could modulate the expression of UPP1 in osteo-lineage cells and examined the sensitivity of these cells to 5-FU mediated apoptosis.