Simple differentiation method using FBS identifies DUSP6 as a marker for fine-tuning of FGF-ERK signaling activity in human pluripotent stem cells.

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.

Generation of a CAG-EGFP tagged cell line (KSCBi017-A-2) from human induced pluripotent stem cells using CRISPR/Cas9.

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.

Generation and characterization of GATA6-specific EGFP expressing human induced pluripotent stem cell line, KSCBi017-A-1, using CRISPR/Cas9.

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.

DUSP6 is a memory retention feedback regulator of ERK signaling for cellular resilience of human pluripotent stem cells in response to dissociation.

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.

Generation of integration-free induced pluripotent stem cell line (KSCBi012-A) from urinary epithelial cells of a healthy male individual.

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.

Generation of integration-free induced pluripotent stem cell line (KSCBi017-A) from peripheral blood mononuclear cells of a healthy male individual.

A human induced pluripotent stem cell (hiPSC) line, KSCBi017-A, was generated from a 50-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.

Korea National Stem Cell Bank.

The Korea National Stem Cell Bank has been banking pluripotent stem cell (PSC) lines since 2012. Quality-controlled and ethically sourced cell lines were developed for distribution. Currently (as of 2020), among the 69 deposited lines, 4 research-grade human embryonic stem cell (hESC) lines and 19 induced pluripotent stem cell (iPSC) lines have been distributed. Good manufacturing practices (GMP)-compliant homozygous iPSC lines for regenerative medicine with homozygous HLA haplotypes that cover 51% of the Korean population have been deposited as well. To ensure the quality of the cell lines, we performed eighteen different quality tests on the identity, sterility, consistency, stability and safety of the cell lines. Regarding genetic stability, we are collecting SNPchip, WES, Methyl-seq, and RNA-seq data, which are open to the public.

Generation of induced pluripotent stem cells (KSCBi009-A) from a patient with Prader-Willi syndrome (PWS) featuring deletion of the paternal chromosome region 15q11.2-q13.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by the loss of paternally expressed genes in an imprinted region of chromosome 15q11.2-q13. We generated a human-induced pluripotent stem cell line, designated KSCBi009-A, from peripheral blood mononuclear cells of a 13-year-old male PWS patient exhibiting deletion of the paternal chromosome 15q11.2-q13 region. The deletion was confirmed via methylation-specific multiplex ligation probe amplification assay (MS-MLPA) of genomic DNA. The hiPSC line expressed pluripotency markers and differentiated into three germ layers. The cell line may serve as a valuable model of an imprinting PWS disorder useful in terms of drug discovery and development.

Functional in vivo and in vitro effects of 20q11.21 genetic aberrations on hPSC differentiation.

Human pluripotent stem cells (hPSCs) have promising therapeutic applications due to their infinite capacity for self-renewal and pluripotency. Genomic stability is imperative for the clinical use of hPSCs; however, copy number variation (CNV), especially recurrent CNV at 20q11.21, may contribute genomic instability of hPSCs. Furthermore, the effects of CNVs in hPSCs at the whole-transcriptome scale are poorly understood. This study aimed to examine the functional in vivo and in vitro effects of frequently detected CNVs at 20q11.21 during early-stage differentiation of hPSCs. Comprehensive transcriptome profiling of abnormal hPSCs revealed that the differential gene expression patterns had a negative effect on differentiation potential. Transcriptional heterogeneity identified by single-cell RNA sequencing (scRNA-seq) of embryoid bodies from two different isogenic lines of hPSCs revealed alterations in differentiated cell distributions compared with that of normal cells. RNA-seq analysis of 22 teratomas identified several differentially expressed lineage-specific markers in hPSCs with CNVs, consistent with the histological results of the altered ecto/meso/endodermal ratio due to CNVs. Our results suggest that CNV amplification contributes to cell proliferation, apoptosis, and cell fate specification. This work shows the functional consequences of recurrent genetic abnormalities and thereby provides evidence to support the development of cell-based applications.

Differential expression of stromal cell-derived factor 1 in human brain microvascular endothelial cells and pericytes involves histone modifications.

Stromal cell-derived factor 1 (SDF-1) regulates neovascularization, which is coordinately controlled by endothelial cells (EC) and their surrounding cells, pericytes or smooth muscle cells. In the basal state, SDF-1 expression is much lower in EC than in their surrounding cells. In this study, we evaluated epigenetic regulation to determine if it is involved in the mechanism responsible for the differential expression of SDF-1 in two types of vascular cells, brain microvascular EC (HBMEC) and pericytes (HBMP). We found that HBMEC did not express SDF-1, but that HBMP did. Furthermore, treatment of EC with 5-aza-2'-dexoycytidine and trichostatin A resulted in a remarkable restoration of SDF-1 expression. Additionally, bisulfite-sequencing analysis revealed no differences in the methylation state of SDF-1 promoter between HBMEC and HBMP. Finally, a chromatin immunoprecipitation assay revealed reduced levels of histone H3 lysine 9 (H3K9) acetylation and H3K4 trimethylation with concomitant enhancement of H3K9 trimethylation in HBMEC relative to HBMP, which suggests that histone modifications are involved in the cell-specific expression of SDF-1.

Generation of a NESTIN-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-1, using CRISPR/Cas9 nuclease.

NESTIN, an intermediate filament, is a neuroectodermal marker involved in induced pluripotent stem cell (iPSC) differentiation toward neural lineages. Here, we introduced an EGFP reporter into the C-terminus of NESTIN in KSCBi005-A hiPSCs through homologous recombination using CRISPR/Cas9 nuclease. The successfully edited line was confirmed by sequencing and had a normal karyotype. It expressed EGFP upon induction of neural differentiation and exhibited potential for differentiation into three germ layers. KSCBi005-A-1 cells could be used to monitor the expression of NESTIN in differentiated cell types. This cell line is available at the National Stem Cell Bank, Korea National Institute of Health.

Generation of patient-specific induced pluripotent stem cells (KSCBi007-A) derived from a patient with Prader-Willi syndrome retain maternal uniparental disomy (UPD).

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by loss of paternally expressed genes in an imprinted region of 15q11.2-q13. We established a human-induced pluripotent stem cell (hiPSC) line, KSCBi007-A, from the peripheral blood mononuclear cells of a 5-month-old girl with PWS that retained maternal uniparental disomy (UPD). Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of genomic DNA revealed the maternal UPD in the hiPSCs. The generated hiPSC line expressed pluripotency markers and showed the ability to differentiate into three germ layers in vitro. This hiPSC line could be used as a cellular model of an imprinting disorder in humans.

Generation of a PDX1-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-3, using the CRISPR/Cas9 system.

PDX1 plays a crucial role in the development and maintenance of ß-cells and directly regulates pancreatic ß-cell-specific transcription factors by binding to the insulin gene. Here, we introduced an EGFP reporter into the C-terminus of PDX1 in KSCBi005-A human induced pluripotent stem cells through homologous recombination using CRISPR/Cas9 nuclease. The cells had a normal karyotype, expressed several pluripotency markers, and maintained their differentiation potential. KSCBi005-A-3 cells can be used to monitor PDX1 expression in live cells during ß-cell differentiation; the cell line has been registered at the National Stem Cell Bank, Korea National Institute of Health.

Dexamethasone coordinately regulates angiopoietin-1 and VEGF: a mechanism of glucocorticoid-induced stabilization of blood-brain barrier.

Glucocorticoids stabilize the blood-brain barrier (BBB), leading to attenuation of vasogenic brain edema. However, the action mechanism of glucocorticoids has been poorly elucidated. To elucidate the mechanism, we investigated whether dexamethasone (Dex), a synthetic glucocorticoid hormone, regulates the levels of key permeability regulating factors such as angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor (VEGF) in the three types of cells comprising BBB. Dex increased the level of angiopoietin-1 mRNA and protein and decreased VEGF mRNA and protein in brain astrocytes and pericytes, but not in endothelial cells. The mRNA and protein of angiopoietin-2 were detected only in endothelial cells and not regulated by Dex. The Dex-induced regulation of angiopoietin-1 and VEGF was inhibited by RU486, suggestive of glucocorticoid receptor mediation. The mRNA stability of angiopoietin-1 and VEGF was not changed by Dex treatment, implying that Dex increases angiopoietin-1 and decreases VEGF through transcriptional regulation. This is the first study showing the coordinate regulation of angiopoietin-1 and VEGF by glucocorticoids, suggesting a novel mechanism underlying glucocorticoids-induced stabilization of BBB.

A Standard Nomenclature for Referencing and Authentication of Pluripotent Stem Cells.

Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines.

Desumoylation of homeodomain-interacting protein kinase 2 (HIPK2) through the cytoplasmic-nuclear shuttling of the SUMO-specific protease SENP1.

The modification of homeodomain-interacting protein kinase 2 (HIPK2) by small ubiquitin-like modifier 1 (SUMO-1) plays an important role in its targeting into the promyelocytic leukemia body, as well as in its differential interaction with binding partner, but the desumoylation of HIPK2 by SUMO-specific proteases is largely unknown. In this study, we show that HIPK2 is a desumoylation target for the SUMO-specific protease SENP1 that shuttles between the cytoplasm and the nucleus. Mutation analyses reveal that SENP1 contains the nuclear export sequence (NES) within the extreme carboxyl-terminal region, and SENP1 is exported to the cytoplasm in a NES-dependent manner. Sumoylated HIPK2 are deconjugated by SENP1 both in vitro and in cultured cells, and the desumoylation is enhanced either by the forced translocation of SENP1 into the nucleus or by the SENP1 NES mutant. Concomitantly, desumoylation induces dissociation of HIPK2 from nuclear bodies. These results demonstrate that HIPK2 is a target for SENP1 desumoylation, and suggest that the desumoylation of HIPK2 may be regulated by the cytoplasmic-nuclear shuttling of SENP1.

Registration of human embryonic stem cell lines: Korea, 2010.

In an effort to increase the credibility of human embryonic stem cell (hESC) lines established in Korea, obligatory registration was introduced by the Bioethics and Safety Act 2008, effective as of January 1, 2010. The DNA fingerprint, chromosome stability, expression of pluripotency markers, and contamination of mycoplasma of the submitted lines were analyzed by Korea Centers for Disease Control and Prevention (KCDC). The characterization data and ethical aspects, such as informed consent for donation of surplus embryos, were reviewed by a 10-member advisory review committee for stem cell registry. A total of 55 domestic hESC lines were submitted for registration in 2010; among them 51 were registered. Among these submitted lines, 26 were additionally characterized by KCDC, while 25 lines previously characterized by the Ministry of Education, Science and Technology were not additionally analyzed by KCDC. Registration completed an oversight system for embryo research by registering the products of licensed embryo research projects, making embryo research more transparent in Korea. Information about hESC lines is available at the website of the Korea Stem Cell Registry (kscr.nih.go.kr).

Coordinated regulation of angiopoietin-1 and vascular endothelial growth factor by arsenite in human brain microvascular pericytes: implications of arsenite-induced vascular dysfunction.

Arsenite is an environmental toxicant that is associated with vascular disease; however, the underlying mechanism of its toxicity has yet to be elucidated. Vascular stability appears to be tightly regulated by several vasoactive proteins produced by two adjacent vascular cells, endothelial cells (EC) and pericytes. The disruption of vascular stability may be involved in arsenite toxicity. The roles of angipoietins (Ang) and vascular endothelial growth factor (VEGF) in this process have been evaluated, but these studies have mostly been limited to EC. In this study, we used human brain microvascular pericytes (HBMP) to evaluate the effects of arsenite on Ang-1 and VEGF regulation. Ang-2 was reported to be not detected in HBMP. Arsenite decreased Ang-1 secretion in a time and dose-dependent manner, while it increased VEGF secretion. Although arsenite did not alter Ang-1 mRNA expression, it increased intracellular Ang-1 protein levels in a dose-dependent manner, suggesting a role for arsenite in the intracellular trapping of Ang-1. Contrary to Ang-1, the expression of VEGF mRNA was dose-dependently up-regulated by arsenite. Treatment with N-actyl-l:-cysteine (NAC) alone decreased the release of Ang-1, but failed to attenuate the arsenite-induced decrease in Ang-1 secretion, while NAC completely blocked the arsenite-stimulated VEGF secretion. These results indicate that reactive oxygen species are involved in the regulation of VEGF, but not of Ang-1, secretion in response to arsenite treatment in pericytes. Furthermore, immunocytochemical analysis using confocal microscopy revealed a colocalization of Ang-1 with actin filaments that occurred independently of tubulin. In conclusion, arsenite decreases Ang-1 secretion and increases VEGF secretion, which may offer new insight into understanding the arsenite toxicity associated with vascular instability and subsequent development of vascular disease.

Isolation of a ventricle-specific promoter for the zebrafish ventricular myosin heavy chain (vmhc) gene and its regulation by GATA factors during embryonic heart development.

We investigated chamber-specific gene expression by isolating a 2.2-kb polymerase chain reaction product containing the 5'-flanking region of the zebrafish ventricular myosin heavy-chain gene (vmhc). Promoter analysis revealed that the fragment, consisting of nucleotides from -301 to +26, is sufficient for vmhc promoter activity. Among several putative cis-acting elements in the region, a GATA-binding site was identified to be crucial for the activity of the promoter, as evidenced by the complete abolishment of promoter activity by a single nucleotide substitution of GATA-binding site (-287, C-->T). Knockdown of GATA-binding proteins 4 and 6 (GATA4 and -6) by their antisense morpholino oligonucleotides resulted in reduced green fluorescent protein (GFP) reporter gene and endogenous vmhc expression. These findings suggest that GATA4 and -6 play a key role in the regulation of vmhc expression in the ventricle. In addition, the vmhc promoter and the transgenic zebrafish (vmhc:gfp) are useful tools to study the formation and function of the ventricle. Developmental Dynamics 238:1574-1581, 2009. (c) 2009 Wiley-Liss, Inc.

Phosphorylation by the DHIPK2 protein kinase modulates the corepressor activity of Groucho.

Groucho function is essential for Drosophila development, acting as a corepressor for specific transcription factors that are downstream targets of various signaling pathways. Here we provide evidence that Groucho is phosphorylated by the DHIPK2 protein kinase. Phosphorylation modulates Groucho corepressor activity by attenuating its protein-protein interaction with a DNA-bound transcription factor. During eye development, DHIPK2 modifies Groucho activity, and eye phenotypes generated by overexpression of Groucho differ depending on its phosphorylation state. Moreover, analysis of nuclear extracts fractionated by column chromatography further shows that phospho-Groucho associates poorly with the corepressor complex, whereas the unphosphorylated form binds tightly. We propose that Groucho phosphorylation by DHIPK2 and its subsequent dissociation from the corepressor complex play a key role in relieving the transcriptional repression of target genes regulated by Groucho, thereby controlling cell fate determination during development.