Generation and characterization of human umbilical cord blood-derived induced pluripotent stem cells (KRIBBi005-A).

Induced pluripotent stem cells (iPSCs) hold great promise as a potential source for regenerative medicine. Umbilical cord blood (UCB) cells possess a high level of stem cells, exhibit less immune rejection, and have fewer DNA mutations and, thus, are an easily accessible and valuable cell source for iPSC generation for basic research and clinical applications. Here, we reprogrammed CD34+-UCB cells with the group O/D-negative (RhD-) blood type as a universal source for red blood cell (RBC) generation. The resulting iPSCs, with features typical of pluripotent stem cells, will further advance the development of various therapeutics, including blood transfusion.

Generation of induced pluripotent stem cell line (KRIBBi004-A) from adult bone marrow CD34+ cells from a patient carrying 46,XX,t(1;5)(p31.1;35.1) karyotype.

Induced pluripotent stem cell (iPS) technology may be advantageous for the study of genetic aberrations in terms of recapitulating the full manifestation of pathological features in vitro, identifying underlying pathways, and developing personalized therapeutics rather than procuring somatic cells from patients. Here, we derived an iPSC line from a patient with reciprocal chromosome translocation, t(1;5)(p31.1;35.1), as a novel alternative model to identify clinical phenotypes induced by genetic instability. The resulting iPSC line generated from somatic cells with an existing instability showed representative characteristics of PSCs, and might serve as an unparalleled cellular resource for the development of a custom remedy.

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.

Establishment of iPSC (KRIBBi001-A) from CD34+ group O D-negative bone marrow blood.

Human induced pluripotent stem cells with indefinite propagation in vitro provide a potential donor source of cells including erythroid cells for human therapy. Since group O D-negative (RhD-) blood cells are considered as universal donors for transfusion, it is compelling to derive iPSC line from group O/RhD- sample as a new cellular source to generate universal RBCs. The resulting iPSC line derived from group O/RhD- somatic source showed typical features of pluripotent stem cells and could provide an unprecedented cellular tool to develop universal therapeutics for blood transfusion.

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.

Generation of a human induced pluripotent stem cell line, KSCBi003-A, from human adipose tissue-derived mesenchymal stem cells using a chromosomal integration-free system.

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.

Recent progress of national banking project on homozygous HLA-typed induced pluripotent stem cells in South Korea.

Induced pluripotent stem cells (iPSCs) can be generated by introducing several factors into mature somatic cells. Banking of iPSCs can lead to wider application for treatment and research. In an economical view, it is important to store cells that can cover a high percentage of the population. Therefore, the use of homozygous human leukocyte antigen-iPSCs (HLA-iPSCs) is thought as a potential candidate for effective iPSC banking system for further clinical use. We screened the database stored in the Catholic Hematopoietic Stem Cell Bank of Korea and sorted the most frequent homozygous HLA types of the South Korean population. Blood cells with the selected homozygous HLA types were obtained and transferred to the GMP facility in the Catholic Institute of Cell Therapy. Cells were reprogrammed to iPSCs inside the facility and went through several quality controls. As a result, a total of 13 homozygous GMP-grade iPSC lines were obtained in the facility. The generated iPSCs showed high pluripotency and normal karyotype after reprogramming. Five HLA-homozygous iPSCs had the type that was included in the top five most frequent HLA types. Homozygous HLA-iPSCs can open a new opportunity for further application of iPSCs in clinical research and therapy.

Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a modified RNA system.

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.

Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a non-integration system.

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.

Generation of human induced pluripotent stem cells from urinary cells of a healthy donor using a non-integration system.

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.

Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method.

BACKGROUND: Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. METHODS: Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. RESULTS: Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34+CD43+ hematopoietic progenitor cells (HPCs) and CD34+CD45+ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro. CONCLUSION: In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

The use of pluripotent stem cell for personalized cell therapies against neurological disorders.

Although there are a number of weaknesses for clinical use, pluripotent stem cells are valuable sources for patient-specific cell therapies against various diseases. Backed-up by a huge number of basic researches, neuronal differentiation mechanism is well established and pluripotent stem cell therapies against neurological disorders are getting closer to clinical application. However, there are increasing needs for standardization of the sourcing pluripotent stem cells by establishing stem cell registries and banking. Global harmonization will accelerate practical use of personalized therapies using pluripotent stem cells.

JNK/stress-activated protein kinase associated protein 1 is required for early development of telencephalic commissures in embryonic brains.

We previously reported that mice lacking JSAP1 (jsap1-/-) were lethal and the brain of jsap1-/- at E18.5 exhibited multiple types of developmental defects, which included impaired axon projection of the corpus callosum and anterior commissures. In the current study, we examined whether the early telencephalic commissures were formed abnormally from the beginning of initial development or whether they arose normally, but have been progressively lost their maintenance in the absence of JSAP1. The early corpus callosum in the brain of jsap1+/+ at E15.5-E16.5 was found to cross the midline with forming a distinct U-shaped tract, whereas the early axonal tract in jsap1-/- appeared to cross the midline in a diffuse manner, but the lately arriving axons did not cross the midline. In the brain of jsap1-/- at E17.5, the axon terminals of lately arriving collaterals remained within each hemisphere, forming an early Probst's bundle-like shape. The early anterior commissure in the brain of jsap1+/+ at E14.5-E15.5 crossed the midline, whereas the anterior commissure in jsap1-/- developed, but was deviated from their normal path before approaching the midline. The axon tracts of the corpus callosum and anterior commissure in the brain of jsap1-/- at E16.5-E17.5 expressed phosphorylated forms of FAK and JNK, however, their expression levels in the axonal tracts were reduced compared to the respective controls in jsap1+/+. Considering the known scaffolding function of JSAP1 for the FAK and JNK pathways, these results suggest that JSAP1 is required for the pathfinding of the developing telencephalic commissures in the early brains.

Chemicals that modulate stem cell differentiation.

Important cellular processes such as cell fate are likely to be controlled by an elaborate orchestration of multiple signaling pathways, many of which are still not well understood or known. Because protein kinases, the members of a large family of proteins involved in modulating many known signaling pathways, are likely to play important roles in balancing multiple signals to modulate cell fate, we focused our initial search for chemical reagents that regulate stem cell fate among known inhibitors of protein kinases. We have screened 41 characterized inhibitors of six major protein kinase subfamilies to alter the orchestration of multiple signaling pathways involved in differentiation of stem cells. We found that some of them cause recognizable changes in the differentiation rates of two types of stem cells, rat mesenchymal stem cells (MSCs) and mouse embryonic stem cells (ESCs). Among many, we describe the two most effective derivatives of the same scaffold compound, isoquinolinesulfonamide, on the stem cell differentiation: rat MSCs to chondrocytes and mouse ESCs to dopaminergic neurons.

Morphogenetic lung defects of JSAP1-deficient embryos proceeds via the disruptions of the normal expressions of cytoskeletal and chaperone proteins.

Recent studies have shown that JNK/stress-activated protein kinase-associated protein 1 (JSAP1)-deficient mice die from respiratory failure shortly after birth. To understand the underlying mechanism, we investigated the histological appearances and cell type changes in developing jsap1(-/-) lungs between E12.5 and E18.5. At the light microscopic level, no overt abnormality was detected in jsap1(-/-) until E16.5. However, alveoli and airway formations that normally occur after E16.5 were poorly advanced in jsap1(-/-). Despite these morphological defects, surfactant secreting cells labeled by anti-SP-B or anti-SP-C were present in normal ranges in jsap1(-/-) lungs. Smooth muscle alpha-actin expressing cells were also developed in jsap1(-/-) lungs, although actin expression was decreased. The expressions of transcriptional factors, such as, nuclear factor Ib (Nfib), N-myc, and octamer transcriptional factor 1 (Oct-1), which play a critical role in lung morphogenesis, were found to be down-regulated, whereas signal transducer and activator of transcription 3 (Stat3), sonic hedgehog (Shh), and smoothened (Smo) were up-regulated, in jsap1(-/-) lungs at E17.5-E18.5 compared with those in jsap1(+/+) lungs. Proteomics analysis of E17.5 lung identified 39 proteins with altered expressions, which included actin, tropomyosin, myosin light chain, vimentin, heat shock protein (Hsp27), and Hsp84. These results suggest that JSAP1 is required for the normal expressions of cytoskeletal and chaperone proteins in the developing lung, and that impaired expressions of these proteins might cause morphogenetic defects observed in jsap1(-/-) lungs.

Inhibition of the TPA-induced cutaneous inflammation and hyperplasia by EC-SOD.

This study reports the roles of extracellular superoxide dismutase (EC-SOD) in the cutaneous inflammation and hyperplasia with 12-O-tetradecanoylphorbol-3-acetate (TPA) application in EC-SOD transgenic mice (Tg EC-SOD). Topical double TPA treatment induced the various inflammatory changes including the epidermal thickness, elevated the PCNA-labeling index, the edema formation, and increased production of hydrogen peroxide (H2O2) in wild type mice (WT). These changes were markedly suppressed in TPA-treated Tg EC-SOD. The expressions of the inflammatory cytokines, IL-1alpha and IL-1beta, were reduced in the TPA-treated Tg EC-SOD compared with those in TPA-treated WT. The expression of IL-1alpha was significantly increased in the skin of TPA-treated WT, especially in the basal and suprabasal layers, but it was restricted focally in basal layer of the skin of TPA-treated Tg EC-SOD. The number of infiltrating inflammatory cells and the IL-1beta expressing cells was obviously reduced in TPA-treated Tg EC-SOD in comparison with TPA-treated WT. The result suggests that EC-SOD might play an important role in the suppression of TPA-induced cutaneous inflammation and epidermal hyperplasia by regulating the expression of IL-1alpha and IL-1beta, although the mechanisms remain to be elucidated.

JSAP1 is required for the cell adhesion and spreading of mouse embryonic fibroblasts.

The roles of JSAP1 and JIP1 in cell adhesion and spreading were examined using mouse embryonic fibroblasts (MEFs) deficient in JIP1 (JIP1-KO), JSAP1 (JSAP1-KO), and in both JIP1 and JSAP1 (double-KO), and by using their wild type. After being plated on fibronectin-coated culture plates, wild type MEFs rapidly adhered and differentiated to typical longitudinal fibroblasts in 4 h. JSAP1-KO MEFs showed a similar sequence of adhesion and cell spreading, but their adhesion was weak, and cell spreading sequence proceeded in a delayed manner compared with the wild type. In spreading JSAP1-KO MEFs, adhesion-triggered actin cytoskeleton reorganization and FAK activation proceeded at a slower pace than in wild type MEFs. The cellular properties of double-KO MEFs and JIP1-KO MEFs were similar to those of JSAP1-KO MEFs and wild type MEFs, respectively. These results suggest that JSAP1 plays a role in adhesion and cell spreading by regulating the rapid reorganization of the actin cytoskeleton.

Progressive neuronal loss and behavioral impairments of transgenic C57BL/6 inbred mice expressing the carboxy terminus of amyloid precursor protein.

The beta-secretase cleaved Abeta-bearing carboxy-terminal fragments (betaCTFs) of amyloid precursor protein (APP) in neural cells have been suggested to be cytotoxic. However, the functional significance of betaCTFs in vivo remains elusive. We created a transgenic mouse line Tg-betaCTF99/B6 expressing the human betaCTF99 in the brain of inbred C57BL/6 strain. Tg-betaCTF99/B6 mouse brain at 12-16 months showed severely down-regulated calbindin, phospho-CREB, and Bcl-xL expression and up-regulated phospho-JNK, Bcl-2, and Bax expression. Neuronal cell density in the Tg-betaCTF99/B6 cerebral cortex at 16-18 months was lower than that of the non-transgenic control, but not at 5 months. At 11-14 months, Tg-betaCTF99/B6 mice displayed cognitive impairments and increased anxiety, which were not observed at 5 months. These results suggest that increased betaCTF99 expression is highly detrimental to the aging brain and that it produces a progressive and age-dependent AD-like pathogenesis.

The axon guidance defect of the telencephalic commissures of the JSAP1-deficient brain was partially rescued by the transgenic expression of JIP1.

The JNK interacting protein, JSAP1, has been identified as a scaffold protein for mitogen-activated protein kinase (MAPK) signaling pathways and as a linker protein for the cargo transport along the axons. To investigate the physiological function of JSAP1 in vivo, we generated mice lacking JSAP1. The JSAP1 null mutation produced various developmental deficits in the brain, including an axon guidance defect of the corpus callosum, in which phospho-FAK and phospho-JNK were distributed at reduced levels. The axon guidance defect of the corpus callosum in the jsap1-/- brain was correlated with the misplacement of glial sling cells, which reverted to their normal position after the transgenic expression of JNK interacting protein 1(JIP1). The transgenic JIP1 partially rescued the axon guidance defect of the corpus callosum and the anterior commissure of the jsap1-/- brain. The JSAP1 null mutation impaired the normal distribution of the Ca+2 regulating protein, calretinin, but not the synaptic vesicle marker, SNAP-25, along the axons of the thalamocortical tract. These results suggest that JSAP1 is required for the axon guidance of the telencephalic commissures and the distribution of cellular protein(s) along axons in vivo, and that the signaling network organized commonly by JIP1 and JSAP1 regulates the axon guidance in the developing brain.

Chronic restraint stress massively alters the expression of genes important for lipid metabolism and detoxification in liver.

Repeatedly delivered psychological, chemical or physical stress leads to drastic changes of physiology in various regions of body including liver. The underlying hepatic mechanisms whereby body copes with the stress conditions, however, are poorly understood. In the present study, we performed a genome-wide screen for the altered gene expression in liver of mice that were exposed to chronic restraint stress. cDNA microarray analysis followed by RT-PCR techniques led us to identify >235 genes that were up- or down-regulated by >1.8-fold in their expression levels. Among the wide ranges of genes identified from the screen, the elevated expression of a group of genes important for lipid metabolism and detoxification were particularly notable. The results of this study suggest that similar to the effects of genetically predisposed or externally-delivered environmental chemicals, diets, and drugs, the chronic restraint stress potently alters the gene expression profile in liver.