Evaluation of hormonal and circulating inflammatory biomarker profiles in the year following bariatric surgery.

Background: Bariatric surgery (BS) has a superior effect on reducing body weight and fat in patients with morbid obesity. As a result, BS mitigates obesity-related complications such as type 2 diabetes (T2D). However, few studies have shown the mechanism underlying diabetes remission after surgery. This study aimed to investigate the differences in serum hormone and inflammatory cytokine levels related to diabetes before surgery and during 12 months of follow-up in Korean patients with obesity. Methods: The study participants were patients with morbid obesity (n=63) who underwent sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB) between 2016 - 2017 at seven tertiary hospitals in Korea. The patients were followed for 1 year after surgery. Results: Sixty-three patients had significant weight loss after surgery and showed improvements in clinical parameters and hormonal and inflammatory profiles. Among them, 23 patients who were diabetic preoperatively showed different remission after surgery. The levels of inflammation-related clinical parameters changed significantly in the remission group, and serum inflammatory cytokine and hormones significantly decreased at certain points and showed an overall decreasing trend. Conclusions: Our study found postoperative changes of factors in blood samples, and the changes in hormones secreted from the three major metabolic tissue (pancreas, adipose, and gut) along with the differences in multi-origin inflammatory cytokines between remission and non-remission groups provide a path for understanding how the effect of BS in improving glucose metabolism is mediated.

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.

Modelling Toxoplasma gondii infection in human cerebral organoids.

Pluripotent stem cell-derived cerebral organoids have the potential to recapitulate the pathophysiology of in vivo human brain tissue, constituting a valuable resource for modelling brain disorders, including infectious diseases. Toxoplasma gondii, an intracellular protozoan parasite, infects most warm-blooded animals, including humans, causing toxoplasmosis. In immunodeficient patients and pregnant women, infection often results in severe central nervous system disease and fetal miscarriage. However, understanding the molecular pathophysiology of the disease has been challenging due to limited in vitro model systems. Here, we developed a new in vitro model system of T. gondii infection using human brain organoids. We observed that tachyzoites can infect human cerebral organoids and are transformed to bradyzoites and replicate in parasitophorous vacuoles to form cysts, indicating that the T. gondii asexual life cycle is efficiently simulated in the brain organoids. Transcriptomic analysis of T. gondii-infected organoids revealed the activation of the type I interferon immune response against infection. In addition, in brain organoids, T. gondii exhibited a changed transcriptome related to protozoan invasion and replication. This study shows cerebral organoids as physiologically relevant in vitro model systems useful for advancing the understanding of T. gondii infections and host interactions.

Generation of a human induced pluripotent stem cell line from a patient with Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that is characterized by severe visual impairment in early infancy. We generated a human induced pluripotent stem cell (hiPSC) line, DKHi090-A, from peripheral blood mononuclear cells (PBMCs) of a patient with LCA, by using a Sendai virus-based gene delivery system. We confirmed that DKHi090-A has a nicotinamide mononucleotide adenyltransferase 1 (NMNAT1) mutation and normal karyotype. DKHi090-A line is pluripotent and is capable of multilineage differentiation. This cell line is registered and is available at the National Stem Cell Bank, Korea National Institute of Health.

Generation of human induced pluripotent stem cells from peripheral blood mononuclear cells of a Senior-Loken syndrome patient.

Senior-Loken syndrome (SLS) is a rare disorder primarily associated with kidney and retinal dysfunction. We generated a human induced pluripotency stem cell (hiPSC) line, designated DKHi005-A, from peripheral blood mononuclear cells of a patient with SLS using a Sendai virus reprogramming method. We confirmed that DKHi005-A cells harbor the same mutation as the patient and show a normal karyotype. DKHi005-A also has pluripotency and the capacity for differentiation into the three germ layers. This cell line is registered and available at the National Stem Cell Bank, Korea National Institute of Health.

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.

Genetic analysis of parathyroid and pancreatic tumors in a patient with multiple endocrine neoplasia type 1 using whole-exome sequencing.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) syndrome is an autosomal dominant hereditary disorder characterized by the presence of endocrine tumors affecting the parathyroid, pancreas, and pituitary. A heterozygous germline inactivating mutation in the MEN1 gene (first hit) may be followed by somatic loss of the remaining normal copy or somatic mutations in the MEN1 gene (second hit). Whole-exome sequencing has been successfully used to elucidate the mutations associated with the different types of tumors. CASE PRESENTATION: We performed whole-exome sequencing (WES) on three parathyroid tumors, one pancreatic insulinoma, and a blood sample taken from the same patient with MEN1 to study tumor heterogeneity in MEN1 originating from different tumors. We identified a novel frame-shift deletion (c.1382_1383delAG, p.E461GfsX69) in the MEN1 gene using WES, which was confirmed by Sanger sequencing. WES and the SNP array revealed somatic LOH on chromosome 11 in parathyroid tumors (left upper, left lower, and right upper parathyroid). However, we did not detect a somatic MEN1 gene mutation or LOH in the pancreatic insulinoma. WES revealed two somatic functional variants outside the MEN1 gene in the pancreatic insulinoma. CONCLUSIONS: This study revealed heterogeneity among tumors in the same patient with MEN1, suggesting that different tumor-specific tumorigenic mechanisms may contribute to the pathogenesis of MEN1 tumors. The present study supports the clinical applicability of the WES strategy to research on multiple tumor samples and blood.

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting.

Kelch-like ECH-associated protein 1 (keap1) is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a). Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC). The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential.

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.

Patterns of gene expression associated with Pten deficiency in the developing inner ear.

In inner ear development, phosphatase and tensin homolog (PTEN) is necessary for neuronal maintenance, such as neuronal survival and accurate nerve innervations of hair cells. We previously reported that Pten conditional knockout (cKO) mice exhibited disorganized fasciculus with neuronal apoptosis in spiral ganglion neurons (SGNs). To better understand the genes and signaling networks related to auditory neuron maintenance, we compared the profiles of differentially expressed genes (DEGs) using microarray analysis of the inner ear in E14.5 Pten cKO and wild-type mice. We identified 46 statistically significant transcripts using significance analysis of microarrays, with the false-discovery rate set at 0%. Among the DEGs, expression levels of candidate genes and expression domains were validated by quantitative real-time RT-PCR and in situ hybridization, respectively. Ingenuity pathway analysis using DEGs identified significant signaling networks associated with apoptosis, cellular movement, and axon guidance (i.e., secreted phosphoprotein 1 (Spp1)-mediated cellular movement and regulator of G-protein signaling 4 (Rgs4)-mediated axon guidance). This result was consistent with the phenotypic defects of SGNs in Pten cKO mice (e.g., neuronal apoptosis, abnormal migration, and irregular nerve fiber patterns of SGNs). From this study, we suggest two key regulatory signaling networks mediated by Spp1 and Rgs4, which may play potential roles in neuronal differentiation of developing auditory neurons.

Recessive C10orf2 mutations in a family with infantile-onset spinocerebellar ataxia, sensorimotor polyneuropathy, and myopathy.

Recessive mutations in chromosome 10 open reading frame 2 (C10orf2) are relevant in infantile-onset spinocerebellar ataxia (IOSCA). In this study, we investigated the causative mutation in a Korean family with combined phenotypes of IOSCA, sensorimotor polyneuropathy, and myopathy. We investigated recessive mutations in a Korean family with two individuals affected by IOSCA. Causative mutations were investigated using whole exome sequencing. Electrophysiological analyses and muscle and nerve biopsies were performed, along with magnetic resonance imaging (MRI) of the brain and lower extremities. Compound heterozygous mutations c.1460C>T and c.1485-1G>A in C10orf2 were identified as causative of IOSCA. Skeletal muscle showed mitochondrial DNA (mtDNA) deletions. Both patients showed a period of normal development until 12-15 months, followed by ataxia, athetosis, hearing loss, and intellectual disability. Electrophysiological findings indicated motor and sensory polyneuropathies. Muscle biopsy revealed variations in the size and shape of myofibers with scattered, small, and angulated degenerating myofibers containing abnormal mitochondria; these observations are consistent with myopathy and may be the result of mtDNA deletions. Sural nerve biopsy revealed an axonal neuropathy. High-signal-intensity lesions in the middle cerebellar peduncles were correlated with clinical severity, and MRI of the lower legs was compatible with the hypothesis of length-dependent axonal degeneration. We identified novel compound heterozygous mutations of the C10orf2 gene as the cause of IOSCA with sensorimotor polyneuropathy and myopathy. Signs of motor neuropathy and myopathy were discovered for the first time in IOSCA patients with C10orf2 mutations. These results suggest that the clinical spectrum of IOSCA caused by C10orf2 mutations may be more variable than previously reported.

Identification of CDH23 mutations in Korean families with hearing loss by whole-exome sequencing.

BACKGROUND: Patient genetic heterogeneity renders it difficult to discover disease-cause genes. Whole-exome sequencing is a powerful new strategy that can be used to this end. The purpose of the present study was to identify a hitherto unknown mutation causing autosomal recessive nonsyndromic hearing loss (ARNSHL) in Korean families. METHODS: We performed whole-exome sequencing in 16 individuals from 13 unrelated small families with ARNSHL. After filtering out population-specific polymorphisms, we focused on known deafness genes. Pathogenic effects of the detected mutations on protein structure or function were predicted via in silico analysis. RESULTS: We identified compound heterozygous CDH23 mutations in hearing-loss genes of two families. These include two previously reported pathological mutations, p.Pro240Leu and p.Glu1595Lys, as well as one novel mutation, p.Asn342Ser. The p.Pro240Leu mutation was found in both families. We also identified 26 non-synonymous variants in CDH23 coding exons from 16 hearing-loss patients and 30 Korean exomes. CONCLUSION: The present study is the first to show that CDH23 mutations cause hearing loss in Koreans. Although the precise contribution made by such mutations needs to be determined using a larger patient cohort, our data indicate that mutations in the CDH23 gene are one of the most important causes of non-syndromic hearing loss in East Asians. Further exome sequencing will identify common mutations or polymorphisms and contribute to the molecular diagnosis of, and development of new therapies for, hereditary hearing loss.

Whole-exome sequencing identifies MYO15A mutations as a cause of autosomal recessive nonsyndromic hearing loss in Korean families.

BACKGROUND: The genetic heterogeneity of hearing loss makes genetic diagnosis expensive and time consuming using available methods. Whole-exome sequencing has recently been introduced as an alternative approach to identifying causative mutations in Mendelian disorders. METHODS: To identify the hidden mutations that cause autosomal recessive nonsyndromic hearing loss (ARNSHL), we performed whole-exome sequencing of 13 unrelated Korean small families with ARNSHL who were negative for GJB2 or SLC26A4 mutations. RESULTS: We found two novel compound heterozygous mutations, IVS11 + 1 and p.R2146Q, of MYO15A in one (SR903 family) of the 13 families with ARNSHL. In addition to these causative mutations, 13 nonsynonymous variants, including variants with uncertain pathogenicity (SR285 family), were identified in the coding exons of MYO15A from Korean exomes. CONCLUSION: This is the first report of MYO15A mutations in an East Asian population. We suggest that close attention should be paid to this gene when performing genetic testing of patients with hearing loss in East Asia. The present results also indicate that whole-exome sequencing is a valuable method for comprehensive medical diagnosis of a genetically heterogeneous recessive disease, especially in small-sized families.

Conditional deletion of pten leads to defects in nerve innervation and neuronal survival in inner ear development.

All cellular phenomena and developmental events, including inner ear development, are modulated through harmonized signaling networks. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a major signaling component involved in cross talk with key regulators of development; i.e., Wnt, Notch, and bone morphogenetic proteins. Although Pten function has been studied in various systems, its role in inner ear development is poorly understood. Here, we used inner ear-specific Pten conditional knockout mice and examined the characteristics of the inner ear. In a detailed analysis of the phenotype, reduced cochlear turning and widened epithelia were observed. Phalloidin staining of sensory epithelium revealed that hair cell patterns were disturbed; i.e., additional rows of hair cells were discovered. The neural abnormality revealed a reduction in and disorganization of nerve fibers, including apoptosis at the neural precursor stage. Pten deficiency induced increased phosphorylation of Akt at Ser473. The elevation of inhibitory glycogen synthase kinase 3ß Ser9 phosphorylation (pGSK3ß) was sustained until the neuronal differentiation stage at embryonic day 14.5, instead of pGSK3ß downregulation. This is the first report on the influence of Pten/Akt/GSK3ß signaling on the development of spiral ganglia. These results suggest that Pten is required for the maintenance of neuroblast number, neural precursors, and differentiation in the inner ear.

Identification and functional analysis of cystathionine beta-synthase gene mutations in patients with homocystinuria.

Homocystinuria is an autosomal recessive inborn error of metabolism that is most often caused by mutation in the cystathionine beta-synthase (CBS) gene. Patients may develop serious clinical manifestations such as lens dislocation, mental retardation, osteoporosis, and atherothrombotic vascular disease. Over 100 mutations have been reported, but so far, none have been reported in Korea. Mutation analysis of the CBS gene in six Korean patients with homocystinuria was performed by direct sequencing. Eight mutations were identified, including four known mutations (T257M, R336C, T353M, and G347S) and four novel mutations (L154Q, A155V, del234D, and A288T). All patients were compound heterozygotes. To characterize these mutations, normal or mutated forms of CBS were cloned into pcDNA3.1 expression vector followed by transfection into mammalian cells for transient expression. Whereas the expression levels of mutant proteins were comparable to that of normal control, enzyme activities of all the mutant forms were significantly decreased. In addition, a novel single nucleotide polymorphism, R18C, was identified, which showed one-third to two-thirds the enzyme activity of wild type and 1% of the allele frequency in normal control. The spectrum of mutations observed in Korean patients bears less resemblance to those observed in Western countries.

Reversal of gene expression profile in the phenylketonuria mouse model after adeno-associated virus vector-mediated gene therapy.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by phenylalanine hydroxylase (PAH) deficiency. Accumulation of phenylalanine leads to severe mental and psychomotor retardation, and hypopigmentation of skin and hair. We have demonstrated the cognitive outcome of biochemical and phenotypic reversal by the adeno-associated virus vector-mediated gene delivery of a human PAH transgene. In this study, we identified the expression of genes related to pathologic abnormalities of the PKU-affected brain, in which the symptoms of PKU are mainly manifest, and transcriptional changes in effective gene therapy treatment using oligonucleotide array. Therapeutic effectiveness was verified by change in enzyme activity (15+/-5.84%), phenylalanine plasma level (261+/-108 microM), and coat color. Our data indicated that 12 genes were significantly up-regulated in PKU. Four are involved in defense and inflammatory responses of neutrophils (NE, MPO, NGP, and CRAMP), three other overexpressed genes are related to extracellular matrix organization and degradation (COL1A1, COL1A2, and MMP13); the remainder were a nociceptor in sensory neurons (MrgA1), a structural gene of P lysozyme (Lzp-s), an immunoglobulin alpha heavy chain constant region gene (Igh-2), an osteocalcin-related protein precursor (Bglap-rs1), and a membrane-spanning 4 domain, subfamily A, member 3 (Ms4a3). Data demonstrated that elevated genes in the PKU-affected brain could be normalized by human PAH gene delivery. Although we could not precisely link transcript level changes and neurologic pathogenesis, this study provides a more comprehensive understanding of the PKU-affected brain at the molecular level, possibly resulting in better therapeutic approaches.

Mesomelic dwarfism in pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is associated with mutations in the cartilage oligomeric matrix protein (COMP) gene and the clinical characteristics include short stature, deformities of the extremities involving the epiphyses and metaphyses, early onset arthritis, and ligament laxity. PSACH has been considered a rhizomelic form of dwarfism. So far no previous report has described mesomelic shortening of the limbs in PSACH. We reviewed nine patients with a diagnosis of PSACH based on clinical and radiographic examination and mutation analysis of the COMP gene. The mean height in the adults was 116 cm. All patients showed mesomelic dwarfism. The average ratios of radial length to humeral length and tibial length to femoral length were 0.62 and 0.63, respectively. The tibia and the radius showed more severe bony deformity than the femur and humerus. The degree of short stature was related to the site of the mutation in the COMP gene, but there was no correlation between bony deformity and height or gene mutation.

Identification of cartilage oligomeric matrix protein (COMP) gene mutations in patients with pseudoachondroplasia and multiple epiphyseal dysplasia.

Mutations in the cartilage oligomeric matrix protein (COMP) gene are responsible for two dominantly inherited skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). Mutation analysis of the COMP gene in Korean patients with PSACH and MED was performed. All nine patients with PSACH had mutations in the COMP gene, while three of the five patients with MED had detectable COMP mutations. Eight mutations, including three novel mutations, were identified in the COMP gene in the patients with PSACH and MED. Six mutations were found within the calmodulin-like repeats (CLRs) domain, especially in the seventh CLR and the other two mutations were in exon 16 outside of CLRs, which encode the C-terminal globular domain. Among the three novel mutations, two were missense mutations (Asp473Tyr, Asp482His) and one was a consecutive two-codon deletion, delAspAsp(469-473) in the five consecutive aspartic acid residues. All three novel mutations produced the PSACH phenotype.

In vivo differentiation of mouse embryonic stem cells into hepatocytes.

Embryonic stem (ES) cells have been regarded as a powerful resource for cell replacement therapy. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. Another type of disease that could benefit from the availability of stem cell therapy is liver disease. However, for this potential to be realized, it is necessary to demonstrate the differentiation of ES cells into hepatocytes. To demonstrate the in vivo differentiation potential of mouse ES cells, we injected ES cells into the spleen of immunosuppressed nude mice. Histological analysis of teratomas derived from injected ES cells revealed that some areas contained typical hepatocytes arranged in a sinusoidal structure. The hepatic nature of these cells was further confirmed by showing that transcripts of liver-specific genes were present in the differentiated teratoma using reverse transcriptase-polymerase chain reaction and immunohistochemistry using several liver-specific antibodies including HEP-PAR, phenylalanine hydroxylase, and mouse N-system aminotransferase to identify the respective proteins in the differentiated hepatocytes. This is the first demonstration that mouse ES cells can differentiate in vivo into a mixed population of hepatocytes of varying maturity. This finding extends the potential use of ES cells in the cell replacement therapy by including its possible application for treating liver diseases.