Ahlm Kwon

Tissue-specific Differentiation Potency of Mesenchymal Stromal Cells from Perinatal Tissues

Human perinatal tissue is an abundant source of mesenchymal stromal cells(MSCs) and lacks the ethical concerns. Perinatal MSCs can be obtained from various tissues as like amnion, chorion, and umbilical cord. Still, little is known of the distinct nature of each MSC type. In this study, we successfully isolated and cultured MSCs from amnion(AMSCs), chorion(CMSCs), and umbilical cord(UC-MSCs). Proliferation potential was different among them, that AMSCs revealed the lowest proliferation rate due to increased Annexin V and senescence-associated β-galactosidase positive cells. We demonstrated distinct characteristic gene expression according to the source of the original tissue using microarray. In particular, genes associated with apoptosis and senescence including CDKN2A were up-regulated in AMSCs. In CMSCs, genes associated with heart morphogenesis and blood circulation including HTR2B were up-regulated. Genes associated with neurological system processes including NPY were up-regulated in UC-MSCs. Quantitative RT-PCR confirmed the gene expression data. And in vitro differentiation of MSCs demonstrated that CMSCs and UC-MSCs had a more pronounced ability to differentiate into cardiomyocyte and neural cells, respectively. This study firstly demonstrated the innate tissue-specific differentiation potency of perinatal MSCs which can be helpful in choosing more adequate cell sources for better outcome in a specific disease.

Isolation and Characterization of Chorionic Mesenchymal Stromal Cells from Human Full Term Placenta

This study focused on the characterization of mesenchymal stromal cells (MSCs) from the chorion of human full term placenta from 15 donors. Chorionic MSCs revealed homologous fibroblast-like morphology and expressed CD73, CD29, CD105, and CD90. The hematopoietic stem cell markers including HLA DR, CD11b, CD34, CD79a, and CD45 were not expressed. The growth kinetics of their serial passage was steady at the later passages (passage 10). The multilineage capability of chorionic MSCs was demonstrated by successful adipogenic, osteogenic and chondrogenic differentiation and associated gene expression. Chorionic MSCs expressed genes associated with undifferentiated cells (NANOG, OCT4, REX1) and cardiogenic or neurogenic markers such as SOX2, FGF4, NES, MAP2, and NF. TERT was negative in all the samples. These findings suggest that chorionic MSCs undifferentiated stem cells and less likely to be transformed into cancer cells. A low HLA DR expression suggests that chorionic MSCs may serve as a great source of stem cells for transplantation because of their immune-privileged status and their immunosuppressive effect. Based on these unique properties, it is concluded that chorionic MSCs are pluripotent stem cells that are probably less differentiated than BM-MSCs, and they have considerable potential for use in cell-based therapies.

Genetic and epigenetic alterations of bone marrow stromal cells in myelodysplastic syndrome and acute myeloid leukemia patients

We evaluated the characteristics of bone marrow stromal cells (BMSCs) and hematopoietic cells (HCs) from patients of myelodysplastic syndrome (MDS, n=21) and acute myeloid leukemia (AML, n=58), and compared the results with control BMSCs derived from healthy donors (n=8). The patient BMSCs had lower proliferative activity than that of the controls due to increased senescence. This retarded proliferation induced failure to obtain enough metaphase cells for karyotyping in patient BMSCs (10%). Patient BMSCs were genetically altered which was demonstrated by chromosome abnormalities in 5% of the patients (one MDS and three AML), whereas no clonal abnormalities were detected in the controls. The most common abnormality of the BMSCs was an extra chromosome 5, followed by an extra chromosome 7 and balanced translocations. The proportion of the abnormal metaphase cells was low (17.8%). We also analyzed the epigenetic changes of long interspersed nucleotide element 1 (LINE-1) repetitive element and CDKN2B using pyrosequencing. The quantitative measurement of global LINE-1 methylation demonstrated that patient BMSCs revealed global hypomethylation (68.2±3.8) compared with controls (72.9±3.4, P<0.001) and that the global hypomethylation of BMSCs were more significant in AML than in MDS patients (67.9±3.8, 69.4±4.2, respectively). These findings seem worthy of further evaluation of their association with ineffective hematopoiesis and leukemogenesis.

Impact of genetic abnormalities on the prognoses and clinical parameters of patients with multiple myeloma.

Background We reviewed patients with multiple myeloma (MM) in order to assess the incidence of genetic abnormalities and their associations with clinical parameters, risk groups, and prognosis. Methods A total of 130 patients with MM were enrolled. The incidences of genetic abnormalities were determined in all patients. The relationships of the genetic abnormalities and clinical parameters were investigated. In addition, a survival analysis was performed. Results Abnormal karyotypes were detected in 42.3% (N=55) of the patients, and this was increased to 63.1% (N=82) after including the results determined with interphase FISH. Hypodiploidy was observed in 7.7% (N=10) of the patients, and all were included in the group with complex karyotypes (30.8%, N=40). The 14q32 rearrangements were detected in 29.2% (N=38) of the patients, and these most commonly included t(11;14), which was followed by t(4;14) and t(14;16) (16.2%, 11.5%, and 0.8%, respectively). Abnormal karyotypes and complex karyotypes were associated with disease progression markers, including low hemoglobin levels, low platelet counts, high plasma cell burden, high β2-microglobulin, and high international staging system stages. A high free light chain (FLC) ratio and FLC difference were associated with abnormal karyotypes, complex karyotypes, and higher plasma cell burden. Hypodiploidy and low platelet counts were significant independent prognostic factors and were more important in patient outcome than any single abnormality. Conclusions Genetic abnormalities were associated with disease progression markers and prognosis of MM patients.

Mutational characteristics of ANK1 and SPTB genes in hereditary spherocytosis

The aim of this study was to describe the mutational characteristics in Korean hereditary spherocytosis (HS) patients. Relevant literatures including genetically confirmed cases with well‐documented clinical summaries and relevant information were also reviewed to investigate the mutational gene‐ or domain‐specific laboratory and clinical association. Twenty‐five HS patients carried one heterozygous mutation of ANK1 (n = 13) or SPTB (n = 12) but not in SPTA1, SLC4A1, or EPB42. Deleterious mutations including frameshift, nonsense, and splice site mutations were identified in 91% (21/23), and non‐hotspot mutations were dispersed across multiple exons. Genotype–phenotype correlation was clarified after combined analysis of the cases and the literature review; anemia was most severe in HS patients with mutations on the ANK1 spectrin‐binding domain (p < 0.05), and SPTB mutations in HS patients spared the tetramerization domain in which mutations of hereditary elliptocytosis and pyropoikilocytosis are located. Splenectomy (17/75) was more frequent in ANK1 mutant HS (32%) than in HS with SPTB mutation (10%) (p = 0.028). Aplastic crisis occurred in 32.0% of the patients (8/25; 3 ANK1 and 5 SPTB), and parvovirus B19 was detected in 88%. The study clarifies ANK1 or SPTB mutational characteristics in HS Korean patients. The genetic association of laboratory and clinical aspects suggests comprehensive considerations for genetic‐based management of HS.

Mutational spectrum of Korean patients with corneal dystrophy.

Corneal dystrophy typically refers to a group of rare hereditary disorders with a heterogeneous genetic background. A comprehensive molecular genetic analysis was performed to characterize the genetic spectrum of corneal dystrophies in Korean patients. Patients with various corneal dystrophies underwent thorough ophthalmic examination, histopathologic examination, and Sanger sequencing. A total of 120 probands were included, with a mean age of 50 years (SD = 18 years) and 70% were female. A total of 26 mutations in five genes (14 clearly pathogenic and 12 likely pathogenic) were identified in 49 probands (41%). Epithelial–stromal TGFBI dystrophies, macular corneal dystrophy and Schnyder corneal dystrophy (SCD) showed 100% mutation detection rates, while endothelial corneal dystrophies showed lower detection rates of 3%. Twenty six non‐duplicate mutations including eight novel mutations were identified and mutations associated with SCD were identified genetically for the first time in this population. This study provides a comprehensive characterization of the genetic aberrations in Korean patients and also highlights the diagnostic value of molecular genetic analysis in corneal dystrophies.

Genetic Profiles of Korean Patients With Glucose-6-Phosphate Dehydrogenase Deficiency.

Background We describe the genetic profiles of Korean patients with glucose-6-phosphate dehydrogenase (G6PD) deficiencies and the effects of G6PD mutations on protein stability and enzyme activity on the basis of in silico analysis. Methods In parallel with a genetic analysis, the pathogenicity of G6PD mutations detected in Korean patients was predicted in silico. The simulated effects of G6PD mutations were compared to the WHO classes based on G6PD enzyme activity. Four previously reported mutations and three newly diagnosed patients with missense mutations were estimated. Results One novel mutation (p.Cys385Gly, labeled G6PD Kangnam) and two known mutations [p.Ile220Met (G6PD São Paulo) and p.Glu416Lys (G6PD Tokyo)] were identified in this study. G6PD mutations identified in Koreans were also found in Brazil (G6PD São Paulo), Poland (G6PD Seoul), United States of America (G6PD Riley), Mexico (G6PD Guadalajara), and Japan (G6PD Tokyo). Several mutations occurred at the same nucleotide, but resulted in different amino acid residue changes in different ethnic populations (p.Ile380 variant, G6PD Calvo Mackenna; p.Cys385 variants, Tomah, Madrid, Lynwood; p.Arg387 variant, Beverly Hills; p.Pro396 variant, Bari; and p.Pro396Ala in India). On the basis of the in silico analysis, Class I or II mutations were predicted to be highly deleterious, and the effects of one Class IV mutation were equivocal. Conclusions The genetic profiles of Korean individuals with G6PD mutations indicated that the same mutations may have arisen by independent mutational events, and were not derived from shared ancestral mutations. The in silico analysis provided insight into the role of G6PD mutations in enzyme function and stability.

Genetic–pathologic characterization of myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineages. The current study demonstrates that three driver mutations were detected in 82.6% of 407 MPNs with a mutation distribution of JAK2 in 275 (67.6%), CALR in 55 (13.5%) and MPL in 6 (1.5%). The mutations were mutually exclusive in principle except in one patient with both CALR and MPL mutations. The driver mutation directed the pathologic features of MPNs, including lineage hyperplasia, laboratory findings and clinical presentation. JAK2-mutated MPN showed erythroid, granulocytic and/or megakaryocytic hyperplasia whereas CALR- and MPL-mutated MPNs displayed granulocytic and/or megakaryocytic hyperplasia. The lineage hyperplasia was closely associated with a higher mutant allele burden and peripheral cytosis. These findings corroborated that the lineage hyperplasia consisted of clonal proliferation of each hematopoietic lineage acquiring driver mutations. Our study has also demonstrated that bone marrow (BM) fibrosis was associated with disease progression. Patients with overt fibrosis (grade ⩾2) presented an increased mutant allele burden (P<0.001), an increase in chromosomal abnormalities (P<0.001) and a poor prognosis (P<0.001). Moreover, among patients with overt fibrosis, all patients with wild-type JAK2/CALR/MPL (triple-negative) showed genomic alterations by genome-wide microarray study and revealed the poorest overall survival, followed by JAK2-mutated MPNs. The genetic–pathologic characteristics provided the information for understanding disease pathogenesis and the progression of MPNs. The prognostic significance of the driver mutation and BM fibrosis suggests the necessity of a prospective therapeutic strategy to improve the clinical outcome.

Ubiquitin C decrement plays a pivotal role in replicative senescence of bone marrow mesenchymal stromal cells

Human bone marrow-mesenchymal stromal cells (hBM-MSCs) undergo cellular senescence during in vitro culture. In this study, we defined this replicative senescence as impaired proliferation, deterioration in representative cell characteristics, accumulated DNA damage, and decreased telomere length and telomerase activity with or without genomic abnormalities. The UBC gene expression gradually decreased during passaging along with the reduction in series of molecules including hub genes; CDK1, CCNA2, MCM10, E2F1, BRCA1, HIST1H1A and HIST1H3B. UBC knockdown in hBM-MSCs induced impaired proliferation in dose-dependent manner and showed replicative senescence-like phenomenon. Gene expression changes after UBC knockdown were similar to late passage hBM-MSCs. Additionally, UBC overexpession improved the proliferation activity of hBM-MSCs accompanied by increased expression of the hub genes. Consequently, UBC worked in higher-order through regulation of the hub genes controlling cell cycle and proliferation. These results indicate that the decrement of UBC expression plays a pivotal role in replicative senescence of hBM-MSCs.

Buccal swab as a suitable sample for a microarray-based rapid detection assay using a warfarin genotyping kit.

Genetic testing for common variants in the CYP2C9 and VKORC1 genes may provide useful clinical information to guide dosing for patients receiving oral warfarin. Although methods for sequencing the regions of the CYP2C9 and VKORC1 genes containing the clinically significant SNPs could potentially be used in a clinical setting, most molecular diagnostic laboratories choose alternate methods for routine genotyping applications due to regulatory and quality assurance issues, as well as financial constraints and a need for rapid TATs [1]. The Verigene® Warfarin Metabolism Nucleic Acid Test (Verigene® Warfarin assay, Nanosphere, Inc., Northbrook, IL, USA) is one alternate method, which has been approved by the Food and Drug Administration (FDA) [2]. This microarray-based rapid detection assay employs signal amplification rather than DNA amplification and is suitably designed to genotype CYP2C9*2, CYP2C9*3 and VKORC1 1173CNT [3,4]. Venous blood is a commonly used specimen in the pharmacogenomic era. However, in certain situations, such as after allogeneic hematopoietic stem cell transplantation (HSCT), genotype results from blood samples do not reflect the patients pharmacogenomic status because the leukocytes in the blood originated from the donors hematopoietic cells. Saliva or buccal swab sampling provides a good, replicable sample source [5,6]. In this study, we evaluated the feasibility of a buccal swab specimen as an alternative to peripheral blood for use in amicroarray-based, rapid detection assay, the Verigene® Warfarin assay. Fifteen buccal swab specimens were obtained using a cotton-tipped applicator in a conical