ABSTRACT
OBJECTIVE: To investigate the effect of endogenous and exogenous oestrogen exposure on hearing levels in postmenopausal women. STUDY DESIGN: Retrospective cross-sectional study. SETTING: Population-based survey data collected by the Korean National Health and Nutrition Survey between 1 January 2010 and 31 December 2012. SUBJECTS AND METHODS: Participants comprised 3653 postmenopausal women. Detailed histories for reproductive factors and data on the use of hormone replacement therapy were obtained through health questionnaires and otologic examinations, including pure-tone audiogram and otoscopic findings. Complex-sample linear regression models controlling for confounding factors were generated to determine whether hormone-related factors were associated with hearing loss. RESULTS: Women who experienced a longer duration of oestrogen exposure had better hearing compared to those who do not in multivariate model adjusting for confounding factors with a lower adjusted beta coefficient of hearing threshold (ß = -0.18, 95% confidence interval = -0.3 to -0.07, P = .002). The results also suggested that hormone replacement therapy may be beneficial for attenuating hearing loss (ß = -1.22, 95% confidence interval = -2.19 to -0.25, P = .014), particularly in the high-frequency range from 3 to 6 KHz. CONCLUSION: A longer duration of lifetime oestrogen exposure (LEE) and the use of hormone replacement therapy are likely to attenuate hearing loss. These epidemiologic data provide evidence that oestrogen may be beneficial for attenuating age-related hearing decline.
Subject(s)
Estrogens/physiology , Estrogens/therapeutic use , Hearing Loss/drug therapy , Postmenopause , Aged , Cross-Sectional Studies , Estrogen Replacement Therapy , Female , Humans , Middle Aged , Nutrition Surveys , Republic of Korea , Retrospective Studies , Surveys and QuestionnairesABSTRACT
The hair follicle goes through repetitive cycles including anagen, catagen, and telogen. The interaction of dermal papilla cells (DPCs) and keratinocytes regulates the hair cycle and hair growth. Humanin was discovered in the surviving brain cells of patients with Alzheimer's disease. HNG, a humanin analogue, activates cell growth, proliferation, and cell cycle progression, and it protects cells from apoptosis. This study was performed to investigate the promoting effect and action mechanisms of HNG on hair growth. HNG significantly increased DPC proliferation. HNG significantly increased hair shaft elongation in vibrissa hair follicle organ culture. In vivo experiment showed that HNG prolonged anagen duration and inhibited hair follicle cell apoptosis, indicating that HNG inhibited the transition from the anagen to catagen phase mice. Furthermore, HNG activated extracellular signal-regulated kinase (Erk)1/2, Akt, and signal transducer and activator of transcription (Stat3) within minutes and up-regulated vascular endothelial growth factor (VEGF) levels on DPCs. This means that HNG could induce the anagen phase longer by up-regulating VEGF, which is a Stat3 target gene and one of the anagen maintenance factors. HNG stimulated the anagen phase longer with VEGF up-regulation, and it prevented apoptosis by activating Erk1/2, Akt, and Stat3 signaling.
Subject(s)
Dermis/growth & development , Hair Follicle/growth & development , Hair/growth & development , Intracellular Signaling Peptides and Proteins/pharmacology , Animals , Apoptosis , Cell Cycle , Cell Proliferation , Cells, Cultured , Dermis/drug effects , Dermis/metabolism , Female , Hair/drug effects , Hair/metabolism , Hair Follicle/drug effects , Hair Follicle/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Male , Mice , Mice, Inbred C57BL , Rats , Rats, Wistar , Skin Physiological PhenomenaABSTRACT
Human embryonic stem cell (hESC) culture system has been changing culture conditions from conventional to xeno-free for therapeutic cell applications, and N-glycolylneuraminic acid (Neu5Gc) could be a useful indicator of xenogeneic contaminations in hESCs because human cells can no longer produce it genetically. We set up the humanized culture condition using commercially available humanized materials and two different adaptation methods: sequential or direct. SNUhES4 and H1 hESC lines, previously established in conventional culture conditions, were maintained using the humanized culture condition and were examined for the presence of Neu5Gc. The hESCs showed the same morphology and character as those of the conventional culture condition. Moreover, they were negative for Neu5Gc within two passages without loss of pluripotency. This study suggested that this method can effectively cleanse previously established hESC lines, bringing them one step closer to being clinical-grade hESCs.
ABSTRACT
Chromosomal translocation of 2q37.1 just distal to the NPPC gene coding for C-type natriuretic peptide (CNP) and subsequent overproduction of CNP have been reported to cause a skeletal overgrowth syndrome. Loeys-Dietz syndrome (LDS) is one of marfanoid overgrowth syndromes, of which subtype IV is caused by haploinsufficiency of transforming growth factor beta 2 (TGFB2). We report on a girl with clinical phenotypes of overgrowth syndrome, including long and slim body habitus, macrodactyly of the big toe, scoliosis, ankle valgus deformity, coxa valga, slipped capital femoral epiphysis, and aortic root dilatation. Karyotyping revealed a balanced chromosomal translocation between 1q41 and 2q37.1, and the breakpoints could be mapped by targeted resequencing analysis. On chromosome 2q37.1, the translocation took place 200,365 bp downstream of NPPC, and serum level of the amino terminal of CNP was elevated. The contralateral site of translocation on chromosome 1q41 disrupted TGFB2 gene, presumed to cause its haploinsufficiency. This case supports the concept that NPPC is overexpressed because of the loss of a specific negative regulatory control in the normal chromosomal location, and demonstrates the effectiveness of targeted resequencing in the mapping of breakpoints.
Subject(s)
Loeys-Dietz Syndrome/genetics , Natriuretic Peptide, C-Type/biosynthesis , Translocation, Genetic/genetics , Adolescent , Chromosomes, Human, Pair 1/genetics , Chromosomes, Human, Pair 2/genetics , Female , Gene Expression Regulation , Haploinsufficiency , Humans , Karyotyping , Loeys-Dietz Syndrome/physiopathology , Natriuretic Peptide, C-Type/blood , Natriuretic Peptide, C-Type/genetics , Phenotype , Transforming Growth Factor beta2/geneticsABSTRACT
UNLABELLED: Monochorionic (MC) pregnancy in humans is usually considered to be associated only with monozygotic twinning. However, several reports have revealed that dizygotic (DZ) twins can also share a chorion during pregnancy. A chimera is defined as an organism that contains different cells derived from two or more distinct zygotes. As artificial reproductive techniques develop, it can be predicted that the occurrence of MC DZ twins will increase, and DNA-fingerprinting methods, such as short tandem repeat (STR) analysis, will be essential for their accurate diagnosis. We report the first Korean case of MC DZ twins with blood chimerism, 46,XX/46,XY, as a consequence of in vitro fertilization/embryo transfer. The clinical phenotypes of the twins' genitalia were complete female and male, respectively. Monochorionicity was confirmed by pathological analysis of the placenta after delivery. The dizygosity and confined blood chimerism of the twins were confirmed by STR analysis using their peripheral lymphocytes and skin fibroblasts. The confined blood chimerism of the twins can be considered similar to the status of the hematopoietic system in patients after allogenic bone marrow transplantation. CONCLUSION: When MC twins with discordant sex are expected during pregnancy, it is important to consider the possibility of DZ twins showing normal sexual development, especially in twins who were fertilized using artificial reproductive techniques.
Subject(s)
Chimerism , Chorion/blood supply , Embryo Transfer/adverse effects , Fertilization in Vitro/adverse effects , Twins, Dizygotic/genetics , Adult , Female , Humans , Infant, Newborn , Male , Placenta/blood supply , Pregnancy , Pregnancy, Twin , Ultrasonography, PrenatalABSTRACT
The voltage-gated sodium channel genes and HOXD genes are clustered on chromosome 2q, and duplication of this region is associated with 2 clinical phenotypes: early-onset epilepsy and mesomelic dysplasia Kantaputra type, respectively. We report a case involving 2q24.3-2q32.1 duplication encompassing both the voltage-gated sodium channel and HOXD gene clusters, which were detected by a comparative genomic hybridization array. The associated clinical features were early-infantile-onset epilepsy, hypoplastic left heart syndrome, and global developmental delay. However, no features of mesomelic dysplasia were found. A fluorescent in situ hybridization study showed that the noncontiguous insertion of the duplicated chromosome 2q segment into chromosome 6q was inherited from the father, who has a balanced insertional translocation. The unique genotype-phenotype correlation in the present case suggests that dosage-sensitive effects might apply only to the voltage-gated sodium channel genes.
Subject(s)
Aicardi Syndrome/genetics , Spasms, Infantile/genetics , Trisomy/genetics , Aicardi Syndrome/physiopathology , Brain/physiopathology , Chromosomes, Human, Pair 2/genetics , Comparative Genomic Hybridization , Developmental Disabilities/genetics , Developmental Disabilities/physiopathology , Electroencephalography , Fathers , Humans , Hypoplastic Left Heart Syndrome/genetics , Hypoplastic Left Heart Syndrome/physiopathology , In Situ Hybridization, Fluorescence , Infant , Male , Phenotype , Spasms, Infantile/physiopathology , Translocation, Genetic , Trisomy/physiopathologyABSTRACT
Langer-Giedion syndrome (LGS; MIM 150230), also called trichorhinophalangeal syndrome type II (TRPS2), is a contiguous gene syndrome caused by a one-copy deletion in the chromosome 8q23-q24 region, spanning the genes TRPS1 and EXT1. We identified an LGS family with two affected and two unaffected siblings from unaffected parents. To investigate the etiology of recurrence of LGS in this family, array CGH was performed on all family members. We identified a 7.29 Mb interstitial deletion at chromosome region 8q23-q24 in the two affected siblings, but no such deletion in the unaffected family members. However, the mother and one of the two unaffected siblings carried a 1.29 Mb deletion at chromosome region 8q24.1, sharing the distal breakpoint with the larger deleted segment found in the affected siblings. Another unaffected sibling had a 6.0 Mb duplication, sharing the proximal breakpoint of the deletion in the affected siblings. Karyotypic and FISH analyses in the unaffected mother revealed an insertional translocation of 8q23-q24 genomic material into chromosome 13: 46,XX,ins(13;8)(q33;q23q24). This insertional translocation in the mother results in the recurrence of LGS in this family, highlighting the importance of submicroscopic rearrangements in the genetic counseling for LGS.
Subject(s)
Langer-Giedion Syndrome/genetics , Mutagenesis, Insertional , Abnormal Karyotype , Adolescent , Base Sequence , Chromosome Breakage , Chromosomes, Human, Pair 8/genetics , Comparative Genomic Hybridization , DNA Mutational Analysis , Female , Genetic Association Studies , Humans , Langer-Giedion Syndrome/diagnostic imaging , Male , Polymorphism, Single Nucleotide , Radiography , Sequence Deletion , Young AdultABSTRACT
Monozygotic twins, developed from a single zygote, are almost identical in clinical phenotype and concordant karyotypes. Monozygotic twins with discordant karyotypes are thought to be quite rare. Here, we report monochorionic-diamniotic twins discordant for Down syndrome. On findings of prenatal ultrasonography, nuchal translucency thickness was different between twins, and suggested that one of the twins was at high risk for having chromosomal abnormalities including Down syndrome. The twins were monochorionic-diamniotic; therefore, chorionic villi sampling of the common placenta was performed. The karyotype of the chorionic villi cells was 46,XX, and pregnancy was maintained. After delivery, dysmorphic clinical features suggesting Down syndrome were found in one of the twins, while the other twin showed a morphologically normal appearance. Karyotypes of peripheral blood leukocytes were repeatedly normal in the dysmorphic twin; however, the karyotype of skin fibroblasts from the dysmorphic twin indicated Down syndrome mosaicism; 47,XX,+21[99]/46,XX[2]. The karyotype of skin fibroblasts from the morphologically normal twin was 46,XX. Monozygosity of the twins was confirmed by a short tandem repeat analysis using 16 polymorphic markers. A mitotic nondisjunction followed by the twinning would explain the discordant karyotypes between monozygotic twins.
Subject(s)
Diseases in Twins/genetics , Down Syndrome/genetics , Mosaicism , Twins, Monozygotic/genetics , Chromosome Aberrations , Diseases in Twins/diagnosis , Down Syndrome/diagnosis , Female , Humans , Infant , Infant, Newborn , Karyotyping , Pregnancy , Prenatal Diagnosis , Ultrasonography, PrenatalABSTRACT
OBJECTIVE: The aim of the present study was to examine the relationship among male age, strict morphology, and sperm chromatin structure and condensation. METHODS: Sperm samples from a total of 100 men underwent semen analysis, and sperm chromatin structure and condensation were assessed with toluidine blue (TB) and aniline blue (AB) tests. RESULTS: Prevalence of strict morphology of less than 4%, and abnormal sperm chromatin structure and condensation did not show any statistically significant differences according to male age (p=0.605, p=0.235, and p=0.080). No significant correlation was demonstrated among age of male partners, strict morphology, and abnormal sperm chromatin structure using TB and AB tests. However, abnormal sperm chromatin condensation was positively associated with sperm chromatin structure (r=0.594, p=0.000) and showed negative correlation with strict morphology (r=-0.219, p=0.029). CONCLUSION: The tests for sperm chromatin condensation showed a significant association with strict morphology. Further study is needed to elucidate the relationship between clinical outcome and sperm chromatin tests.
ABSTRACT
Hypochondroplasia (HCH) is an autosomal dominant inherited skeletal dysplasia, usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3). A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for preimplantation genetic diagnosis (PGD). We confirmed the mutation in the proband (FGFR3:c.1620C>A, p.N540K), and established a nested allele-specific PCR and sequence analysis for PGD using single lymphocyte cells. We performed this molecular genetic analysis to detect the presence of mutation among 20 blastomeres from 18 different embryos, and selected 9 embryos with the wild-type sequence (FGFR3:c.1620C). A successful pregnancy was achieved through a frozen-thawed cycle and resulted in the full-term birth of a normal neonate. To the best of our knowledge, this is the first report of a successful pregnancy and birth using single-cell allele-specific PCR and sequencing for PGD in an HCH patient.
ABSTRACT
We report a case of isodicentric chromosome 15 (idic(15) chromosome), the presence of which resulted in uncontrolled seizures, including epileptic spasms, tonic seizures, and global developmental delay. A 10-month-old female infant was referred to our pediatric neurology clinic because of uncontrolled seizures and global developmental delay. She had generalized tonic-clonic seizures since 7 months of age. At referral, she could not control her head and presented with generalized hypotonia. Her brain magnetic resonance imaging scans and metabolic evaluation results were normal. Routine karyotyping indicated the presence of a supernumerary marker chromosome of unknown origin (47, XX +mar). An array-comparative genomic hybridization (CGH) analysis revealed amplification from 15q11.1 to 15q13.1. Subsequent fluorescence in situ hybridization analysis confirmed a idic(15) chromosome. Array-CGH analysis has the advantage in determining the unknown origin of a supernumerary marker chromosome, and could be a useful method for the genetic diagnosis of epilepsy syndromes associated with various chromosomal aberrations.
ABSTRACT
Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.
ABSTRACT
Although previous studies showed that cardiomyocytes (CMs) can be generated from human embryonic stem cells (hESCs), the protocols for cryopreservation of hESC-derived CMs is not available to date. Here, we report on the efficient generation of hESC-derived CMs by direct differentiation using BMP2 in a serum-free condition, along with successful cryopreservation of derived CMs using Rho-associated kinase (ROCK) inhibitor. To induce differentiation, hESCs were treated with activin A and BMP2 for 5 days. A mesodermal gene, Brachyury, was expressed from day 3, and cardiac-specific markers such as Nkx2.5 and cTnI were detected at day 14. Furthermore, these cardiac progenitors expressed ion channel-related transcripts such as HCN1 and HCN2 from day 10. Beating clusters were observed from 14 days of differentiation for up to 35 days. Using mass cryopreservation, we froze hESC-derived CMs at 2 stages, at day 12 and 16 (prebeating and postbeating), after treating with ROCK inhibitor, Y-27632. Postthaw survival of CMs was higher in day 12 group compared to day 16, and some cell clusters from day 12 group recovered their contraction. From transmission electron microscope (TEM) analysis, less ultrastructural alterations were observed in day 12 group. Our results provide an insight into the use of BMP2 for cardiac lineage differentiation in a serum-free condition and a possibility of long-term storage of hESC-derived CMs.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Cryopreservation/methods , Embryonic Stem Cells , Myocytes, Cardiac , Amides/pharmacology , Cell Differentiation/drug effects , Cell Line , Enzyme Inhibitors/pharmacology , Humans , Immunohistochemistry , Pyridines/pharmacologyABSTRACT
Human embryonic stem cells (hESCs) have capacities to self-renew and differentiate into all cell types in vitro. Red ginseng (RG) is known to have a wide range of pharmacological effects in vivo; however, the reports on its effects on hESCs are few. In this paper, we tried to demonstrate the effects of RG on the proliferation and differentiation of hESCs. Undifferentiated hESCs, embryoid bodies (EBs), and hESC-derived cardiac progenitors (CPs) were treated with RG extract at 0.125, 0.25, and 0.5 mg/mL. After treatment of undifferentiated hESCs from day 2 to day 6 of culture, BrdU labeling showed that RG treatment increased the proliferation of hESCs, and the expression of Oct4 and Nanog was increased in RG-treated group. To find out the effects of RG on early differentiation stage cells, EBs were treated with RG extract for 10 days and attached for further differentiation. Immunostaining for three germ layer markers showed that RG treatment increased the expressions of Brachyury and HNF3ß on EBs. Also, RG treatment increased the expression of Brachyury in early-stage and of Nkx2.5 in late-stage hESC-derived CPs. These results demonstrate facilitating effects of RG extract on the proliferation and early differentiation of hESC.
ABSTRACT
Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.
Subject(s)
Cell Differentiation/drug effects , Cell Lineage/drug effects , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Endoderm/cytology , Mesoderm/cytology , Reactive Oxygen Species/pharmacology , Biomarkers/metabolism , Cell Line , Cells, Cultured , Down-Regulation/drug effects , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Embryonic Stem Cells/enzymology , Endoderm/drug effects , Enzyme Activation/drug effects , Free Radical Scavengers/pharmacology , Humans , Mesoderm/drug effects , Mitogen-Activated Protein Kinases/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Reactive Oxygen Species/metabolism , Up-Regulation/drug effectsABSTRACT
Application of human embryonic stem cells (hESCs) to stem-cell therapy is not feasible because of the risk of tumorigenicity and rejection. In contrast, human mesenchymal stem cells (hMSCs) are free from the risk of tumorigenicity and also have immune privilege. However, hMSCs obtained from adults have infinite variety in terms of the biological characteristics and functionality. We report here a new derivation method of hMSCs from hESCs. The derivation of hMSCs from three different hESC lines (SNUhES3, CHA3-hESC, and H9) was performed by embryoid bodies formation and subsequent culture with stage-different media without using inductive xenogenic feeder and mechanical selection procedure. The derived cells were morphologically similar to the unique fingerprint-like pattern of hMSCs and grew stably for at least 35 passages in vitro. These cells had hMSCs-like immunophenotypes: negative for CD34 and CD45; positive for CD29, CD44, CD73, CD90, and CD105. They could be differentiated into multiple lineages including osteocytes, chondrocytes, adipocytes, and myocytes. They maintained normal karyotype during the long-term cultivation and did not show tumorigenicity when transplanted into the immunodeficient mice. In conclusion, the new embryoid body-based derivation method of hMSCs from hESCs is simple, safe, and reproducible in three different hESC lines. We expect that this method will provide a more effective and powerful tool to derive hMSCs from various hESC lines.
Subject(s)
Cell Culture Techniques/methods , Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Mesenchymal Stem Cells/cytology , Adult , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Cell Shape , Electrocardiography , Embryonic Stem Cells/metabolism , Feasibility Studies , Flow Cytometry , Humans , Karyotyping , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Mice , Myocardium/pathologyABSTRACT
Human embryonic stem cells (hESCs) are pluripotent and hold great promise as useful tools in basic scientific research and in the field of regenerative medicine. However, several studies have recently reported chromosomal abnormalities such as gains of chromosomes 12, 17 and X in hESCs. This genetic instability presents an obstacle in the application of hESCs as sources of cell therapies. We found that trisomy 12 was correlated with changes in hESC colony morphology during hESC maintenance. In this study, we investigated whether normal and trisomy 12 cells could be separated in hESC cultures displaying trisomy 12 mosaicism with two types of colony morphology using a mechanical transfer technique. Eight sublines were cultured from eight hESC colonies displaying normal or abnormal morphology. Four sublines with normal morphology had normal chromosome 12 numbers, whereas the four sublines with abnormal morphology displayed trisomy 12. These results indicate that a hESC colony with a minor degree of chromosomal mosaicism and normal morphology could proceed to a colony with normal chromosomes after prolonged cultures with mechanical transfer. Therefore, analysis of cultures for chromosomal abnormalities when changes in colony morphology are observed during culture is essential for maintaining normal hESC lines.
Subject(s)
Cell Separation/methods , Cell- and Tissue-Based Therapy/methods , Chromosomes, Human, Pair 12/genetics , Embryonic Stem Cells/cytology , Mosaicism , Trisomy , Alkaline Phosphatase/metabolism , Cell Line , Humans , In Situ Hybridization, Fluorescence , KaryotypingABSTRACT
PURPOSE: Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs. MATERIALS AND METHODS: hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed. RESULTS: Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and a-myosin heavy chain (aMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and a-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS. CONCLUSION: We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Cell Differentiation , Embryonic Stem Cells/drug effects , Myocytes, Cardiac/cytology , Cell Culture Techniques , Cell Line , Cell Proliferation , Embryonic Stem Cells/cytology , Humans , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Signal TransductionABSTRACT
The roles of Notch signaling in cardiac differentiation from murine embryonic stem cells have been well documented. We investigated whether Notch signaling plays a similar role in human embryonic stem cells (hESCs). Although, as previously reported, blocking Notch signaling via the addition of gamma-secretase inhibitor (GSI) alone failed to affect hESC differentiation, we found that GSI plus reduced-volume culture medium (GSI/RVCM) accelerated mesodermal differentiation. GSI/RVCM conditions simultaneously suppressed commitment toward neuroectodermal lineages. Furthermore, sustained inhibition of Notch signaling further enhanced differentiation into cardiac mesoderm. Spontaneous beating activity was typically observed from 12 days after initiation of GSI treatment in RVCM. Moreover, hESC-derived cardiomyocytes expressed connexin 43 and possessed spontaneous calcium oscillations and cardiomyocyte beats coupled to neonatal rat cardiomyocytes when cocultured. These findings strongly suggest a distinct role for Notch signaling in the induction and specification of hESC-derived cardiac mesoderm in vitro. Disclosure of potential conflicts of interest is found at the end of this article.
Subject(s)
Embryonic Stem Cells/cytology , Mesoderm/cytology , Myocardium/cytology , Myocytes, Cardiac/metabolism , Oligopeptides/pharmacology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Animals , Animals, Newborn , Cell Differentiation , Cells, Cultured , Coculture Techniques , Humans , Mesoderm/metabolism , Rats , Rats, Sprague-Dawley , Signal TransductionABSTRACT
Human embryonic stem cells (hESCs) are considered to be able to stably maintain their characteristics in vitro for prolonged periods, but we had previously encountered changes in proliferative ability and differentiation potential during extended culture of hESCs. Therefore, we investigated the proliferative ability and differentiation potential of hESCs during long-term culture. The hESCs, SNUhES3, were used to analyze population-doubling time, proliferation rate and differentiation potential. We classified hESCs into three groups according to culture period. Ten colonies of hESCs for each group were daily measured colony area and population-doubling time was assessed by the changes of colony area. Proliferation rate of hESCs was measured by 5-bromo-2'-deoxyuridine (BrdU) assay and telomerase activity. To evaluate differentiation potentials for hESCs, expression levels of undifferentiated and/or differentiated hESCs markers were examined by FACS, RT-PCR and immunostaining. Population-doubling time of early passage hESCs was longer than those of middle or late passage. Proliferative ability of hESCs was accelerated depending on culture periods. Cellular morphologies and the expression level of each three germ layer markers were obviously different from each passage of reattached embryoid bodies (EBs) after spontaneous differentiation. Differentiated cells of late passage expressed higher levels of undifferentiated markers such as Oct4 and SSEA4 than those of early and middle passage. But differentiated cells of early and middle passage expressed higher level of differentiated state markers, Nestin (ectoderm), Brachyury (mesoderm), HNF3beta (endoderm). From these results, it can be inferred that hESCs show higher proliferative abilities and reduced differentiation potentials as the passage number increased. Therefore, we conclude that early passage hESCs could be more suitable than middle and late passage hESCs in differentiation studies.