Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells

Human embryonic stem cells (hESCs) need feeder cells for their maintenance in an undifferentiated state. In conventional culture systems, mouse embryonic fibroblasts (MEFs) serve as feeder cells to maintain hESCs. However, the use of MEFs elevates the risk of transmitting mouse pathogens and thus limits the potential of hESCs in cell replacement therapy. Consequently, the use of human feeder cells would be an important step forward in this in vitro technology. To address this issue, we used fibroblast-like cells differentiated from the Miz-hES6 hESC line (Diff (Miz-hES6)) as feeder cells to support the in vitro growth of three hESC lines. Immunofluorescence microscopy and reverse transcription-PCR assessing the expression of undifferentiated hESC markers revealed all three hESC lines were maintained in an undifferentiated state. In vitro proliferation proceeded as efficiently as when the hESCs were cultured on MEFS. Moreover, karyotype analysis revealed the chromosomal normality of the hESC lines and the Diff (Miz-hES6) feeders themselves after even 50 passages. Furthermore, the hESC lines maintained their pluripotency since they remained capable of forming embryoid bodies (EBs) in vitro. Thus, hESC-derived fibroblast-like cells successfully support in vitro hESC propagation.

X-chromosome Inactivation Patterns in Korean Women with Idiopathic Recurrent Spontaneous Abortion

Recurrent spontaneous abortion (RSA) defines as two or more consecutive losses at or =90%) and 4 (8.9%) had mild skewed inactivation (> or =85%). In 54 heterozygous control subjects, 5 (9.3%) women showed extreme skewed X inactivation and 7 (13.0%) had mild one. The frequency of skewed X inactivation between RSA patients and control group was not significantly different (p>0.05). This finding suggests that skewed x romosome be not associated with unexplained RSA patients.

De novo Chromosomal Aberrations in the Fetus; Genetic Counseling and Clinical Outcome

The aim of this study was to examine the incidence and clinical outcome of de novo chromosomal aberrations retrospectively and provide useful data for genetic counseling in the prenatal cytogenetic diagnosis. We found 17 cases of de novo chromosomal aberrations in 5,501 cases of prenatal cytogenetic analysis and reviewed the karyotype, further study, medical records, fetal ultrasound findings and clinical outcomes. Out of the 17 de novo chromosomal aberrations, 5 had balanced reciprocal translocations and 12 had unbalanced translocations characterized as deletion, addition, or marker. In the case of the five balanced reciprocal translocations, 3 cases without abnormal ultrasound findings were carried to term after comprehensive genetic counseling. Neonates were phenotypically normal and clinical examinations were normal. Two cases with abnormal ultrasound findings were terminated therapeutically. Twelve cases of unbalanced translocations were terminated except one case with a mosaic marker chromosome. High resolution fetal ultrasound and detailed cytogenetic and molecular study will be adjunctive tools for predicting the karyotype/phenotype correlations of fetuses with de novo chromosomal aberrations, although they have limitation to find all phenotypic effects.

Amniotic Fluid Interphase Fluorescence in situ Hybridization (FISH) for Detection of Aneuploidy;Experiences in 130 Prenatal Cases

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, 21, and sex chromosomes X and Y. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. We retrospectively reviewed 130 amniotic fluid interphase FISH analyses from January 1997 to December 2001. The review was done in order to assess the role of interphase FISH among the patients who were at the risk of fetal aneuploidies. The sample was considered to be aneuploid when 70% of or more than the total number of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. All of 130 cases but one met the criteria. The results were considered as informative and they were obtained in 24-48 hr. The overall detection rate for aneuploidies was 100% (2 cases of trisomy 21, 2 cases of trisomy 18, and 1 case of Turner syndrome). In comparison to cytogenetics, the rates of both sensitivity and specificity were 100%. The experiment demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies. The experiment can also serve as an adjunctive test to help cytogenetics to reduce significant amount of emotional stress of patients and physicians through early decision making process.

Parental Decisions of Prenatally Detected Sex Chromosome Abnormality

Because of the widespread use of amniocentesis, the prenatal recognition of sex chromosome abnormality (SCA) has become increasingly common. Recent literature provided an insight into the understanding of the natural history and prognosis for individuals with SCA. Our study was designed to review the parental decision on pregnancy with SCA. Over the last 10 yr, we diagnosed 38 cases (0.50%) with SCA out of 7,498 prenatal cases. We reviewed the records and the results of the pregnancies. We included the cases (n=25) of apparently normal anatomic fetus to analyze the factors influencing parental decision. We excluded 13 cases with obvious anomaly or presumably bad outcome. Fifteen (60%) couples continued their pregnancies and ten (40%) terminated theirs. Nine couples (64%) out of fourteen mosaicism cases continued their pregnancies. All five pregnancies assisted by reproductive technique continued their pregnancies. More pregnancies were continued when counseling was done by an MD geneticist rather than by an obstetrician. A significant trend was observed with a higher rate of pregnancy continuation in recent years. The genetic counseling is important to give appropriate information to the parents. Establishing guidelines and protocols will help both obstetricians and parents to make a decision.

Frequencies of Fetal Chromosomal Abnormalities at Prenatal Diagnosis: 10 years experiences in a single institution

We present frequencies of fetal chromosomal abnormalities in 4,907 prenatal cytogenetic examinations at Samsung Cheil Hospital from 1988 to 1997 for 10 yr duration. Prenatal karyotypes were undertaken in 3,913 amniotic fluid samples, 800 chorionic villi samples, and 194 percutaneous umbilical blood samples. The frequency of fetal abnormal karyotypes was 3.1% (150 cases). Numerical chromosome abnormalities were 87 cases (1.8%) and structural aberrations of chromosomes were 63 cases (1.3%). In the numerical chromosomal abnormalities, the frequency of trisomy 21 was by far the highest (36 cases), followed by trisomy 18 in 22 cases and sex chromosome aneuploidies in 19 cases. In the structural chromosomal aberrations, 5 cases had the inversions in chromosome 2, 7, 17, and Y. Chromosomal deletions in 6 cases and additions in 4 cases were analysed. Of the remaining 47 translocation in abnormal fetuses, reciprocal translocation was in 26 cases and Robertsonian translocation in 21 cases. Among them, 41 cases were balanced translocation and 6 were unbalanced. Thirty five cases of translocation were inherited from one of the parents. Four had de novo chromosome rearrangements, and 8 cases were unknown.

Detection of cryptic Y chromosome mosaicism by coamplification PCR with archived cytogenetic slides of suspected Turner syndrome

Turner syndrome is one of the most common cytogenetic abnormalities. It is known that the Y chromosome or Y derived material is present in 6-9% of TS patient and it may develop a high risk of gonadoblastoma in 15-25%. So it is crucial to carry out cyto genetic analysis and Y-specific probe studies for all persons with gonadal dysgenesis to rule out mosaicism with Y-bearing cell line; eg 45,X/46,XY. In this study, 26 archival slides previously analyzed cytogenetically as 45,X, 45,X/46,X,i(X), 45,X/46,X,r(X), and 45,X/46,XX were examined. Coamplification PCR, having the advantage of providing rapid result and confirming PCR failure, was performed with the slide samples in the regions of dystrophin gene in Xp21and DYZ3 in the Y centromeric region. All of archived slides were positive for X-specific gene and one slide of 45,X was found to have the cryptic Y chromosome material. Our result suggests that the archived cytogenetic slides could be applied for the detection of Y chromosome rapidly and efficiently in TS patients.

Retroactive DNA analysis for sex determination and dystrophin gene by polymerase chain reaction with archived cytogenetic slides

We describe a rapid and efficient diagnostic method for sex determination and the dystrophin gene by the polymerase chain reaction (PCR) using archived cytogenetic slides. Archived cytogenetic slides stored for about 4 years at room temperature were used. To confirm whether DNA analysis is possible using the archived cytogenetic slides, we extracted the DNA from the slides and amplified the Y centromeric region (DYZ3), the X centromeric region (DXZ1) and the exon 46 of the dystrophin gene. Of the 50 cases, 24 were peripheral bloods, 13 were amniotic fluid cells, 5 were chorionic villus samplings and 8 were cord bloods. The PCR related sex determination in 22 females and 28 males, showed 100% concordance with the results of chromosome analysis, and all cases showed positive band for the exon 46 of the dystrophin gene. Of the 50 cases of the archived cytogenetic slides, we were fortunate enough to obtain the fresh blood sample from one fetus whose karyotype showed 45,X[34]/46,X,+mar[145] to compare the results of the gDNA with that from archived cytogenetic slide. To confirm whether the marker chromosome was derived from Y chromosome, we studied the six loci (PABY, SRY, RPS4Y (SY16, 17), ZFY, DYS14) on the short arm, one locus (DYZ3) on the centromere and one locus (DYZ1) on the long arm. Of the 8 loci studies, all PCR related Y chromosome showed positive band from both gDNA obtained from cord blood and archived cytogenetic slides. We could conclude from the above results that the marker chromosome was derived from the Y chromosome. We believe our experiment is rapid and efficient for studies of over 10 independent loci from a single slide which has been kept in storage for up to 4 years and that archival Giemsa-stained cytogenetic slide repositories represent valuable DNA resources for clinical and forensic studies.

The Therapeutic Effect of Sertraline in Treatment-resistant Schizophrenics / 대한정신약물학회지

OBJECT: This study was undertaken to know whether there is any therapeutic effecets of sertraline in treatment-resistant schizophrenics. METHOD: Seventy seven treatment-resistant schizophrenic patients, to whom the same antipsychotics as usual were administered, were randomly assigned to two groups; to the experimental group, sertraline was and to the controlled group, placebo was administered adjuntively for 6weeks in single blind design. We assessed psychopathology by BPRS, PANSS, YBOCS, HRSD, and SCL-90-R. RESULTS: BPRS, positive scale of PANSS, and HRSD were significantly decreased in experimental group and statistically siginificant differences between the experimental group and placebo group. Negative scale of PANSS and YBOCS were significantly decresed in experimental group but no statistically significant differences between the experimental group and placebo group. The therapeutic effect of sertraline was occurred within 3rd weeks. CONCLUSION: We suggested that sertraline may be useful in the treatment of schizophrenic symtom in treatment-resistent schizophrenia, especially in positive and depressive symptoms.

Comparison of Early and Mid-Second-Trimester Amniocentesis / 대한산부인과학회잡지

We sought to determine whether early amniocentesis is a safe and acceptable method of genetic evaluation in early pregnancy. During the 20-month period from February 1994 to September 1995, 80 consecutive early amniocentesis were performed transabdominally at 12(+3)-14(+6) weeks of gestation and 305 consecutive mid-second-trimester transabdominal amniocenteses were performed at 16(+0)-18(+0) weeks of gestation. All amniotic fluid samples were cultured using flask method. There were no significant differences between the early and mid-second-trimester amniocenteses in failed sampling, ambiguous results, pregnancy loss within 4 weeks after the procedure, pregnancy loss from 4 weeks after procedure to 28 weeks of gestation, preterm birth, and perinatal death. We may conclude that early amniocentesis is a safe and acceptable method for prenatal diagnosis.