Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells.

BACKGROUND: Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. METHODS AND RESULTS: To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2 weeks. CONCLUSION: This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

Assessment of Potential In vitro Genotoxic and Cytotoxic Effects of Bupropion Hydrochloride (Wellbutrin) in Human Peripheral Lymphocytes and Human Cortical Neuron.

INTRODUCTION: Wellbutrin (bupropion hydrochloride; WB), an anti-depressant of the aminoketone class is new highly selective norepinephrine and dopamine reuptake inhibitor; it is effective in the treatment of patients with major depression. MATERIALS AND METHODS: To investigate the in vitro effects of WB in human cultured peripheral blood lymphocytes and human cortical neural (HCN2) cell lines, micronucleus, sister chromatid exchange analysis, cellular viability, and comet assays were employed. The present study is to our knowledge, the first report on WB genotoxicity in cultured human peripheral blood lymphocytes and its cytotoxicity in the HCN2 cell line. We have also investigated the genotoxic potential of WB to induce chromosomal aberrations. RESULTS: WB-induced cytotoxicity (measured as reduction of the nuclear division index) possibly prevented the division of damaged cells. CONCLUSION: We conclude that although, WB exerts potential genotoxic effects in cultured lymphocytes, its cytogenetic effects are very unlikely to occur in blood cultures of WB-administered subjects.

Complex rearrangement in acute myeloid leukemia M2 with RUNX1/RUNX1T1 fusion involving chromosomes 8, 17 and 21.

BACKGROUND: The translocation t(8;21)(q22;q22) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML) sub type M2. About 3-5 % of cases with additional chromosomal abnormalities, including structural and numerical ones, are reported to include a complex translocation t(8;21;N). CASE PRESENTATION: Here we report a chromosome rearrangement observed in a 19 years-old female diagnosed with AML-M2. When subjected to (molecular) cytogenetic analyses a complex three-way translocation involving chromosomes 8, 17 and 21 was detected, forming not a t(8;21;17) as one would expect. Real time-polymerase chain reaction analysis using 6 AML specific markers showed the presence of RUNX1/RUNX1T1 fusion gene transcripts identical to those found in classical translocation t(8;21) coupled with presence of FLT3-ITD mutation identified by fragment analysis. CONCLUSIONS: The present case highlights importance of complex rearrangements rarely encountered in AML, suggesting that all involved regions harbor critical candidate genes regulating the pathogenesis of AML, leading to novel as well as well-known leukemia associated chromosomal aberrations.

Clinical Manifestations of Chromosomal Anomalies and Polymorphic Variations in Patients Suffering from Reproductive Failure.

BACKGROUND: Human reproduction is the most intricate event as ~ 20% of human pregnancies end in miscarriages for which chromosomal anomalies are a common factor. The chromosomal variations associated with reproductive failures include translocations, inversions, supernumerary marker chromosomes, heterochromatic polymorphisms, etc., Till date, the significance of heteromorphic variants in reproductive failures is unclear. AIM: The aim of this study is to investigate the role of chromosomal anomalies and polymorphic variations in reproductive failure. MATERIALS AND METHODS: Chromosomal analysis using GTG banding was performed on 638 couples (1276 individuals). RESULTS: In the present study, 138 of 1276 individuals showed chromosomal variations with respect to heterochromatic variants and Robertsonian translocations. The most common variants observed across the population studied were the pericentric inversion of the chromosome 9 [inv(9)(p11q13), 3.68%] followed by pstk + on the short arm of chromosome 15 (15pstk+, 1.95%) and Robertsonian translocation of chromosomes 13 and 14 [rob(13;14)(q10;q10), 1.25%]. The maximum percentage of heterochromatic variation was observed in females with recurrent pregnancy loss (Groups A, 4.78%) and males with wives having recurrent miscarriages (Group B, 3.68%) and the minimum was recorded in patients with in vitro fertilization (IVF) failures (Group C, 0.23%) and couples having a history of the malformed child (Group F, 0.23%). CONCLUSIONS: High level of chromosomal polymorphic variations in patients with reproductive failures warrants their in-depth analysis to nail down the causative factors. Hence, cytogenetic analysis coupled with genetic counseling becomes indispensable for patients suffering from infertility, reproductive failures and pregnancy losses before IVF treatment to rule out the carrier status.

A rare case of mosaic 45,X/47,XX,+13 in 28-year-old women with secondary amenorrhoea: A case report and literature review.

In the present paper we report an extremely rare case of mosaicism of 45,X/47,XX,+13 in a 28-year-old women. The patient was referred for cytogenetic evaluation for secondary amenorrhoea. The patient was found to have some mild characteristic features of Turner syndrome such as wide carrying angle and short stature. Ultrasound examination revealed the presence of a small sized uterus and bilateral streak ovaries. G-banded chromosome analysis revealed a mosaic female karyotype involving two different cell lines. One cell line (72% of analysed metaphases) presented monosomy of X while the remaining 28% of cells showed trisomy of chromosome 13. Fluorescence in situ hybridization (FISH) with locus specific probe for trisomy 13 and CEP X for monosomy X substantiated the results obtained from karyotyping.

Genotoxic assessment in peripheral blood lymphocytes of post-polio individuals using sister chromatid exchange analysis and micronucleus assay.

Environmental pollution is a complex issue because of the diversity of anthropogenic agents, both chemical and physical, that have been detected and catalogued. The consequences to biota from exposure to genotoxic agents present an additional problem because of the potential for these agents to produce adverse change at the cellular and organism levels. Past studies in virus have focused on structural damage to the DNA of environmental species that may occur after exposure to genotoxic agents and the use of this information to document exposure and to monitor remediation. In an effort to predict effects at the population, community and ecosystem levels, in the present study, we attempt to characterize damage occurring through genotoxic agents like 5-bromo-2-deoxyuridine, BrdU, using sister chromatid exchange technique and the formation of micronuclei (MN) in the peripheral lymphocytes of the post-polio syndrome sequelae affected by poliovirus. Analysis of structural chromosomal aberrations (CAs) and involvement of the specific chromosome break were pursued in this study. They revealed a significantly higher incidence of CAs (chromatid and chromosome breaks) in patients compared with controls, where the specific chromosome break has emerged as specific. Also, the maximum numbers of breaks were found to be in chromosome 1 at the position 1p36.1. The results also suggest a correlation between CAs and content of MN.