G. Ambartsumyan

Induced Pluripotent Stem Cells and Embryonic Stem Cells Are Distinguished by Gene Expression Signatures

Induced pluripotent stem cells (iPSCs) outwardly appear to be indistinguishable from embryonic stem cells (ESCs). A study of gene expression profiles of mouse and human ESCs and iPSCs suggests that, while iPSCs are quite similar to their embryonic counterparts, a recurrent gene expression signature appears in iPSCs regardless of their origin or the method by which they were generated. Upon extended culture, hiPSCs adopt a gene expression profile more similar to hESCs; however, they still retain a gene expression signature unique from hESCs that extends to miRNA expression. Genome-wide data suggested that the iPSC signature gene expression differences are due to differential promoter binding by the reprogramming factors. High-resolution array profiling demonstrated that there is no common specific subkaryotypic alteration that is required for reprogramming and that reprogramming does not lead to genomic instability. Together, these data suggest that iPSCs should be considered a unique subtype of pluripotent cell.

Aneuploidy and early human embryo development

Human embryo development occurs through a process that encompasses reprogramming, sequential cleavage divisions and mitotic chromosome segregation and embryonic genome activation. Chromosomal abnormalities may arise during germ cell and/or pre-implantation embryo development, and are a major cause of spontaneous miscarriage or birth defects. Nonetheless, model systems suitable for the study of human germ cell and embryo development have been limited until recently. We suggest that human embryonic stem cells may provide a valuable human cell-based model for genetic studies of human pre-implantation pluripotent cells. Here, we review the current literature on diagnosing chromosomal abnormalities in the pre-implantation embryo, and the importance of provisions from the human oocyte in establishing and maintaining the human embryonic genome during the first 3 days post-conception. We focus on transcriptional analysis of human oocytes and embryos and the unique cell cycle and checkpoint requirements in the early embryo. Taken together, data suggest that the unique programs of the early human embryo, including management of aneuploid cells, may paradoxically promote embryo development but contribute to the high rate of spontaneous miscarriages in human pregnancies.

The effect of timing of embryonic progression on chromosomal abnormality

OBJECTIVE To evaluate the relationship between aneuploidy and timing of blastocyst formation. DESIGN Historical cohort study. SETTING Private IVF clinic. PATIENT(S) Ninety-four couples undergoing IVF treatment in combination with chromosomal screening of embryos. The mean maternal age was 39.2 years and average number of embryos per patient 5.3. INTERVENTION(S) A total of 530 embryos were biopsied on day 3 and underwent chromosome screening with microarray-based comparative genomic hybridization. MAIN OUTCOME MEASURE(S) Effect of day of embryo blastulation and morphologic grade on aneuploidy rate. RESULT(S) Day 5 morulas that progressed to blastocysts on day 6 were significantly less likely to be aneuploid (79.8%) than day 5 morulas that did not progress to blastocysts (92.9%). However, there was no significant difference in aneuploidy rates when embryos that became blastocysts on day 5 were directly compared with embryos that became blastocysts on day 6. CONCLUSION(S) Delayed blastulation is not associated with increased aneuploidy rates, but absence of blastulation is associated with increased aneuploidy. Therefore, we conclude that when choosing a morula for transfer on day 5, there may be a benefit in waiting an extra day for the possibility of blastulation to occur.

Relative expression of genes encoding SMAD signal transduction factors in human granulosa cells is correlated with oocyte quality

PurposeTo determine the expression of SMAD transcripts in human granulosa cells.MethodsLuteinized mural granulosa cells were harvested from forty women undergoing oocyte retrieval, and RNAs were isolated. SMAD expression levels were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (q-RTPCR).ResultsSMAD1-7 and 9 are expressed in human granulosa cells, with SMAD2, 3 and 4 showing the highest expression levels. Peak estradiol (E2) levels correlated with the number of oocytes retrieved during IVF. Oocyte number showed no correlation with SMAD expression levels or ratios. Fertilization rates also did not correlate with the expression levels of individual SMADs, but did correlate with higher SMAD4:SMAD3 ratios (p = 0.0062) and trended with SMAD4:SMAD2 (p = 0.0698).ConclusionsSMAD transcripts are differently expressed in human granulosa cells, where they may mediate TGF-beta superfamily signaling during folliculogenesis and ovulation. Further, the relative expression ratios of SMAD2, 3 and 4 may differentially affect fertilization rate.

Centromere protein A dynamics in human pluripotent stem cell self-renewal, differentiation and DNA damage

Human pluripotent stem cells (hPSCs) hold significant promise for use in regenerative medicine, or as a model to understand human embryo development. However, the basic mechanisms required for proliferation and self-renewal of hPSCs have not been fully uncovered. Proliferation in all eukaryotes is dependent upon highly regulated expression of the histone H3 variant Centromere protein A (CENP-A). In the current study, we demonstrate that hPSCs have a unique messenger ribonucleic acid (mRNA) reserve of CENP-A not found in somatic fibroblasts. Using short hairpin RNA technology to reduce but not ablate CENP-A, we show that CENP-A-depleted hPSCs are still capable of maintaining a functional centromeric mark, whereas fibroblasts are not. However, upon induction of differentiation or DNA damage, hPSCs with depleted CENP-A arrest in G2/M and undergo apoptosis. Analysis of CENP-A dynamics following DNA damage in hPSCs reveals that 60 min after irradiation, CENP-A is found in multiple small nuclear foci that are mutually exclusive to γH2AX as well as CENP-C. Furthermore, following irradiation, hPSCs with depleted CENP-A mount a normal apoptotic response at 6 h; however at 24 h, apoptosis is significantly increased in CENP-A-depleted hPSCs relative to control. Taken together, our results indicate that hPSCs exhibit a unique mechanism for maintaining genomic integrity by possessing the flexibility to reduce the amount of CENP-A required to maintain a functional centromere under self-renewing conditions, and maintaining a reserve of CENP-A mRNA to rebuild the centromere following differentiation or DNA damage.