Gamete failure-derived infertility affects millions of people worldwide; for many patients, gamete donation by unrelated donors is the only available treatment. Embryonic stem cells (ESCs) can differentiate in vitro into germ-like cells, but they are genetically unrelated to the patient. Using an in vitro protocol that aims at recapitulating development, we have achieved, for the first time, complete differentiation of human induced pluripotent stem cells (hiPSCs) to postmeiotic cells. Unlike previous reports using human ESCs, postmeiotic cells arose without the over-expression of germline related transcription factors. Moreover, we consistently obtained haploid cells from hiPSCs of different origin (keratinocytes and cord blood), produced with a different number of transcription factors, and of both genetic sexes, suggesting the independence of our approach from the epigenetic memory of the reprogrammed somatic cells. Our work brings us closer to the production of personalized human gametes in vitro.

Diagnosis of homogeneous KS based on lymphocyte karyotyping should be contrasted in other tissues. Mucosa cells could help to better approximate the degree of germ cell mosaicism. Our results indicate that 47,XXY germ cells are not meiotically competent. Increased post-reductional aneuploidy rate is related to meiotic errors in 46,XY cells. Appropriate genetic counselling is recommended in KS.

SUMMARYIt has been described that aneuploidies trigger cell cycle checkpoints leading to apoptosis. The aim of this study was to assess the relationship between the presence of chromosomal abnormalities and apoptosis in germ cells and in Sertoli cells. Fourteen diagnostic testicular biopsies from infertile patients were processed following a sequential methodology, which included enzymatic disaggregation, apoptotic staining, cell sorting, cell fixation, and fluorescent in situ hybridization analysis. The chromosome constitution of germ cells (interphase pre-meiotic germ cells, meiotic figures, post-reductional germ cells, and spermatozoa) and Sertoli cells was evaluated in non-sorted and flow-sorted cell populations (apoptotic and viable). The mean percentage of aneuploidy was compared between the three fractions in each cell type using a Kruskal-Wallis test. If significant results were obtained, a two-by-two Chisquared test was performed. There were significant differences between the apoptotic fraction and the viable and non-sorted fractions in the pre-meiotic germ cells (p < 0.01). In the remaining cell types, no association between the presence of aneuploidy and apoptotic processes was observed, even in the case of post-reductional germ cells in which we detected the highest rates of aneuploidy regardless of the fraction analyzed. From our data, it can be inferred that most of the aneuploid post-reductional germ cells are efficiently removed from the testicular epithelium without differentiating into spermatozoa. Our results suggest that the elimination of aneuploid testicular epithelial cells is triggered by different mechanisms. Accordingly, the cellular elimination of aneuploid germ cells beyond the blood-testis barrier does not involve phosphatidylserine externalization.

One of the most severe complications after successful cancer therapy is the development of therapy-related myeloid neoplasms (t-MN). Constitutional genetic variation is likely to impact on t-MN risk. We aimed to evaluate if polymorphisms in the p53 pathway can be useful for predicting t-MN susceptibility. First, an association study revealed that the Pro variant of the TP53 Arg72Pro polymorphism and the G allele of the MDM2 SNP309 were associated with t-MN risk. The Arg variant of TP53 is more efficient at inducing apoptosis, whereas the Pro variant is a more potent inductor of cell cycle arrest and DNA repair. As regards MDM2 SNP309, the G allele is associated with attenuation of the p53 apoptotic response. Second, to evaluate the biological effect of the TP53 polymorphism, we established Jurkat isogenic cell lines expressing p53Arg or p53Pro. Jurkat p53Arg cells presented higher DNA damage and higher apoptotic potential than p53Pro cells, after treatment with chemotherapy agents. Only p53Pro cells presented t(15;17) translocation and del(5q). We suggest that failure to repair DNA lesions in p53Arg cells would lead them to apoptosis, whereas some p53Pro cells, prone to cell cycle arrest and DNA repair, could undergo misrepair, generating chromosomal abnormalities typical of t-MN.

A sequential methodology that allows apoptotic cell sorting and FISH analysis in human testicular cells, Systems Biology in Reproductive Medicine, 58:6, 354-361, DOI: 10.3109/19396368.2012.717163 To link to this article: https://doi.org/10. 3109/19396368.2012.717163 The objective of this study was to develop a methodology that permits the detection and separation of apoptotic cells in human testicular tissue and their subsequent cytogenetic analysis by fluorescence in situ hybridization (FISH). The sequential methodology consisted of five steps: 1) enzymatic disaggregation of testicular tissue, 2) specific staining of apoptotic cells, 3) cell sorting by flow cytometry, 4) cell fixation, and 5) FISH. Enzymatic disaggregation yielded cell counts that ranged from 1.7x10 5 to 5x10 6 cells, and viability values greater than 72%. The apoptotic (mean ± SD: 22% ± 5.3%) and viable (45.5% ± 7.3%) populations were identified and selected by flow cytometry and demonstrated purity values ranging between 62% and 100%. The paraformaldehyde fixation of the selected fractions resulted in cell loss values of less than 10%. The application of three treatments before FISH (membrane permeabilization, elimination of cytoplasmic components, and re-fixation of the sample) resulted in hybridization frequencies of greater than 98%. In both selected fractions, cells of all spermatogenic stages and Sertoli cells were identified. The methodology developed has enabled the preparation of a cellular suspension with optimal viability and counting, the efficient selection of the apoptotic population, and its analysis by cytogenetic techniques. The application of this methodology in testicular cells should help establish whether there is a direct relationship between chromosome anomalies and apoptosis.