Global Proteomic and Methylome Analysis in Human Induced Pluripotent Stem Cells Reveals Overexpression of a Human TLR3 Affecting Proper Innate Immune Response Signaling.

When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488.

Karyotyping of human synaptonemal complexes by cenM-FISH.

The purpose of this work was to adapt the recently described centromere-specific multicolour (cenM-) FISH technique to human meiotic cells, and evaluate the usefulness of this multiplex fluorescence method for karyotyping human synaptonemal complex (SC), previously analysed by immunocytogenetic approaches. The results obtained demonstrate that cenM-FISH is a reliable one-single-step method, which allows for the identification of all SC present in pachytene spreads. Moreover, when cenM-FISH is applied after immunocytogenetic analysis, the number and distribution of MLH1 foci per chromosome can be established and recombination analysis for each chromosome can be performed easily.

Micro-array analyses decipher exceptional complex familial chromosomal rearrangement.

Recently there has been an increased interest in large-scale genomic variation and clinically in the consequences of haploinsufficiency of genomic segments or disruption of normal gene function by chromosome rearrangements. Here, we present an extraordinary case in which both mother and daughter presented with unexpected chromosomal rearrangement complexity, which we characterized with array-CGH, array painting and multicolor large insert clone hybridizations. We found the same 12 breakpoints involving four chromosomes in both mother and daughter. In addition, the daughter inherited a microdeletion from her father. We mapped all breakpoints to the resolution level of breakpoint spanning clones. Genes were found within 7 of the 12 breakpoint regions, some of which were disrupted by the chromosome rearrangement. One of the rearrangements disrupted a locus, which has been discussed as a quantitative trait locus for fetal hemoglobin expression in adults. Interestingly, both mother and daughter show persistent fetal hemoglobin levels. We detail the most complicated familial complex chromosomal rearrangement reported to date and thus an extreme example of inheritance of chromosomal rearrangements without error in meiotic segregation.

Synapsis and meiotic recombination analyses: MLH1 focus in the XY pair as an indicator.

BACKGROUND: Anomalies in meiotic prophase I have been related to partial or total meiotic arrest. These anomalies include an abnormal synaptic process, resulting in disorders in meiotic recombination. METHODS: In the present study, we analyse primary spermatocytes from 12 infertile men (four with non-obstructive azoospermia, six with oligoastenoteratozoospermia, one with astenoteratozoospermia and one normozoospermic) and five control fertile donors using immunocytological techniques for synaptonemal complex, meiotic recombination and centromeric proteins. RESULTS: Mean numbers of MLH1 foci per cell, frequencies of cells presenting an MLH1 focus in the XY pair and percentages of cells affected by abnormal synaptic patterns (gaps and splits) are reported for each of the infertile patients and control men. A positive correlation between the frequency of cells showing a recombination focus in the XY pair and the number of autosomal recombination foci per cell is found. CONCLUSIONS: Reduced recombination in the XY pair and an increased number of cells affected by gaps may explain some idiopathic male infertility cases. The results suggest that recombination in the XY pair could be an indicator for general recombination frequency and for a successful meiotic process.