Simona Baronchelli

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The potential use of human embryonic stem cells (hESCs) in cell-based therapies points out the critical importance of epigenomic evaluation for cell-based therapies. Specifically, DNA methylation appears to be a crucial player in establishing cell fate commitment and lineage choices. In this study, we report the global changes observed on the CpG islands distributed in promoters, gene bodies, and intergenic regions and the major biochemical pathways and genes involved in methylation changes as H9-hESCs turn into a neuronal culture containing medium-sized spiny striatal neurons (MSNs). Using an ontogeny-recapitulating protocol of striatal neuron differentiation, we analyzed DNA methylation profiles during the conversion from pluripotency to neuropotency up to the acquisition of a mature neuronal phenotype. H9-hESCs changed the methylation pattern both through de novo methylation and hypomethylation of specific gene promoters. Bioinformatic analysis allowed us to identify a panel of striatal-associated genes, which were regulated by DNA methylation and differentially expressed during striatal commitment. Importantly, DNA methylation analysis revealed that H9-hESCs did not acquire methylation-based oncogenic properties after differentiation. Indeed, hypermethylation of cancer-associated genes that characterize transformed cells, such as Polycomb repressive complex-associated genes, was not detected in the neuronal cultures. However, the oncosuppressor gene, BCL2L11 , became hypermethylated in H9-hESC-derived mature neurons. Whole-genome DNA methylation profiling could become a technological platform to predict the differentiative potential of hESC-derived cultures and establish further biosafety assessment quality control tools of the cell-based products.

The shape of the global health care system is changing rapidly to an approach that is much more patient-centered and focused on “precision medicine.” This is especially due to the development of large-scale “omics” biology results that rely on using and sharing sample collections and databases contained within bioresource facilities. “Personalized medicine” or “precision medicine” is the premise to help individuals to get the “right medicine for the right problem at the right time.” For several decades, tissues, body fluids, and cells obtained from patients with selected diseases have been cryopreserved in hospital-based biobanks, but samples were not accessible worldwide. Instead, the value of biobanks relies on the availability, at a necessary scale, of high-quality biospecimens and related data in order to respond to specific biological questions. However, the next generation of biobanks needs to face a major challenge – the costs related to the collection and processing of a large number of samples. Here, we describe the shift of biobanks from conventional repositories to functional infrastructures able to respond to specific medical demands.

Glioblastoma multiforme (GBM), the most common and malignant type of glioma, is characterized by a poor prognosis and the lack of an effective treatment, which are due to a small sub-population of cells with stem-like properties, termed glioma stem cells (GSCs). The term “multiforme” describes the histological features of this tumor, that is, the cellular and morphological heterogeneity. At the molecular level multiple layers of alterations may reflect this heterogeneity providing together the driving force for tumor initiation and development. In order to decipher the common “signature” of the ancestral GSC population, we examined six already characterized GSC lines evaluating their cytogenomic and epigenomic profiles through a multilevel approach (conventional cytogenetic, FISH, aCGH, MeDIP-Chip and functional bioinformatic analysis). We found several canonical cytogenetic alterations associated with GBM and a common minimal deleted region (MDR) at 1p36.31, including CAMTA1 gene, a putative tumor suppressor gene, specific for the GSC population. Therefore, on one hand our data confirm a role of driver mutations for copy number alterations (CNAs) included in the GBM genomic-signature (gain of chromosome 7- EGFR gene, loss of chromosome 13- RB1 gene, loss of chromosome 10-PTEN gene); on the other, it is not obvious that the new identified CNAs are passenger mutations, as they may be necessary for tumor progression specific for the individual patient. Through our approach, we were able to demonstrate that not only individual genes into a pathway can be perturbed through multiple mechanisms and at different levels, but also that different combinations of perturbed genes can incapacitate functional modules within a cellular networks. Therefore, beyond the differences that can create apparent heterogeneity of alterations among GSC lines, there’s a sort of selective force acting on them in order to converge towards the impairment of cell development and differentiation processes. This new overview could have a huge importance in therapy.

Introduction: Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles. Methods: In this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors. Results: Our data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages. Conclusions: Because we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on hBM-MSC malignant transformation in the literature.

The importance of the genetic factor in the aetiology of premature ovarian failure (POF) is emphasized by the high percentage of familial cases and X chromosome abnormalities account for 10% of chromosomal aberrations. In this study, we report the detailed analysis of 4 chromosomal abnormalities involving the X chromosome and associated with POF that were detected during a screening of 269 affected women. Conventional and molecular cytogenetics were valuable tools for locating the breakpoint regions and thus the following karyotypes were defined: 46,X,der(X)t(X;19)(p21.1;q13.42)mat, 46,X,t(X;2)(q21.33;q14.3)dn, 46,X,der(X)t(X;Y)(q26.2;q11.223)mat and 46,X,t(X;13)(q13.3;q31)dn. A bioinformatic analysis of the breakpoint regions identified putative candidate genes for ovarian failure near the breakpoint regions on the X chromosome or on autosomes that were involved in the translocation event. HS6ST1, HS6ST2 and MATER genes were identified and their functions and a literature review revealed an interesting connection to the POF phenotype. Moreover, the 19q13.32 locus is associated with the age of onset of the natural menopause. These results support the position effect of the breakpoint on flanking genes, and cytogenetic techniques, in combination with bioinformatic analysis, may help to improve what is known about this puzzling disorder and its diagnostic potential.

The importance of X chromosome in the aetiology of premature ovarian failure (POF) is well‐known but in many cases POF still remains idiopathic. Chromosome aneuploidy increase is a physiological phenomenon related to aging, but the role of low‐level sex chromosome mosaicism in ovarian function is still undiscovered. Standard cytogenetic analysis was carried out in a total of 269 patients affected by POF: 27 chromosomal abnormalities were identified, including X chromosome and autosomal structural and numerical abnormalities. In 47 patients with 46,XX karyotype we performed interphase FISH using X alpha‐satellite probe in order to identify X chromosome mosaicism rate. Aneuploidy rate in the patient group was significantly higher than the general population group. These findings underline the importance of X chromosome in the aetiology of POF and highlight the potential role of low‐level sex chromosome mosaicism in ovarian aging that may lead to a premature onset of menopause.