Simona Baronchelli

From cytogenomic to epigenomic profiles: monitoring the biologic behavior of in vitro cultured human bone marrow mesenchymal stem cells

IntroductionBone 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.MethodsIn 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.ResultsOur 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.ConclusionsBecause 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.

Cytogenetics of Premature Ovarian Failure: An Investigation on 269 Affected Women

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.

Investigating the role of X chromosome breakpoints in premature ovarian failure

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.

Delineating the Cytogenomic and Epigenomic Landscapes of Glioma Stem Cell Lines

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.

Down-modulation of SEL1L, an Unfolded Protein Response and Endoplasmic Reticulum-associated Degradation Protein, Sensitizes Glioma Stem Cells to the Cytotoxic Effect of Valproic Acid

Background: Valproic acid is considered as a promising anti-cancer therapeutic agent acting on unfolded protein response. SEL1L is an UPR-responsive gene. Results: SEL1L interference synergy enhances VPA cytotoxic effects on glioma stem cells. Conclusion: VPA treatment combined with SEL1L depletion may influence GSC pharmacological response. Significance: Targeting SEL1L in association with valproic acid treatment may improve glioma treatment. Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments.

Epigenetic targeting of glioma stem cells: Short-term and long-term treatments with valproic acid modulate DNA methylation and differentiation behavior, but not temozolomide sensitivity

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system. GBM is a fatal tumor, incurable by conventional therapies. One of the factors underlying tumor recurrence and poor long-term survival is the presence of a cancer stem-like cell population, termed glioma stem cells (GSCs), which is particularly resistant to chemotherapy and radiotherapy and supports tumor self-renewal. The aim of the present study was to evaluate the impact and difference in effects of short-term and long‑term treatments with valproic acid (VPA), a histone deacetylase inhibitor, on seven GSC lines. We investigated for the first time the changes in the genome-wide DNA methylation profile and the differentiation behavior of GSCs induced by short-term and long-term VPA treatments. Moreover, we verified VPA sensitivity after long-term VPA pretreatment and, notably, the results provide evidence of a subpopulation more resistant to further VPA treatments. Finally, since short-term VPA treatment induced a reversal of the MGMT methylation status, we aimed to sensitize GSCs to temozolomide, the drug commonly used for this tumor, using this regimen. The overall data highlighted the heterogeneous behavior of GSC lines that is representative of tumor heterogeneity in GBM. The VPA effects were variable among these cell lines in terms of pro‑differentiating ability and DNA methylation switch. Here, we attempted to identify a suitable therapy for the eradication of the stem cell subpopulation, which is mandatory to achieve an effective treatment for this tumor. Differentiation-inducing and epigenetic therapies are the most promising approaches to affect the multiple properties of GSCs and, finally, defeat GBM.

Cell Line Macroarray: An Alternative High-Throughput Platform to Analyze hiPSC Lines

In the past decade, tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly, enormous amount of data can be obtained from the cell line macroarray (CLMA) technology, which developed from the TMA using formalin-fixed, paraffin-embedded cell pellets. Here, we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones, which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here, we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer, TissueQuest, as a reliable tool to quickly select the best clones, based upon the level of expression of multiple pluripotent biomarkers.

Biobanking shifts to “precision medicine”

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Journal of Biorepository Science for Applied Medicine 2014:2 11–15 Journal of Biorepository Science for Applied Medicine Dovepress

A Review of Research-Grade Human Induced Pluripotent Stem Cells Qualification and Biobanking Processes

Human induced pluripotent stem cell (hiPSC) biobanks are invaluable resources for basic and clinical research, since they provide a sustainable supply of accessible cell lines that meet high quality and safety standards. hiPSCs are particularly useful for understanding disease mechanisms, creating cell models for drug development, and generating novel clinical therapies. For clinical applications and drug discovery, it is fundamental that the acquired pluripotent cell lines never touch animal-derived products nor xenogeneic reagents (Good Manufacturing Practice-grade); whereas for research grade, it is sufficient to operate under Good Laboratory Practice conditions. However, regardless of the end use, it is important that every step in the whole process, starting from the original cells throughout expansion and manipulation, must be performed and recorded rigorously. Here, we describe our biobanking management system that is applied specifically to human pluripotent stem cells.