Morphological and Molecular Changes of Human Granulosa Cells Exposed to 5-Azacytidine and Addressed Toward Muscular Differentiation

Abstract

Converting adult cells from one cell type to another is a particularly interesting idea for regenerative medicine. Terminally differentiated cells can be induced to de-differentiate in vitro to become multipotent progenitors. In mammals these changes do not occur naturally, however exposing differentiated adult cells to synthetic molecules capable of selectively reverting cells from their lineage commitment to a more plastic state makes it possible to re-address their fate. Only scattered information are available on the morphological changes and ultrastructural remodeling taking place when cells convert into a different and specific type. To better clarify these aspects, we derived human granulosa cell (GC) primary cultures and analyzed the morphological changes taking place in response to the exposure to the epigenetic modifier 5-azacytidine (5-aza-CR) and to the treatment with VEGF, as a stimulus for inducing differentiation into muscle cells. Ultrastructural modifications and molecular marker expression were analyzed at different intervals during the treatments. Our results indicate that the temporary up regulation of pluripotency markers is accompanied by the loss of GC-specific ultrastructural features, mainly through autophagocitosis, and is associated with a temporary chromatin decondensation. After exposure to VEGF the induction of muscle specific genes was combined with the appearance of multinucleated cells with a considerable quantity of non-spatially organized filaments. The detailed analysis of the morphological changes occurring in cells undergoing lineage re-addressing allows a better understanding of these process and may prove useful for refining the use of somatic cells in regenerative medicine and tissue replacement therapies.

Mountain high and valley deep: epigenetic controls of pluripotency and cell fate

“…They confirmed that specific chemical compounds can push cells to a transient less committed state, increasing cell plasticity for a relative short time-window, but sufficient to re-address an adult mature cell into another differentiated cell type (Harris et al, 2011;Pennarossa et al, 2013Pennarossa et al, , 2014Brevini et al, 2014;Mirakhori et al, 2015;Chandrakanthan et al, 2016).…”

“…It is interesting to consider that this condition was transient and reversible, and, if returned to their standard culture medium, cells reverted to their original phenotype. Expression of pluripotency related genes decreased gradually within a few days (Pennarossa et al, 2013Brevini et al, 2014).…”

“…In particular, adult skin fibroblasts, derived from different species namely human (Pennarossa et al, 2013;Brevini et al, 2014), porcine , and dog (Brevini et al, 2016), were converted into pancreatic beta-cells through a three step pancreatic induction protocol. At the end of the epigenetic conversion, cells exhibited mature endocrine phenotype, expressing the main hormone and glucose sensor genes specific of the pancreatic tissue (Pennarossa et al, 2013Brevini et al, 2016).…”

The quest for an effective and safe personalized cell therapy using epigenetic tools

“…Among these, DNA methylation plays a fundamental role during both early embryonic development and cell lineage specification, causing silencing of large fraction of the genome and subsequent expression of gene essential for the maintenance of the differentiated and tissue-specific phenotype. Based on this, 5-azacytidine (5-aza-CR), a well-characterized DNMT inhibitor, was selected in order to remove the epigenetic “blocks” that are responsible for tissue specification [3–5, 7]. This drug is a chemical analog of cytosine, it can be incorporated into DNA and RNA, causing an increased effect in resting as well as in dividing cells, and it is known to be a direct inhibitor of methylation in newly synthesized DNA by blocking DNMT function [56].…”

“…Indeed, it has been demonstrated that malignant transformations as well as several disorders, such as autism, bipolar disorder, familial hypertrophic cardiomyopathy, schizophrenia, and syndromes, namely Prader-Willi, Angelman, Beckwith-Wiedemann, and Silver-Russell, are directly or indirectly caused by epigenetic alterations in form of mutation of DNA methylation or incorrect histone modifications [2–5]. In particular, DNA methyltransferase (DNMT) inhibiting nucleoside analogs, non-nucleoside analogs, and histone deacetylase (HDAC) inhibitors have been proposed as potential anti-cancer drugs.…”