Adding a dimension to cell fate.

Cell fate specification, gene expression and spatial restriction are process finely tuned by epigenetic regulatory mechanisms. At the same time, mechanical forces have been shown to be crucial to drive cell plasticity and boost differentiation. Indeed, several studies have demonstrated that transitions along different specification states are strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, that can modulate both cell potency and differentiation, through the activation of specific mechanosensing-related pathways. An overview of small molecule ability to modulate cell plasticity and define cell fate is here presented and results, showing the possibility to erase the epigenetic signature of adult dermal fibroblasts and convert them into insulin-producing cells (EpiCC) are described. The beneficial effects exerted on such processes, when cells are homed on an adequate substrate, that shows "in vivo" tissue-like stiffness are also discussed and the contribution of the Hippo signalling mechano-transduction pathway as one of the mechanisms involved is examined. In addition, results obtained using a genetically modified fibroblast cell line, expressing the enhanced green fluorescent protein (eGFP) under the control of the porcine insulin gene (INS) promoter (INS-eGFP transgenic pigs), are reported. This model offers the advantage to monitor the progression of cell conversion in real time mode. All these observations have a main role in order to allow a swift scale-up culture procedure, essential for cell therapy and tissue engineering applied to human regenerative medicine, and fundamental to ensure an efficient translation process from the results obtained at the laboratory bench to the patient bedside. Moreover, the creation of reliable in vitro model represents a key point to ensure the development of more physiological models that, in turn, may reduce the number of animals used, implementing non-invasive investigations and animal welfare and protection.

Use of a Super-hydrophobic Microbioreactor to Generate and Boost Pancreatic Mini-organoids.

Cell remarkable ability to self-organize and rearrange in functional organoids has been greatly boosted by the recent advances in 3-D culture technologies and materials. This approach can be presently applied to model human organ development and function "in a dish" and to predict drug response in a patient specific fashion.Here we describe a protocol that allows for the derivation of functional pancreatic mini-organoids from skin biopsies. Cells are suspended in a drop of medium and encapsulated with hydrophobic polytetrafluoroethylene (PTFE) powder particles, to form microbioreactors defined as "Liquid Marbles," that stimulate cell coalescence and 3-D aggregation. The PTFE shell ensures an optimal gas exchange between the interior liquid and the surrounding environment. It also makes it possible to scale down experiments and work in smaller volumes and is therefore amenable for higher throughput applications.

Use of a PTFE Micro-Bioreactor to Promote 3D Cell Rearrangement and Maintain High Plasticity in Epigenetically Erased Fibroblasts.

Phenotype definition is driven by epigenetic mechanisms as well as directly influenced by the cell microenvironment and by biophysical signals deriving from the extracellular matrix. The possibility to interact with the epigenetic signature of an adult mature cell, reversing its differentiated state and inducing a short transient high plasticity window, was previously demonstrated. In parallel, in vitro studies have shown that 3D culture systems, mimicking cell native tissue, exert significant effects on cell behavior and functions. Here we report the production of "PTFE micro-bioreactors" for long-term culture of epigenetically derived high plasticity cells. The system promotes 3D cell rearrangement, global DNA demethylation and elevated transcription of pluripotency markers, that is dependent on WW domain containing transcription regulator 1 (TAZ) nuclear accumulation and SMAD family member 2 (SMAD2) co-shuttling. Our findings demonstrate that the use of 3D culture strategies greatly improves the induction and maintenance of a high plasticity state.

Safety and Efficacy of Epigenetically Converted Human Fibroblasts Into Insulin-Secreting Cells: A Preclinical Study.

Type 1 Diabetes Mellitus (T1DM) is a chronic disease that leads to loss of insulin secreting ß-cells, causing high levels of blood glucose. Exogenous insulin administration is not sufficient to mimic the normal function of ß-cells and, consequently, diabetes mellitus often progresses and can lead to major chronic complications and morbidity. The physiological control of glucose levels can only be restored by replacing the ß-cell mass.We recently developed a new strategy that allows for epigenetic conversion of dermal fibroblasts into insulin-secreting cells (EpiCC), using a brief exposure to the demethylating agent 5-aza-cytidine (5-aza-CR), followed by a pancreatic induction protocol. This method has notable advantages compared to the alternative available procedures and may represent a promising tool for clinical translation as a therapy for T1DM. However, a thought evaluation of its therapeutic safety and efficacy is mandatory to support preclinical studies based on EpiCC treatment.We here report the data obtained using human fibroblasts isolated from diabetic and healthy individuals, belonging the two genders. EpiCC were injected into 650 diabetic severe combined immunodeficiency (SCID) mice and demonstrated to be able to restore and maintain glycemic levels within the physiological range. Cells had the ability to self-regulate and not to cause hypoglycemia, when transplanted in healthy animals. Efficacy tests showed that EpiCC successfully re-established normoglycemia in diabetic mice, using a dose range that appeared clinically relevant to the concentration 0.6 × 106 EpiCC. Necropsy and histopathological investigations demonstrated the absence of malignant transformation and cell migration to organs and lymph nodes.The present preclinical study demonstrates safety and efficacy of human EpiCC in diabetic mice and supports the use of epigenetic converted cells for regenerative medicine of diabetes mellitus.

Epigenetic Erasing and Pancreatic Differentiation of Dermal Fibroblasts into Insulin-Producing Cells are Boosted by the Use of Low-Stiffness Substrate.

Several studies have demonstrated the possibility to revert differentiation process, reactivating hypermethylated genes and facilitating cell transition to a different lineage. Beside the epigenetic mechanisms driving cell conversion processes, growing evidences highlight the importance of mechanical forces in supporting cell plasticity and boosting differentiation. Here, we describe epigenetic erasing and conversion of dermal fibroblasts into insulin-producing cells (EpiCC), and demonstrate that the use of a low-stiffness substrate positively influences these processes. Our results show a higher expression of pluripotency genes and a significant bigger decrease of DNA methylation levels in 5-azacytidine (5-aza-CR) treated cells plated on soft matrix, compared to those cultured on plastic dishes. Furthermore, the use of low-stiffness also induces a significant increased up-regulation of ten-eleven translocation 2 (Tet2) and histone acetyltransferase 1 (Hat1) genes, and more decreased histone deacetylase enzyme1 (Hdac1) transcription levels. The soft substrate also encourages morphological changes, actin cytoskeleton re-organization, and the activation of the Hippo signaling pathway, leading to yes-associated protein (YAP) phosphorylation and its cytoplasmic translocation. Altogether, this results in increased epigenetic conversion efficiency and in EpiCC acquisition of a mono-hormonal phenotype. Our findings indicate that mechano-transduction related responsed influence cell plasticity induced by 5-aza-CR and improve fibroblast differentiation toward the pancreatic lineage.

Methylation mechanisms and biomechanical effectors controlling cell fate.

Mammalian development and cell fate specification are controlled by multiple regulatory mechanisms that interact in a coordinated way to ensure proper regulation of gene expression and spatial restriction, allowing cells to adopt distinct differentiation traits and a terminal phenotype. For example, cell potency is modulated by changes in methylation that are under the control of methyltransferases and ten-eleven translocation (TET) enzymes, which establish or erase a phenotype-specific methylation pattern during embryo development and mesenchymal to epithelial transition (MET). Cell plasticity is also responsive to extracellular factors, such as small molecules that interact with cell fate definition and induce a transient pluripotent state that allows the direct conversion of an adult mature cell into another differentiated cell type. In addition, cell-secreted vesicles emerge as powerful effectors, capable of modifying cell function and phenotype and delivering different signals, such as octamer-binding transcription factor-4 (Oct4) and SRY (sex determining region Y)-box 2 (Sox2) mRNAs (implicated in the preservation of pluripotency), thus triggering epigenetic changes in the recipient cells. In parallel, mechanical properties of the cellular microenvironment and three-dimensional rearrangement can affect both cell potency and differentiation through marked effects on cytoskeletal remodelling and with the involvement of specific mechanosensing-related pathways.

5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts.

Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and attractive for therapeutic intervention and for the reactivation of hypermethylated pluripotency genes that facilitate transition to a higher plasticity state. We report the results obtained in human fibroblasts exposed to the epigenetic modifier 5-azacytidine (5-aza-CR), which increases adult cell plasticity and facilitates phenotype change. Although many aspects controlling its demethylating action have been widely investigated, the mechanisms underlying 5-aza-CR effects on cell plasticity are still poorly understood. Our experiments confirm decreased global methylation, but also demonstrate an increase of both Formylcytosine (5fC) and 5-Carboxylcytosine (5caC), indicating 5-aza-CR ability to activate a direct and active demethylating effect, possibly mediated via TET2 protein increased transcription. This was accompanied by transient upregulation of pluripotency markers and incremented histone expression, paralleled by changes in histone acetylating enzymes. Furthermore, adult fibroblasts reshaped into undifferentiated progenitor-like phenotype, with a sparse and open chromatin structure. Our findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state is activated by multiple regulations that accompany the demethylating effect exerted by the modifier.

Erase and Rewind: Epigenetic Conversion of Cell Fate.

The potential of cell therapy in regenerative medicine has greatly expanded thanks to the availability of sources of pluripotent cells. In particular, induced pluripotent stem cells (iPS) have dominated the scenario in the last years for their ability to proliferate and differentiate into specific cell types. Nevertheless, the concerns inherent to the cell reprogramming process, limit iPS use in therapy and pose questions on the long-term behavior of these cells. In particular, despite the development of virus-free methods for their obtainment, a major and persisting drawback, is related to the acquisition of a stable pluripotent state, that is un-physiological and may lead to cell instability. The increased understanding of epigenetic mechanisms has paved the way to the use of "small molecules" and "epigenetic modifiers" that allow the fine tuning of cell genotype and phenotype. In particular, it was demonstrated that an adult mature cell could be directly converted into a different cell type with the use of these chemicals, obtaining a new patient-specific cell, suitable for cell therapy. This approach is simple and direct and may represent a very promising tool for the regenerative medicine of several and diverse degenerative diseases.