Research projects

CELL AND MOLECULAR THERAPY FOR TYPE 1 DIABETES MELLITUS IN PRE-CLINICAL MOUSE MODELS OF THE DISEASE.

The project, physically conducted at the Laboratory for Endocrine and Biohybrid Cell Transplants at the University of Perugia, will address to identify a possible cure for T1D by using different cellular and molecular are approaches, associated with different mechanisms of action but complementary to each other, in achieving the desired result. According to the project lines, we will explore: The possibility of replacing damaged/destroyed insulin producing pancreatic islet beta-cells, which are associated with the autoimmune disease process of type 1 diabetes. Upon damage/destruction, beta-cells are no longer viable to secrete insulin, and will require replacement. The new substitutive cell products, capable of synthesizing and secreting insulin, may have different origins: adult stem cells (exempt from ethical objections) derived from :a) human pancreatic islets (hIDCs); b) Wharton’s Jelly of the post-partum human umbilical cords (hUCMSCs); engineered somatic cells, de-differentiated to the status of pluripotent stem cells (hiPSCs) that, may be re-differentiated back into a cellular phenotype that produces insulin (studies initiated with EU Horizon 2020). Development of biohybrid devices for the treatment of deep trophic ulcerative lesions, typical for instance of the diabetic foot, one of the actual leading causes of peripheral limb amputations. On this purpose, we will employ 3D printed bio-scaffolds to regenerate the deep ulcerations.

Details on cellular models for the treatment of type 1 diabetes mellitus (T1D) used in the project:

1) Stem cells from human adult pancreatic islets (hIDCs).

Adult stem cells derived from human pancreatic islet cells, after expansion in monolayers as described in our studies, express some typical stemness markers, such as OCT4 and CD90, and can therefore be used as an alternative tissue source for the cell therapy of T1D. In fact, these cells are associated with immunomodulatory properties (that is, they are able to counteract the autoimmune attack that leads to type 1 diabetes) but they also are able to produce insulin ex novo after adequate culture maintenance and differentiation. Of particular importance was to evidence the cells ability to differentiate toward the pancreatic beta-cell phenotype, upon their entrapment within our sodium alginate and poly-aminoacidic microcapsules and subsequent graft into immunocompetent diabetic mice. In fact, thanks to the total biocompatibility of the microcapsules, we were able to obtain, after microencapsulation, complete maturation with ex novo production of insulin from the neo-generated beta cells. These results open up new research avenues on the possibility of using insulin-secreting cells as an alternative to pancreatic islet transplantation, which is limited by the scarcity of human donor organs.

2) Stem cells from human post-partum umbilical cord (hUCMS).

Mesenchymal stem cells represent a pluripotent cellular phenotype with high differentiative plasticity toward different cellular phenotypes, including endocrine, and holds significant immuno-regulatory capacity. The use of the hUCMS as a source of mesenchymal stem cells is ethically acceptable, as it does not require invasive procedures for collection, since it is considered medical waste, and does not require informed consent. The use of hUCMS involves two phases: isolation, propagation, and characterization of the cells; characterization of the obtained cells, both under basal conditions and after differentiation stimulation, through screening of critical transcripts in endocrine differentiation (insulin, Glut-2, GK, Pdx-1, NeuroD /Beta2, Nkx6.1) and functional tests (insulin release in response to changes in glucose concentration with which they are stimulated). The same hUCMS show marked immunomodulatory activity thanks to the secretion of Indoleamine 2,3-dioxygenase (IDO) and HLAG-5.

3) Adult induced pluripotent stem cells (hiPSC)

hiPSCs are artificially generated stem cells from a terminally differentiated cell (usually an adult somatic cell, such as a skin fibroblast or a blood cell), by introducing four specific genes encoding certain transcription factors that induce the conversion into a stem cell of a specific cell line; this in turn can develop into the desired cellular phenotype (in our case insulin-secreting cell). Based on these properties, hiPSCs offer great opportunities in the field of regenerative medicine, such as the possibility of inducing their differentiation into most of the cell types present in the body (such as neuronal, pancreatic, cardiac, and hepatic cells), and in the context of tissue or organ regeneration. With respect to hiPSC, our recent participation in the EU Horizon 2020 project has allowed us to validate a specific differentiation protocol to obtain the beta cell insulin-secreting phenotype. The in vitro differentiation methodology leads us to obtain 3D cell aggregates capable of producing insulin on a regulated manner, and therefore constitute a valid tool for management of diabetes by cell therapy.

4) Development of bio-artificial devices (scaffolds) for the treatment of diabetic foot lesions.

This topic has already been preliminarily addressed in a research project funded by the CARIT Foundation of Terni, which utilized a commercially available 3D bioprinter that had been appropriately modified, along with the use of hUCMS.

In recent times, significant technical progress has been made through modifications to the printer, as well as the use of a bio-ink to construct scaffolds. The bio-ink is a solution of a biomaterial or a mixture of different biomaterials in the form of hydrogel, which typically incorporates the desired types of cells to create tissue equivalents.

The scaffolds serve as a synthetic extracellular matrix (ECM) and allow cells to organize into a three-dimensional architecture, similar to that found in physiological tissue, promoting the release of stimulating humoral factors from the cells, which in turn guide the growth and formation of the desired tissue. The production of such scaffolds is achieved through 3D bioprinting, using the so-called bio-inks, which are extruded from the printer to form 3D constructs. The research project involves the printing of scaffolds of various configurations, which can be differentiated through appropriate methodologies into tissues such as cartilage, bone, and skin to repair diabetic foot ulcers.

Il progetto, condotto fisicamente presso il Laboratorio per i Trapianti Cellulari Endocrini ed Organi Bioibridi dell’Università di Perugia, si propone di individuare una terapia valida per il T1D mediante approcci di terapia cellulare e molecolare differenti nei meccanismi di azione, ma complementari ai fini del raggiungimento del risultato.

Potential implications for human public health (social value)

The project targets a disease with significant social and economic impact. In Italy, every diabetic patient requires a total annual cost of €2,738 for direct costs (for a total of over €5 million for the entire diabetic community). The cost of healthcare for a diabetic patient increases by 3-4 times if complications affecting the heart, blood vessels, kidney, eyes, peripheral nervous system, and lower limbs (such as typical diabetic foot ulcers) occur. Hence, intensive and preventive treatment of the disease from the very onset not only improves the quality of life of the patients but also turns to be economically advantageous. The success of the project, even if partial, will provide information to develop future treatments to improve the quality of life of T1D patients.

Potential impact on the economic-industrial field:

The success of the project may also impact on the economic field, albeit in the early stages. The therapeutic combination of insulin-secreting cells and other immunomodulatory factors, within highly biocompatible sodium alginate microcapsules, is approved as a “drug delivery system” (slow-release drug).