Acceleration of Functional Maturation and Differentiation of Neonatal Porcine Islet Cell Monolayers Shortly In Vitro Cocultured with Microencapsulated Sertoli Cells

The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM). Starting from isolated neonatal porcine pancreatic islets (NPIs), we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs) for different time periods (7, 14, 21 days). To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation of β-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs β-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature β-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM

Effects of cadmium on viability and function of porcine pre-pubertal Sertoli cells

Cadmium, an ubiquitous environmental pollutant mainly used for industrial purposes, is highly associated with reproductive toxicity. Sertoli cells (SC), by providing an appropriate microenvironment for the development of germ cells, play a pivotal role on spermatogenesis regulation (Geoffroy-Siraudin et al. 2012). Aim of our investigation was to assess the effects of cadmium on high mammalian SC viability and function. Porcine pre-pubertal SC were isolated, according to previously established methods (Fallarino et al. 2009) and treated with 3 different concentrations (5-10-15 μM) of cadmium chloride. The evaluation of SC function in terms of inhibin B and anti-Müllerian hormone (AMH) secretion showed a significant decrease in all SC treated conditions respect as compared to SC control. On the contrary, evaluation of the FSH-R integrity on SC surface, in terms of 17-b-estradiol production under FSH stimulation, showed no difference between SC control and 5 μM cadmium treated SC monolayers in comparison to 10 and 15μM cadmium treated SC monolayers, where FSH-R was impaired. In addition, the apoptotic test showed a significant increase of early (ANNEXIN V-/Propidium Iodide+) (AV-/PI+) and late apoptotic cells (AV+/ PI+) in all cadmium treated SC conditions in comparison with SC control. In conclusion, our data demonstrate that cadmium, even at low dose, exerts toxic effects on Sertoli cells function possibly adversely affecting the spermatogenesis

Xenograft of free or microencapsulated Sertoli cells as a potential therapy for experimental Type 2 Diabetes Mellitus

Introduction and Aim. Type 2 Diabetes Mellitus (T2DM), one of the world’s most important, common and costly diseases associated with devastating complications, is caused by insulin resistance mainly due to a chronic inflammation of the visceral adipose tissue with local and systemic increases in proinflammatory cytokines and adipokines such as tumor necrosis factor-α (TNF-α) and IL-6. Sertoli cells (SC), considered mere mechanical cell aids, have been recently revisited with respect to their functional competence showing that these cells may provide immunomodulatory and trophic factors that are able to ameliorate survival and development of different cell types and constitute an immuno-protective shield for transplantation in many diseases such as Type 1 Diabetes Mellitus. Aim of this work was to verify if the injection of free (subcutaneously) or microencapsulated (intraperitoneally) SC would reverse hyperglycaemia in db/db mice spontaneous T2DM Materials and Methods. Porcine pre-pubertal SC were isolated, according to previously established methods, after finely chopping the retrieval testicles, with double enzymatic digestion with Collagenase P and a mixed solution of trypsin and DNase I. SC enveloped in Barium alginate-based microcapsules (Ba-SCMCs) were prepared according to our method, by a mono air-jet device system. Free SC and Ba-SCMCs were examined as far as: (a) SC morphology by light microscopy; (b) SC viability, by fluorescence microscopy after staining with ethidium bromide and fluorescein diacetate; (c) SC in vitro function (α-aromatase activity and IGF-I secretion); (d) reversal of T2DM in spontaneous diabetes db/db mice, were concerned. Results. Ba-SCMCs showed excellent features in terms of size, morphology, sphericity and coalescence. SC viability, both in free and microencapsulated SC, was very high (over 90%). Very good α-aromatase activity and IGF-I secretion were associated with the examined SC preparations. Both subcutaneous free SC injection and intraperitoneal transplantation of Ba-SCMCs demonstrated a significant reduction, in 60% of the treated mice, of HbA1c (6.6 % vs 8.8 %) with a normalization of intraperitoneal glucose tolerance test. Conclusions. SC may be enveloped in Ba-SCMCs with no loss of their functional properties and morphology. Xenograft of SC, both free and enveloped in barium alginate microcapsules, induced an important reduction of HbA1c blood levels with a normalization of glucose tolerance test (IPGTT). This result might open new perspectives for the future therapy of human T2DM

Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone

Type I diabetes mellitus is caused by autoimmune destruction of pancreatic β cells, and effective treatment of the disease might require rescuing β cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional β cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-β–dependent emergence of autoantigen-specific regulatory T cells and recovery of β cell function in the diabetic recipients

Effects of hydroxyapatite and Biostite on osteogenic induction of hMSC.

When isolated from the iliac crest human mesenchymal stem cells (hMSC) differentiate into osteoblast-like cells with appropriate stimulation in culture. This in vitro study tested the hypothesis that Biostite and hydroxyapatite (HA) affect proliferation and differentiation of hMSC into osteoblastic cells. Cell proliferation was determined by measuring 3H-thymidine incorporation into DNA and typical markers of osteoblastic phenotype were determined by RT-PCR assay. No differences emerged in cell proliferation cultures with Biostite or hydroxyapatite (HA), but gene expression analysis revealed higher expression of collagen,alkaline phosphatase (ALP), osteopontin and bone sialoprotein (BSP) in the presence of Biostite. TGFb2 production, as assessed by an Elisa kit, and Runx2 expression by RT-PCR, were greater in Biostite cultures, suggesting Biostite provides a better environment for hMSC differentiation into osteoblasts and is, potentially, a more promising bone-filling material than HA

Conformal polymer coatings for pancreatic islets transplantation

The aim of this work was to improve an aqueous two-phase system methodology for fabrication of coherent microcapsules. Simulated microgravity was investigated as tool to improve the cell cluster morphology in order to increase the overall quality of conformal polymer coatings, while the application of two concentric alginate layers and the use of barium instead of calcium as gelling ion was evaluated. Simulated microgravity enabled improvement of neonatal porcine cell cluster sphericity however the freely floating cells, originated during incubation and often found on the capsule surface, raised immunological concerns. Overall, these technical changes translated into improving quality of microcapsules, in terms of either morphologic aspects or the membrane's functional performance. Preparation procedure did not seem to adversely affect viability of the embodied cells. Moreover, the employed alginates high biocompatibility, per se, would promote a good encapsulated cell engraftment. Minimization of last generation microcapsule's size, made of highly purified alginates, represents a further advance on the new horizons of cell therapy for the treatment of a wide variety of chronic disorders, including insulin-dependent diabetes mellitus