Microencapsulation of cells and molecular therapy of type 1 diabetes mellitus: The actual state and future perspectives between promise and progress

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Exploiting the great potential of Sequence Capture data by a new tool, SUPER-CAP

The recent development of Sequence Capture methodology represents a powerful strategy for enhancing data generation to assess genetic variation of targeted genomic regions. Here, we present SUPER-CAP, a bioinformatics web tool aimed at handling Sequence Capture data, fine calculating the allele frequency of variations and building genotype-specific sequence of captured genes. The dataset used to develop this in silico strategy consists of 378 loci and related regulative regions in a collection of 44 tomato landraces. About 14,000 high-quality variants were identified. The high depth (>40×) of coverage and adopting the correct filtering criteria allowed identification of about 4,000 rare variants and 10 genes with a different copy number variation. We also show that the tool is capable to reconstruct genotype-specific sequences for each genotype by using the detected variants. This allows evaluating the combined effect of multiple variants in the same protein. The architecture and functionality of SUPER-CAP makes the software appropriate for a broad set of analyses including SNP discovery and mining. Its functionality, together with the capability to process large data sets and efficient detection of sequence variation, makes SUPER-CAP a valuable bioinformatics tool for genomics and breeding purposes

IGF2 and IGF1R mRNAs Are Detectable in Human Spermatozoa.

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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

Cell encapsulation:Promise and progress

In cell encapsulation, transplanted cells are protected from immune rejection by an artificial, semipermeable membrane, potentially allowing transplantation (allo-or xenotransplantation) without the need for immunosuppression. Yet, despite some promising results in animal studies, the field has not lived up to expectations, and clinical products based on encapsulated cell technology continue to elude the scientific community. This commentary discusses the reasons for this, summarizes recent progress in the field and outlines what is needed to bring this technology closer to clinical application

Stem Cells for the Cell and Molecular Therapy of Type 1 Diabetes Mellitus (T1D) between Lights and Shadows

T1D consists of selective autoimmune killing of pancreatic islet β-cells that physiologically accomplish the task of secreting insulin on a regulated manner, in order to maintain blood glucose levels within normal range (70-140 mg/dl), under any circumstances

'In vitro' Effect of Different Follicle¿Stimulating Hormone Preparations on Sertoli Cells: Toward a Personalized Treatment for Male Infertility

Follicle-stimulating hormone (FSH), a major regulator of spermatogenesis, has a crucial function in the development and function of the testis and it is extensively given as a fertility treatment to stimulate spermatogenesis. We analyzed the effects of different FSH preparations (α-follitropin, β-follitropin, and urofollitropin) in combination with testosterone on porcine pre-pubertal Sertoli cells. To study the effect of the different FSH treatments in the Sertoli cell function we performed Real Time PCR analysis of AMH, inhibin B, and FSH-r, an ELISA assay for AMH and inhibin B, and a high-throughput comparative proteomic analysis. We verified that all three preparations induced a reduction of AMH in terms of mRNA and secreted proteins, and an increase of inhibin B in terms of mRNA in all the FSH formulations, while solely α-follitropin produced an increase of secreted inhibin B in the culture medium. Comparative proteomic analysis of the three FSH preparations identified 46 proteins, 11 up-regulated and 2 down-regulated. Surprisingly, the combination of testosterone with β-follitropin specifically induced an up-regulation of eight specific secreted proteins. Our study, showing that the three different FSH preparations induce different effects, could offer the opportunity to shed light inside new applications to a personalized reproductive medicine

Transplantation of microencapsulated Sertoli cells in a mouse model of Duchenne muscular dystrophy (DMD) reduces inflammation and rescues muscle performance

Duchenne muscular dystrophy (DMD), a progressive muscle degenerative disease associated with chronic inflammation, necrosis and fibrosis, is currently treated with antiinflammatory steroids, despite their limited efficacy and undesired side effects. Testicular Sertoli cells (SCs) have been successfully implanted to treat many experimental diseases due to their ability to secrete trophic, antiinflammatory and immunomodulatory molecules (Mital et al., 2010). We transplanted microencapsulated SCs, within highly biocompatible microcapsules (Luca et al., 2007) into the peritoneal cavity of mdx mice, an animal model of DMD. Three weeks after transplantation, skeletal muscles from SC-treated mice, compared with muscles from mock-treated mice, showed: i) dramatically reduced number of infiltrated cells, including (MAC3+) macrophages; ii) a marked decrease in necrotic myofibers, and an increased number of regenerated (normally sized and centrally nucleated) myofibers; and, iii) a significant decrease in fibrous tissue infiltration. Moreover, SC-treated, but not mock-treated mdx mice showed recovery of muscle performance in treadmill endurance tests and a comparable resistance to exercise-induced muscle damage to that of untreated wildtype mice. These preliminary results suggest that our transplant product creates a suitable microenvironment for muscle regeneration and growth potentially applicable to DMD patients

A new model to study the effects of gonadotropins on an “in vitro” prepubertal artificial porcine mini-testis

At present, there is no reliable experimental model “in vitro” to analyze the complex interactions between gonadotropins on the pre-pubertal Sertoli cells (SC) and Leydig cells (LD). Considering that, in the pre-pubertal period, only the anti-mullerian hormone (AMH) is upregulated by FSH and down-regulated by androgens [1-2], AMH could be considered a potential marker of pre-pubertal testis function. The aim of our work was to study the effects of FSH, LH and HCG on an in-vitro model of “mini-testis”. SC and LD, obtained from 15-20 days old neonatal pigs, were isolated and evaluated in terms of purity by AMH (unique pre-pubertal SCs marker), INSL3 (LD marker), ASMI (peritubular cells marker) and PGP9.5 (gonocytes and spermatogonial cells marker). Finally, purified SC and LD were co-cultured to obtain the “mini-testis” and were stimulated with gonadotropins. We have then evaluated: a) AMH, inhibin B and testosterone levels released in the culture medium (by ELISA), both in basal conditions and after stimulations; b) analysis of the follicle-stimulating hormone receptor (FSHR), MAPkinasi (Erk1/2, AKT) by Real Time PCR. We show an increase in inhibin B levels after FSH and FSH/LH stimulation and a selectively increase in testosterone production after LH treatment. AMH secretion was downregulated by FSH treatment. These data seem to preliminarily suggest that ERK1/ ERK2 expression was up-regulated by FSH and FSH/LH stimulation while FSHreceptor expression was down-regulated by FSH and increased by FSH/LH treatment; AKT was up-regulated in all conditions. The proposed model, by creating an artificial mini- testis, could help better understanding the complex and still partially unknown interactions between human gonadotropins, SC and LD possibly creating a novel background to shed light inside a future therapy of male infertilit