Peribiliary glands as a niche of extra-pancreatic precursors yielding insulin-producing cells in experimental and human diabetes

Peribiliary glands (PBGs) are niches in the biliary tree and containing heterogeneous endodermal stem/progenitors cells that can differentiate, in vitro and in vivo, towards pancreatic islets. The aim of this study was to evaluate, in experimental and human diabetes, proliferation of cells in PBGs and differentiation of the biliary tree stem/progenitor cells (BTSCs) towards insulin-producing cells. Diabetes was generated in mice by intraperitoneal injection of a single dose of 200 mg/kg (N=12) or 120 mg/kg (N=12) of streptozotocin. Liver, pancreas and extrahepatic biliary trees were en bloc dissected and examined. Cells in PBGs proliferated in experimental diabetes, and their proliferation was greatest in the PBGs of the hepato-pancreatic ampulla, and inversely correlated with the pancreatic islet area. In rodents, the cell proliferation in PBGs was characterized by the expansion of Sox9-positive stem/progenitor cells that gave rise to insulin-producing cells. Insulin-producing cells were located mostly in PBGs in the portion of the biliary tree closest to the duodenum, and their appearance was associated with up-regulation of MafA and Gli1 gene expression. In patients with type 2 diabetes, PBGs at the level of the hepato-pancreatic ampulla contained cells showing signs of proliferation and pancreatic fate commitment. In vitro, high glucose concentrations induced the differentiation of human BTSCs cultures towards pancreatic beta cell fates. The cells in PBGs respond to diabetes with proliferation and differentiation towards insulin-producing cells indicating that PBG niches may rescue pancreatic islet impairment in diabetes. These findings offer important implications for the patho-physiology and complications of this disease. This article is protected by copyright. All rights reserved

Adult human biliary tree stem cells differentiate to β-pancreatic islet cells by treatment with a recombinant human Pdx1 peptide

Generation of β-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards β-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the β-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage β-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional β-pancreatic cells

Culture of Human Limbal Epithelial Stem Cells on Tenon's Fibroblast Feeder-Layers: A Translational Approach.

The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential.Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged.Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems

Family expansion and gene rearrangements contributed to the functional specialization of PRDM genes in vertebrates

<p>Abstract</p> <p>Background</p> <p>Progressive diversification of paralogs after gene expansion is essential to increase their functional specialization. However, mode and tempo of this divergence remain mostly unclear. Here we report the comparative analysis of PRDM genes, a family of putative transcriptional regulators involved in human tumorigenesis.</p> <p>Results</p> <p>Our analysis assessed that the PRDM genes originated in metazoans, expanded in vertebrates and further duplicated in primates. We experimentally showed that fast-evolving paralogs are poorly expressed, and that the most recent duplicates, such as primate-specific <it>PRDM7</it>, acquire tissue-specificity. <it>PRDM7 </it>underwent major structural rearrangements that decreased the number of encoded Zn-Fingers and modified gene splicing. Through internal duplication and activation of a non-canonical splice site (GC-AG), <it>PRDM7 </it>can acquire a novel intron. We also detected an alternative isoform that can retain the intron in the mature transcript and that is predominantly expressed in human melanocytes.</p> <p>Conclusion</p> <p>Our findings show that (a) molecular evolution of paralogs correlates with their expression pattern; (b) gene diversification is obtained through massive genomic rearrangements; and (c) splicing modification contributes to the functional specialization of novel genes.</p

T-cell specific deregulation of Notch3 receptor induces alterations in the development of myeloid compartment unveiled by the deletion of NF-kappaB/p50 expression

Purpose/Objective: T-cell specific deregulation of Notch3 in transgenicmice (N3-tg), induces the development of a T-cell acute lymphoblastic leukemia (T-ALL), sustained by the constitutive activation of NF-kB canonical pathway. Besides, Notch signalling modulation in bone-marrow stromal cells or in hematopoietic stem cells, has been related to alterations in differentiation/proliferation processes of myeloid cells. To clarify the Notch/NF-kB relationships in the progression of T-ALL and the effects of a T-cell specific deregulation of Notch on myeloid compartment, we decided to delete NF-kB canonical pathway in N3-tg mice. Materials and methods: We generated N3-tg/p50-/- mice, deleted of the NF-kB/p50 subunit in a Notch3 transgenic background. The follow-up of double mutant versus N3-tg mice versus relative controls was conducted and immunophenotyping of hematopoietic cell subsets was performed at different age and in multiple tissues from the indicated animals by flow-cytometry tecniques. Total RNA and protein extract samples, derived from sorted T- or myeloid-cells of our mice models, were processed for RT-qPCR and Western blotting analysis, respectively, to test the expression of Notch-related molecules. Results: The progression of T-ALL, as defined by the peripheral expansion of immature CD4+ CD8+ T cells, was strongly inhibited in N3-tg/p50-/- versus N3-tg mice. However, the double mutant mice succumb earlier than N3-tg counterparts displaying a dramatic increase of Mac1+ Gr1+ myeloid cells in both spleen and blood, as well as of granulocyte/monocyte progenitors in the bone marrow. The expansion of myeloid subsets was detectable at a lower extent also in N3-tg versus wild-type mice. Preliminary data indicate that Mac1+ Gr1+ cells do not express Notch3, suggesting that this receptor may influence the equilibrium of the myeloid compartment mainly in trans, possibly through its interaction with the Jagged-1 ligand. Conclusions: Our results suggest that the NF-kB canonical pathway deletion inhibits the T-ALL progression, thus unveiling the influence of Notch signalling modulation on the behaviour of myeloid cells. We provide a useful model to extend our understanding of Notch/NF-kB interplay in driving the relationships between lymphoid and myeloid compartments in the context of hematological malignancy

Optimization of the isolation and expansion method of human mediastinal–adipose tissue derived mesenchymal stem cells with virally inactivated GMP-grade platelet lysate

Mesenchymal stem cells (MSCs) are adult multipotent cells currently employed in several clinical trials due to their immunomodulating, angiogenic and repairing features. The adipose tissue is certainly considered an eligible source of MSCs. Recently, putative adipose tissue derived MSCs (ADMSCs) have been isolated from the mediastinal depots. However, very little is known about the properties, the function and the potential of human mediastinal ADMSCs (hmADMSCs). However, the lack of standardized methodologies to culture ADMSCs prevents comparison across. Herein for the first time, we report a detailed step by step description to optimize the isolation and the expansion methodology of hmADMSCs using a virally inactivated good manufacturing practice (GMP)-grade platelet lysate, highlighting the critical aspects of the procedure and providing useful troubleshooting suggestions. Our approach offers a reproducible system which could provide standardization across laboratories. Moreover, our system is time and cost effective, and it can provide a reproducible source of adipose stem cells to enable future studies to unravel new insights regard this promising stem cell population

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon's fibroblasts

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon's fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2-3 cm(2) undamaged Tenon's biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon's fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon's and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes

The Outcome of Notch3-dependent T Cell Leukemia Is Modified By NF-kappaB Deletion

Background: The Notch3 deregulation inside T-cell compartment of transgenic (N3-tg) mice, induces an aggressive form of T-cell acute lymphoblastic leukemia (T-ALL), strongly sustained by an NF-κB constitutive activation, mainly represented by the p50/p65-dependent canonical pathway. To clarify the Notch/NF-κB relationships in the onset/progression of T-ALL, we decided to inhibit NF-κB canonical pathway in N3-tg mice. Methods: We generated N3-tg/p50-/- mice, deleted of the NF-κB/p50 subunit in a Notch3 transgenic background. The follow-up of N3-tg/p50-/- versus N3- tg mice was conducted and hematopoietic cell analysis was performed at different ages and in multiple tissues from the indicated animals by flow-cytometry techniques. Results: The p50 deletion inhibited the progression of T-ALL in N3-tg/p50-/- mice, as defined primarily by the peripheral expansion of immature CD4+CD8+ T cells. Surprisingly, the double mutant mice succumb earlier than N3-tg counterparts. Moribund N3-tg/p50-/- mice display the trait of a myeloproliferative disease, with the dramatic expansion of Mac1+Gr1+ myeloid cells in both spleen and blood, as well as of granulocyte/monocyte progenitors in the bone marrow. Preliminary data indicate that these cells do not express Notch3, suggesting that in the absence of p50 expression, Notch3 is able to mainly influence the equilibrium of the myeloid compartment in trans. Conclusions: The results presented suggest that the ablation of NF-κB canonical pathway may strongly impact on the outcomes of a T cell specific deregulation of Notch signaling. Thus, providing a useful experimental model to extend our understanding of Notch/NF-κB interplay and to unravel novel strategies for the therapy of different hematological malignancies

Deletion of NF-kappa/p50 influences the outcome of Notch3-dependent T cell leukemia

The deregulation of Notch3 signaling inside T-cell compartment of transgenic (N3-tg) mice, induces an aggressive form of T-cell acute lymphoblastic lymphoma (T-ALL), sustained by a constitutive activation of the NF-B canonical pathway, mainly represented by the p50/p65 heterodimer. To clarify the Notch/NF-B relationships in the development of T-ALL, we generated double mutant mice, deleted of the NF-B/p50 subunit in a Notch3 transgenic background (N3-tg/p50-/-). The follow-up of N3-tg/p50-/- versus N3-tg mice revealed that p50 deletion strongly inhibits the development of Notch3-dependent T-ALL. Surprisingly, double mutant succumb earlier than N3-tg mice, displaying the trait of a myeloproliferative disease, with an aberrant accumulation of Mac1+Gr1+ myeloid cells in both spleen and peripheral blood, as well as of granulocyte/monocyte progenitors in the bone marrow. Our preliminary results suggest that Notch3 overexpression in T-cell compartment is able to influence, possibly in trans, the equilibrium of the myeloid compartment and that the ablation of NF-B canonical pathway may impact on the outcomes of a T-cell specific deregulation of Notch signaling. We provide a useful experimental model to extend our understanding of Notch/NF-B interplay in hemopoietic system and to unravel novel relationships between lymphoid and myeloid differentiation