β-cell engineering from human pancreatic duct-derived cells

Pancreatic epithelial cells represent an attractive cell source for replacement therapy of type 1 diabetes. In this context, we designed a protocol for expansion of human pancreatic duct-derived cells (HDDCs) and showed their β-cell engineering potential. We reprogrammed HDDCs into β-cell-like lineage by using two different strategies: cocktails of molecules that recapitulate the embryonic β-cell development and the mRNA-based overexpression of key pancreatic transcription factors (TFs). This second approach showed higher efficiency leading to a mature β-cell phenotype in vitro only after MAFA overexpression within 7 days. Glucose-responsive insulin secretion was detected in both in vitro and in vivo after transplantation in immunosuppressed diabetic mice. Although further studies are still required to observe long-term function and side effects after transplantation, this study suggests the role of HDDCs as promising candidates for diabetes cell therapy.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

New Insights into Diabetes Cell Therapy

Since insulin discovery, islet transplantation was the first protocol to show the possibility to cure patients with type 1 diabetes using low-risk procedures. The scarcity of pancreas donors triggered a burst of studies focused on the production of new β cells in vitro. These were rapidly dominated by pluripotent stem cells (PSCs) demonstrating diabetes-reversal potential in diabetic mice. Subsequent enthusiasm fostered a clinical trial with immunoisolated embryonic-derived pancreatic progenitors. Yet safety is the Achilles' heel of PSCs, and a whole branch of β cell engineering medicine focuses on transdifferentiation of adult pancreatic cells. New data showed the possibility to chemically stimulate acinar or α cells to undergo β cell neogenesis and provide opportunities to intervene in situ without the need for a transplant, at least after weighing benefits against systemic adverse effects. The current studies suggested the pancreas as a reservoir of facultative progenitors (e.g., in the duct lining) could be exploited ex vivo for expansion and β cell differentiation in timely fashion and without the hurdles of PSC use. Diabetes cell therapy is thus a growing field not only with great potential but also with many pitfalls to overcome for becoming fully envisioned as a competitor to the current treatment standards

B-cell replacement sources for type 1 diabetes: a focus on pancreatic ductal cells

Thorough research on the capacity of human islet transplantation to cure type 1 diabetes led to the achievement of 3- to 5-year-long insulin independence in nearly half of transplanted patients. Yet, translation of this technique to clinical routine is limited by organ shortage and the need for long-term immunosuppression, restricting its use to adults with unstable disease. The production of new bona fide β cells in vitro was thus investigated and finally achieved with human pluripotent stem cells (PSCs). Besides ethical concerns about the use of human embryos, studies are now evaluating the possibility of circumventing the spontaneous tumor formation associated with transplantation of PSCs. These issues fueled the search for cell candidates for β-cell engineering with safe profiles for clinical translation. In vivo studies revealed the regeneration capacity of the exocrine pancreas after injury that depends at least partially on facultative progenitors in the ductal compartment. These stimulated subpopulations of pancreatic ductal cells (PDCs) underwent β-cell transdifferentiation through reactivation of embryonic signaling pathways. In vitro models for expansion and differentiation of purified PDCs toward insulin-producing cells were described using cocktails of growth factors, extracellular-matrix proteins and transcription factor overexpression. In this review, we will describe the latest findings in pancreatic β-cell mass regeneration due to adult ductal progenitor cells. We will further describe recent advances in human PDC transdifferentiation to insulin-producing cells with potential for clinical translational studies

Signaling pathway-focused gene expression profiling in pressure overloaded hearts

The β-blocker propranolol displays antihypertrophic and antifibrotic properties in the heart subjected to pressure overload. Yet the underlying mechanisms responsible for these important effects remain to be completely understood. The purpose of this study was to determine signaling pathway-focused gene expression profile associated with the antihypertrophic action of propranolol in pressure overloaded hearts. To address this question, a focused real-time PCR array was used to screen left ventricular RNA expression of 84 gene transcripts representative of 18 different signaling pathways in C57BL/6 mice subjected to transverse aortic constriction (TAC) or sham surgery. On the surgery day, mice received either propranolol (80 mg/kg/day) or vehicle for 14 days. TAC caused a 49% increase in the left ventricular weight-to-body weight (LVW/BW) ratio without changing gene expression. Propranolol blunted LVW/BW ratio increase by approximately 50% while causing about a 3-fold increase in the expression of two genes, namely Brca1 and Cdkn2a, belonging to the TGF-beta and estrogen pathways, respectively. In conclusion, after 2 weeks of pressure overload, TAC hearts show a gene expression profile superimposable to that of sham hearts. Conversely, propranolol treatment is associated with an increased expression of genes which negatively regulate cell cycle progression. It remains to be established whether a mechanistic link between gene expression changes and the antihypertrophic action of propranolol occurs

Liver progenitor cells expressing HLA-E and method for obtaining the latter

The current invention concerns isolated liver progenitor cells, cell lines thereof, cell populations comprising such and compositions comprising such wherein the liver progenitor cells are HLA-E positive. In addition, the invention concerns a method of preparing these liver progenitor cells, wherein the method comprises the step of adding one or more cytokines to the cell medium of a culture of liver progenitor or stem cells

β-Cell Differentiation of Human Pancreatic Duct-Derived Cells After In Vitro Expansion

β-Cell replacement therapy is a promising field of research that is currently evaluating new sources of cells for clinical use. Pancreatic epithelial cells are potent candidates for β-cell engineering, but their large-scale expansion has not been evidenced yet. Here we describe the efficient expansion and β-cell differentiation of purified human pancreatic duct cells (DCs). When cultured in endothelial growth-promoting media, purified CA19-9+ cells proliferated extensively and achieved up to 22 population doublings over nine passages. While proliferating, human pancreatic duct-derived cells (HDDCs) downregulated most DC markers, but they retained low CK19 and SOX9 gene expression. HDDCs acquired mesenchymal features but differed from fibroblasts or pancreatic stromal cells. Coexpression of duct and mesenchymal markers suggested that HDDCs were derived from DCs via a partial epithelial-to-mesenchymal transition (EMT). This was supported by the blockade of HDDC appearance in CA19-9+ cell cultures after incubation with the EMT inhibitor A83-01. After a differentiation protocol mimicking pancreatic development, HDDC populations contained about 2% of immature insulin-producing cells and showed glucose-unresponsive insulin secretion. Downregulation of the mesenchymal phenotype improved β-cell gene expression profile of differentiated HDDCs without affecting insulin protein expression and secretion. We show that pancreatic ducts represent a new source for engineering large amounts of β-like-cells with potential for treating diabetes

Liver progenitor cells expressing HLA-G and method for obtaining these cells

The current invention concerns isolated liver progenitor cells, cell lines thereof, cell populations comprising such and compositions comprising such wherein the liver progenitor cells are HLA-E positive. In addition, the invention concerns a method of preparing these liver progenitor cells, wherein the method comprises the step of adding one or more cytokines to the cell medium of a culture of liver progenitor or stem cell

RNA-based MAFA over-expression is sufficient to drive human pancreatic duct-derived cells toward a B-cell-like phenotype

RNA-based MAFA over-expression is sufficient to drive human pancreatic duct-derived cells toward a B-cell-like phenotype Elisa Corritore1, Erica Dugnani2, Valentina Pasquale2, Lorenzo Piemonti2, A. Vetere3,Susan Bonner-Weir4, Etienne M. Sokal1, Philippe A. Lysy1,5 1Institut de Recherche Expérimentale et Clinique, Pediatric Research Laboratory,Université Catholique de Louvain, Brussels, Belgium; 2San Raffaele Research Institute, Milan, Italy; 3Chemical Biology Program, Broad Institute of Harvard and MIT, Cambridge, USA ; 4Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Harvard Medical School, Boston, USA ; 5Pediatric Endocrinology Unit, Cliniques Universitaires Saint Luc, Université Catholique de Louvain, Brussels, Belgium Pancreatic epithelial cells represent an attractive cell source for replacement therapy of type 1 diabetes. Previously, we designed a protocol for expansion of human pancreatic duct-derived cells (HDDCs) and showed their β-cell engineering potential. In this study, we reprogrammed HDDCs into β-cell-like lineage by over-expressing mRNAs of key pancreatic transcription factors (TFs). Pancreatic duct cells (n=6) were purified and propagated into endothelial growth-promoting media. Synthetic modified (sm) RNAs were manufactured by unidirectional subcloning of PDX1, NGN3 and MAFA into a plasmid containing 5’ and 3’ UTR regions. The UTR-flanked inserts were excised and poly(A)-tailed. The final smRNAs were synthesized through in vitro transcription followed by phosphatase and DNase treatments, before being daily transfected in HDDCs. In all donors, transfection of PDX1, NGN3 and MAFA led to upregulation of endogenous target (ex: NGN3) and β-cell marker (ex: INS, synaptophysin, SLC2A2, GCK) genes with the highest expression levels being reached after MAFA transfection. Co-transfection protocols failed to show significant improvement of β-cell differentiation. Acceptable impact on innate immune response and cell viability was noticed after 7 consecutive daily smRNA transfections, based respectively on minimal IFNA and RIG-1 gene expression and on annexin-V/PI staining. After MAFA transfection, HDDCs stained positive for MAFA and insulin (19.3 ± 3.3 %) proteins, while ELISA assays showed detectable amounts of C-peptide content and release (21.45 ± 2.42 pg/mL/106 cells) under basal conditions. In conclusion, we showed that MAFA RNA over-expression is sufficient to efficiently reprogram HDDCs toward β-cell-like phenotype in a timely manner. Further research is mandatory to demonstrate a controlled insulin secretion capacity after differentiation

V-Maf Musculoaponeurotic Fibrosarcoma Oncogene Homolog A Synthetic Modified mRNA Drives Reprogramming of Human Pancreatic Duct-Derived Cells Into Insulin-Secreting Cells.

: β-cell replacement therapy represents the most promising approach to restore β-cell mass and glucose homeostasis in patients with type 1 diabetes. Safety and ethical issues associated with pluripotent stem cells stimulated the search for adult progenitor cells with endocrine differentiation capacities. We have already described a model for expansion and differentiation of human pancreatic duct-derived cells (HDDCs) into insulin-producing cells. Here we show an innovative and robust in vitro system for large-scale production of β-like cells from HDDCs using a nonintegrative RNA-based reprogramming technique. Synthetic modified RNAs for pancreatic transcription factors (pancreatic duodenal homeobox 1, neurogenin3, and V-Maf musculoaponeurotic fibrosarcoma oncogene homolog A [MAFA]) were manufactured and daily transfected in HDDCs without strongly affecting immune response and cell viability. MAFA overexpression was efficient and sufficient to induce β-cell differentiation of HDDCs, which acquired a broad repertoire of mature β-cell markers while downregulating characteristic epithelial-mesenchymal transition markers. Within 7 days, MAFA-reprogrammed HDDC populations contained 37% of insulin(+) cells and a proportion of endocrine cells expressing somatostatin and pancreatic polypeptide. Ultrastructure analysis of differentiated HDDCs showed both immature and mature insulin granules with light-backscattering properties. Furthermore, in vitro HDDC-derived β cells (called β-HDDCs) secreted human insulin and C-peptide in response to glucose, KCl, 3-isobutyl-1-methylxanthine, and tolbutamide stimulation. Transplantation of β-HDDCs into diabetic SCID-beige mice confirmed their functional glucose-responsive insulin secretion and their capacity to mitigate hyperglycemia. Our data describe a new, reliable, and fast procedure in adult human pancreatic cells to generate clinically relevant amounts of new β cells with potential to reverse diabetes