Human serum versus human serum albumin supplementation in human islet pretransplantation culture: in vitro and in vivo assessment

There is conflicting evidence favoring both the use of human serum (HS) and of human serum albumin (HSA) in human islet culture. We evaluated the effects of HS versus HSA supplementation on 1) in vitro β-cell viability and function and 2) in vivo islet graft revascularization, islet viability, β-cell death, and metabolic outcome after transplantation. Islets isolated from 14 cadaveric organ donors were cultured for 3 days in CMRL 1066 medium supplemented with HS or HSA. After 3 days in culture, β-cell apoptosis was lower in HS group (1.41 ± 0.27 vs. 2.38 ± 0.39%, p = 0.029), and the recovery of islets was 77 ± 11% and 54 ± 1% in HS- and HSA-cultured groups, respectively. Glucose-stimulated insulin secretion (GSIS) was higher in HS group (29.4, range 10.4-99.9, vs. 22.3, range 8.7-70.6, p = 0.031). In vivo viability and revascularization was determined in HS- and HSA-cultured islets transplanted into the anterior chamber of the eye of Balb/c mice (n = 14), and β-cell apoptosis in paraffin-embedded mouse eyes. Islet viability and β-cell apoptosis were similar in both groups. Revascularization was observed in one graft (HS group) on day 10 after transplantation. Islet function was determined in streptozotocin (STZ)-diabetic nude mice (n = 33) transplanted with 2,000 IEQs cultured with HS or HSA that showed similar blood glucose levels and percentage of normoglycemic animals over time. In conclusion, human islets cultured in medium supplemented with HS showed higher survival in vitro, as well as islet viability and function. The higher in vitro survival increased the number of islets available for transplantation. However, the beneficial effect on viability and function did not translate into an improved metabolic evolution when a similar number of HSA- and HS-cultured islets was transplanted

Epithelial To Mesenchymal Transition In Human Endocrine Islet Cells

BACKGROUND: β-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells. OBJECTIVE: To investigate whether EMT takes place in the endocrine non-β cells of human islets. METHODOLOGY: Human islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer. RESULTS: Co-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-β-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers. CONCLUSIONS: In adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a β-cell phenotype

Pancreatic ductal cells may have a negative effect on human islet transplantation.

AimTo evaluate the effect of pancreatic ductal cells on experimental human islet transplantation.Materials and methodsIsolated islets were additionally purified by handpicking. Ductal cells were purified by magnetic cell sorting and then clustered into ductal pancreatospheres (DPS). Islets, DPS, and islets + DPS (100 islets + 75 DPS, or 100 islets + 200 DPS) were cultured and glucose-stimulated insulin secretion, β-cell apoptosis, and gene expression was determined. Islets and islets + DPS preparations (800 islets + 600 DPS) were transplanted to streptozotocin-treated immunodeficient mice and glycemia, graft morphometry, and gene expression were determined.ResultsInsulin stimulation index was higher in islets than in islets co-cultured with DPS (5.59 ± 0.93 vs 4.02 ± 0.46; p0.05), and the ratio β-/endocrine non-β-cell mass was lower in islets + DPS grafts (islets: 2.05 ± 0.18, islets + DPS: 1.35 ± 0.15; pConclusionsIslet preparations enriched with ductal cells have a lower insulin stimulation index in vitro and achieved a worse metabolic outcome after transplantation. Inflammation may mediate the deleterious effects of ductal cells on islet cells

El modelo in vivo pez cebra para evaluar las propiedades probioticas de bacterias en acuicultura

La acuicultura es uno de los sectores de producción de alimentos de más rápido crecimiento con un máximo histórico de producción en 2012, proporcionando casi la mitad del pescado destinado a la alimentación humana. Una delas mayores limitaciones a la expansión de este sector son las pérdidas causadas por brotes infecciosos, generalmente oportunistas, provocados primordialmente por la presión medioambiental, el hacinamiento, etc. Durante las últimas décadas, se han utilizado sobretodo antibióticos para minimizar las pérdidas económicas causadas por enfermedades. Sin embargo, su uso no es aconsejable debido al riesgo potencial de transferir resistencias bacterianas desde el medio ambiente acuático al ser humano. Una alternativa es suplementar la dieta con probióticos que mejoren el bienestar general de los peces, aunque en la actualidad solo hay una cepa probiótica (Pediococcus acidilactici MA 18/5M; BACTOCELL ®) autorizada en la Unión Europea para su uso en acuicultura. El uso del modelo de pez cebra (Danio rerio) es una potente herramienta que puede ayudar a dilucidar las propiedades probióticas de un microrganismo in vivo. Durante los últimos años, hemos establecido varias colaboraciones con el objetivo de optimizar modelos in vivo, en larvas y adultos de pez cebra, para estudiar la capacidad colonizadora de bacterias en sistemas “germ-free”, su efecto inmunomodulador, mejora en la reproducción, capacidad anti-inflamatoria y capacidad protectora frente a una infección oportunista producida por el patógeno Vibrio anguillarum. En esta presentación se expondrán los principales resultados y logros alcanzados en el uso del modelo pez cebra para evaluar la capacidad probiótica de microorganismos

Pancreatic ductal cells may have a negative effect on human islet transplantation. PLoS One. 2019 Jul 19;14(7):e0220064.

Aim: To evaluate the effect of pancreatic ductal cells on experimental human islet transplantation. Materials and methods: Isolated islets were additionally purified by handpicking. Ductal cells were purified by magnetic cell sorting and then clustered into ductal pancreatospheres (DPS). Islets, DPS, and islets + DPS (100 islets + 75 DPS, or 100 islets + 200 DPS) were cultured and glucose-stimulated insulin secretion, β-cell apoptosis, and gene expression was determined. Islets and islets + DPS preparations (800 islets + 600 DPS) were transplanted to streptozotocin-treated immunodeficient mice and glycemia, graft morphometry, and gene expression were determined. Results: Insulin stimulation index was higher in islets than in islets co-cultured with DPS (5.59 ± 0.93 vs 4.02 ± 0.46; p0.05), and the ratio β-/endocrine non-β-cell mass was lower in islets + DPS grafts (islets: 2.05 ± 0.18, islets + DPS: 1.35 ± 0.15; p<0.01). IL1B and IL1RN expression was significantly higher in islets + DPS grafts. Conclusions: Islet preparations enriched with ductal cells have a lower insulin stimulation index in vitro and achieved a worse metabolic outcome after transplantation. Inflammation may mediate the deleterious effects of ductal cells on islet cells

Human serum versus human serum albumin supplementation in human islet pretransplantation culture: in vitro and in vivo assessment

There is conflicting evidence favoring both the use of human serum (HS) and of human serum albumin (HSA) in human islet culture. We evaluated the effects of HS versus HSA supplementation on 1) in vitro β-cell viability and function and 2) in vivo islet graft revascularization, islet viability, β-cell death, and metabolic outcome after transplantation. Islets isolated from 14 cadaveric organ donors were cultured for 3 days in CMRL 1066 medium supplemented with HS or HSA. After 3 days in culture, β-cell apoptosis was lower in HS group (1.41 ± 0.27 vs. 2.38 ± 0.39%, p = 0.029), and the recovery of islets was 77 ± 11% and 54 ± 1% in HS- and HSA-cultured groups, respectively. Glucose-stimulated insulin secretion (GSIS) was higher in HS group (29.4, range 10.4-99.9, vs. 22.3, range 8.7-70.6, p = 0.031). In vivo viability and revascularization was determined in HS- and HSA-cultured islets transplanted into the anterior chamber of the eye of Balb/c mice (n = 14), and β-cell apoptosis in paraffin-embedded mouse eyes. Islet viability and β-cell apoptosis were similar in both groups. Revascularization was observed in one graft (HS group) on day 10 after transplantation. Islet function was determined in streptozotocin (STZ)-diabetic nude mice (n = 33) transplanted with 2,000 IEQs cultured with HS or HSA that showed similar blood glucose levels and percentage of normoglycemic animals over time. In conclusion, human islets cultured in medium supplemented with HS showed higher survival in vitro, as well as islet viability and function. The higher in vitro survival increased the number of islets available for transplantation. However, the beneficial effect on viability and function did not translate into an improved metabolic evolution when a similar number of HSA- and HS-cultured islets was transplanted

Epithelial To Mesenchymal Transition In Human Endocrine Islet Cells

BACKGROUND: β-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells. OBJECTIVE: To investigate whether EMT takes place in the endocrine non-β cells of human islets. METHODOLOGY: Human islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer. RESULTS: Co-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-β-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers. CONCLUSIONS: In adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a β-cell phenotype

β-cell death.

<p>(A) β-cell death in passage 1 (days 0, 4, 8 and 12) and passage 2. A minimum of 1000 β-cells per sample was counted. Data are means ± SEM (n = 6). (B) Representative immunofluorescence image showing double staining for TUNEL (green) and insulin (red) on day 12. Nuclei are stained in blue with DAPI. Arrow indicates a TUNEL⁺/insulin⁺ β-cell. Scale bar = 50μm.</p

Changes in gene expression at passage 1.

<p>Gene expression analysis of the initial p1 period (days 0, 4, 8 and 12). Results are expressed as relative mRNA levels (RQ). Data are means ± SEM (n = 3–4 donors). * P< 0.05 and ** P< 0.01 vs day 0.</p