Microporous Scaffolds Drive Assembly and Maturation of Progenitors into β-Cell Clusters

AbstractHuman pluripotent stem cells (hPSCs) represent a promising cell source for the development of β-cells for use in therapies for type 1 diabetes. Current culture approaches provide the signals to drive differentiation towards β-cells, with the cells spontaneously assembling into clusters. Herein, we adapted the current culture systems to cells seeded on microporous biomaterials, with the hypothesis that the pores can guide the assembly into β-cell clusters of defined size that can enhance maturation. The microporous scaffold culture allows hPSC-derived pancreatic progenitors to form clusters at a consistent size as cells undergo differentiation to immature β-cells. By modulating the scaffold pore sizes, we observed 250-425 µm pore size scaffolds significantly enhance insulin expression and key β-cell maturation markers compared to suspension cultures. Furthermore, when compared to suspension cultures, the scaffold culture showed increased insulin secretion in response to glucose stimulus indicating the development of functional β-cells. In addition, scaffolds facilitated cell-cell interactions enabled by the scaffold design and cell-mediated matrix deposition of extracellular matrix (ECM) proteins associated with the basement membrane of islet cells. We further investigated the influence of ECM on cell development by incorporating an ECM matrix on the scaffold prior to cell seeding; however, their presence did not further enhance maturation. These results suggest the microporous scaffold culture facilitates 3D cluster formation, supports cell-cell interactions, and provides a matrix similar to a basement membrane to drive in vitro hPSC-derived β-cell maturation and demonstrates the feasibility of these scaffolds as a biomanufacturing platform.

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Increase in β-Cell Mass in Transplanted Porcine Neonatal Pancreatic Cell Clusters Is Due to Proliferation of β-Cells and Differentiation of Duct Cells*

Endocrinology ◽

10.1210/endo.142.5.8162 ◽

2001 ◽

Vol 142 (5) ◽

pp. 2115-2122 ◽

Cited By ~ 35

Author(s):

N. Trivedi ◽

J. Hollister-Lock ◽

M. D. Lopez-Avalos ◽

J. J. O’Neil ◽

M. Keegan ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Mass ◽

Insulin Content ◽

Double Immunostaining ◽

Β Cells ◽

Β Cell ◽

Pancreatic Cell ◽

Cell Clusters ◽

Duct Cells ◽

Neonatal Pig

Abstract A 20-fold increase in β-cell mass has been found after transplantation of porcine neonatal pancreatic cell clusters (NPCCs). Here the mechanisms leading to this increased β-cell mass were studied. NPCCs (4000 islet equivalents) generated after 8 days culture of digested neonatal pig pancreas were transplanted beneath the renal capsule of streptozotocin (STZ) diabetic and normoglycemic nude mice. Grafts were removed at 10 days, 6 weeks, and 20 weeks after transplantation for immunostaining and insulin content. Proliferation of β-cells and duct cells was assessed morphometrically using double immunostaining for Ki-67 with insulin or cytokeratin 7 (CK7). Graft maturation was assessed with double immunostaining of CK7 and insulin. Apoptosis was determined using propidium iodide staining. β-cell proliferation in NPCCs was higher after 8 days of culture compared with that found in neonatal pig pancreas. After transplantation, β-cell proliferation remained high at 10 days, decreased somewhat at 6 weeks, and was much lower 20 weeks after transplantation. Diabetic recipients not cured at 6 weeks after transplantation had significantly higherβ -cell proliferation compared with those cured and to normoglycemic recipients. The size of individual β-cells, as determined by cross-sectional area, increased as the grafts matured. Graft insulin content was 20-fold increased at 20 weeks after transplantation compared with 8 days cultured NPCCs. The proliferation index of duct cells was significantly higher in neonatal pig pancreas than in 8 days cultured NPCCs and in 10-day-old grafts. The incidence of apoptosis in duct cells appeared to be low. About 20% of duct cells 10 days post transplantation showed costaining for CK7 and insulin, a marker of protodifferentiation. In conclusion, the increase in β-cell mass after transplantation of NPCCs is due to both proliferation of differentiated β-cells and differentiation of duct cells intoβ -cells.

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Corrigendum to “Dexamethasone suppresses the expansion and transdifferentiation of transplanted porcine neonatal pancreas cell clusters (NPCCs) into β-cell in normal nude mice” [Diabetes Res. Clin. Pract. 66S (2004) S97–S101]

Diabetes Research and Clinical Practice ◽

10.1016/j.diabres.2005.04.001 ◽

2005 ◽

Vol 69 (2) ◽

pp. 207

Author(s):

S.-H. Ko ◽

H.-S. Kwon ◽

S.-H. Suh ◽

J.-H. Yang ◽

S.-R. Kim ◽

...

Keyword(s):

Nude Mice ◽

Β Cell ◽

Cell Clusters ◽

Pancreas Cell

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Accelerated Functional Maturation of Isolated Neonatal Porcine Cell Clusters: In Vitro and In Vivo Results in NOD Mice

Cell Transplantation ◽

10.3727/000000005783983034 ◽

2005 ◽

Vol 14 (5) ◽

pp. 249-261 ◽

Cited By ~ 35

Author(s):

Giovanni Luca ◽

Claudio Nastruzzi ◽

Mario Calvitti ◽

Ennio Becchetti ◽

Tiziano Baroni ◽

...

Keyword(s):

Sertoli Cells ◽

Time Lag ◽

Cell Mass ◽

Nod Mice ◽

Β Cell ◽

Cell Clusters ◽

Porcine Islets ◽

Neonatal Porcine Islets

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the β-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from <3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in β-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional β-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.

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Dimensionality and Calcium Dynamics in Pancreatic β-Cell Clusters

Biophysical Journal ◽

10.1016/j.bpj.2012.11.2710 ◽

2013 ◽

Vol 104 (2) ◽

pp. 491a-492a

Author(s):

Thomas H. Hraha ◽

Abigail B. Bernard ◽

Kristi S. Anseth ◽

Richard K.P. Benninger

Keyword(s):

Calcium Dynamics ◽

Pancreatic Β Cell ◽

Β Cell ◽

Cell Clusters

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Comparison of Size, Viability, and Function of Fetal Pig Islet-Like Cell Clusters after Digestion Using Collagenase or Liberase

Cell Transplantation ◽

10.3727/000000002783985477 ◽

2002 ◽

Vol 11 (6) ◽

pp. 539-545 ◽

Cited By ~ 22

Author(s):

Pauline Georges ◽

Roslyn P. Muirhead ◽

Lindy Williams ◽

Sara Holman ◽

Muhammad Tani Tabiin ◽

...

Keyword(s):

Gestational Age ◽

Renal Capsule ◽

Β Cell ◽

Large White ◽

Cell Clusters ◽

Fluorescent Markers ◽

And Function ◽

Fetal Pig ◽

Methods Of Isolation

Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 ± 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 ± 2 vs. 31 ± 2 min). The size of ICCs was similar for both collagenase (83 ± 0.5 μm) and Liberase (79 ± 0.4 μm) as was the number of ICCs produced per pancreas (7653 ± 1297 vs. 8101 ± 1177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 ± 1% vs. 76 ± 1%, p < 0.05). Responsiveness to β-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at 1 month after transplantation of 1500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.

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Decrease in Ins+Glut2LO β-cells with advancing age in mouse and human pancreas

Journal of Endocrinology ◽

10.1530/joe-16-0475 ◽

2017 ◽

Vol 233 (3) ◽

pp. 229-241 ◽

Cited By ~ 3

Author(s):

Christine A Beamish ◽

Sofia Mehta ◽

Brenda J Strutt ◽

Subrata Chakrabarti ◽

Manami Hara ◽

...

Keyword(s):

Glucose Transporter ◽

Cell Mass ◽

Positive Association ◽

Human Islets ◽

Human Pancreas ◽

Fetal Life ◽

Β Cells ◽

Β Cell ◽

Cell Clusters ◽

Glucose Transporter 2

The presence and location of resident pancreatic β-cell progenitors is controversial. A subpopulation of insulin-expressing but glucose transporter-2-low (Ins+Glut2LO) cells may represent multipotent pancreatic progenitors in adult mouse and in human islets, and they are enriched in small, extra-islet β-cell clusters (<5 β cells) in mice. Here, we sought to identify and compare the ontogeny of these cells in mouse and human pancreata throughout life. Mouse pancreata were collected at postnatal days 7, 14, 21, 28, and at 3, 6, 12, and 18 months of age, and in the first 28 days after β-cell mass depletion following streptozotocin (STZ) administration. Samples of human pancreas were examined during fetal life (22–30 weeks gestation), infancy (0–1 year), childhood (2–9), adolescence (10–17), and adulthood (18–80). Tissues were analyzed by immunohistochemistry for the expression and location of insulin, GLUT2 and Ki67. The proportion of β cells within clusters relative to that in islets was higher in pancreas of human than of mouse at all ages examined, and decreased significantly at adolescence. In mice, the total number of Ins+Glut2LO cells decreased after 7 days concurrent with the proportion of clusters. These cells were more abundant in clusters than in islets in both species. A positive association existed between the appearance of new β cells after the STZ treatment of young mice, particularly in clusters and smaller islets, and an increased proportional presence of Ins+Glut2LO cells during early β-cell regeneration. These data suggest that Ins+Glut2LO cells are preferentially located within β-cell clusters throughout life in pancreas of mouse and human, and may represent a source of β-cell plasticity.

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Single-Cell Transcriptional Profiling of Mouse Islets Following Short-Term Obesogenic Dietary Intervention

Metabolites ◽

10.3390/metabo10120513 ◽

2020 ◽

Vol 10 (12) ◽

pp. 513

Author(s):

Annie R. Piñeros ◽

Hongyu Gao ◽

Wenting Wu ◽

Yunlong Liu ◽

Sarah A. Tersey ◽

...

Keyword(s):

Insulin Resistance ◽

Insulin Secretion ◽

Single Cell ◽

Cell Types ◽

Endoplasmic Reticulum Stress Response ◽

High Fat ◽

Β Cells ◽

Β Cell ◽

Cell Clusters ◽

Ductal Cells

Obesity is closely associated with adipose tissue inflammation and insulin resistance. Dysglycemia and type 2 diabetes results when islet β cells fail to maintain appropriate insulin secretion in the face of insulin resistance. To clarify the early transcriptional events leading to β-cell failure in the setting of obesity, we fed male C57BL/6J mice an obesogenic, high-fat diet (60% kcal from fat) or a control diet (10% kcal from fat) for one week, and islets from these mice (from four high-fat- and three control-fed mice) were subjected to single-cell RNA sequencing (sc-RNAseq) analysis. Islet endocrine cell types (α cells, β cells, δ cells, PP cells) and other resident cell types (macrophages, T cells) were annotated by transcript profiles and visualized using Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) plots. UMAP analysis revealed distinct cell clusters (11 for β cells, 5 for α cells, 3 for δ cells, PP cells, ductal cells, endothelial cells), emphasizing the heterogeneity of cell populations in the islet. Collectively, the clusters containing the majority of β cells showed the fewest gene expression changes, whereas clusters harboring the minority of β cells showed the most changes. We identified that distinct β-cell clusters downregulate genes associated with the endoplasmic reticulum stress response and upregulate genes associated with insulin secretion, whereas others upregulate genes that impair insulin secretion, cell proliferation, and cell survival. Moreover, all β-cell clusters negatively regulate genes associated with immune response activation. Glucagon-producing α cells exhibited patterns similar to β cells but, again, in clusters containing the minority of α cells. Our data indicate that an early transcriptional response in islets to an obesogenic diet reflects an attempt by distinct populations of β cells to augment or impair cellular function and/or reduce inflammatory responses as possible harbingers of ensuing insulin resistance.

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Patterning of Mono- and Multilayered Pancreatic β-Cell Clusters

Langmuir ◽

10.1021/la1004424 ◽

2010 ◽

Vol 26 (12) ◽

pp. 9943-9949 ◽

Cited By ~ 19

Author(s):

Adam D. Mendelsohn ◽

Daniel A. Bernards ◽

Rachel D. Lowe ◽

Tejal A. Desai

Keyword(s):

Pancreatic Β Cell ◽

Β Cell ◽

Cell Clusters

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