Culturing functional pancreatic islets on α5-laminins and curative transplantation to diabetic mice

Sigmundsson K., Ojala J.R.M., Öhman M.K. Österholm A-M., Moreno-Moral A., Domogatskaya A., Yen Chong L., Sun Y., Chai X., Steele J.A.M, George B., Patarroyo M., Nilsson A-S., Rodin S., Ghosh S., Stevens M.M., Petretto E., Tryggvason K.
Matrix Biology, 2018

Here, the authors have developed a novel method to grow and maintain normoxic and functional islets which may significantly enhance the efficacy of islet transplantation treatment for diabetes. A key component of this method is the coating with biologically relevant laminins, found in the peri-islet capsule and BM of islet capillaries. Islets cultured in vitro on α5-laminins adhere and spread to form layers of 1-3 cells in thickness while maintaining cell-to-cell contacts. The cells remained normoxic and functional for at least 7 days in culture. In contrast, spherical islets kept in suspension developed hypoxia and central necrosis within 16 hours. Mouse islets plated on laminin-521 could be cultured in a serum-free mTeSR1 medium for an extended period of time. Approximately 20% of islet cells showed a co-expression of insulin and glucagon. The double-hormone expression was confirmed in histological analyses of mouse and monkey pancreata. The flattened islets start robust cell proliferation after a lag period of approximately two weeks in a serum-free mTeSR1 culture on laminin-521. Transplantation mouse islets cultured on α5-laminin-coated polydimethylsiloxane membranes for 3–7 days normalized blood glucose already within 3 days in mice with streptozotocin-induced diabetes.


Key matrix proteins within the pancreatic islet basement membrane are differentially digested during human islet isolation

Cross S.E., Vaughan R.H., Willcox A.J., McBride A.J., Abraham A.A., Han B., Johnson J.D., Maillard E., Bateman P.A., Ramracheya R.D., Rorsman P., Kadler K.E., Dunne M.J., Hughes S.J., Johnson P.R.V.
Am J Transplant. 2016

Here the authors investigated the impact of islet isolation on basement membrane (BM) integrity in human islet. Collagen IV, panlaminin, perlecan and laminin-α5 in the islet BM were significantly digested by enzyme treatment. Laminin-α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Islet function and survival decreased and islet cytotoxicity increased during culture. The islet basement membrane (BM) influences islet function and survival and an incomplete islet BM has implications for islet integrity and transplanted graft longevity.


Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells

Qu H., Liu X., Ni Y., Jiang Y., Feng X., Xiao J., Guo Y., Kong D., Li A., Li X., Zhuang X., Wang Z., Wang Y., Chang Y., Chen S., Kong F., Zhang X., Zhao S., Sun Y., Xu D., Wang D., Zheng C.
Journal of Translational Medicine, 2014

Efficient induction of differentiation to insulin-producing cells from MSCs. Up-regulated insulin expression at both mRNA and protein levels. Administration of the insulin producing cells in T1 diabetes rats rapidly 1) down-regulated fasting blood glucose levels, 2) significantly reduced the HbA1c concentration and 3) markedly improved the symptoms and survival of the rats.


The Vascular Basement Membrane: A Niche for Insulin Gene Expression and B Cell Proliferation

Nikolova G., Jabs N., Konstantinova I., Domogatskaya A., Tryggvason K., Sorokin L., Fässler R., Gu G., Gerber H-P., Ferrara N., Melton D.A., Lammert E.
Developmental Cell, 2006

Mouse pancreatic islets intimately interact with endothelial cells and differentiation and delamination of insulin-producing b cells from pancreatic epithelium strictly require endothelial cells. Doug Melton and colleagues show that BMs within islets is formed and found exclusively around capillaries but not islet cells. Islet endothelial cells express laminin a4 and a5. Laminins promote insulin gene expression and proliferation in B-cells and B1-intergrin is required for this laminin response. Laminin-411 and -511 worked well but also laminin-111 which shows that the applied laminin does not necessarily have to be endothelial cell-derived. Research on islet transplantation has shown that it takes about 1–2 weeks for transplanted islets to become revascularized in the host and the authors suggest that treating islets with these laminins prior to transplantation will help maintain insulin production until new capillaries are formed in transplanted islets.


Co-culture with extracellular matrix proteins reduces hypoxia-induced human islet cell death

Brandhorst et al., Xenotransplantation, Abstracts of the IPITA-IXA-CTS 2015 Joint Congress November 15–19, 2015, Melbourne, Australia

Islets are experiencing hypoxic conditions after transplantation. The aim of this study was to assess the effect of collagen IV and laminin isoform -521, -511 and -411 on survival and function of isolated human islets exposed to severe hypoxia. Compared with hypoxic controls (100%) all ECMs significantly increased islet recovery after culture at 0.75% oxygen ranging from 163 +/- 12% to 173 +/- 28% (P < 0.05) using collagen IV or laminin-411, respectively. Increased post-culture recovery correlated with decreased islet fragmentation which was lowest using laminin-521 (66%, P < 0.01) and laminin-511 (66% P < 0.05). Islet ROS generation was also lowest after culture with laminin-521 and laminin-511. Islet viability was increased in all experimental groups when compared to controls but was highest using collagen IV and laminin-511. This observation corresponds to the insulin response after glucose challenge that was best preserved when collagen IV or laminin-511 were used for islet incubation.


Human Laminin Isotype Coating for Creating Islet Cell Sheets

Yamashita S., Ohashi K., Utoh R., Okano T., Yamamoto M.
Cell Medicine, 2015

This study describe an experimental approach to create a monolayered islet cell construct (islet cell sheet), followed by transplantation into a subcutaneous pocket. The authors try to identify an optimal human ECM as a coating material on PIPAAm surfaces, which allowed rat islet cells to attach on the surfaces and subsequently to be harvested as a monolithic cell sheet. Dispersed rat islet cells were seeded onto PIPAAm dishes coated with various human laminin isotypes: LN211, LN332, LN411, LN511, and HL-placenta. The highest value of plating efficiency was found in the HL-332-PIPAAm group (83.1 ± 0.7%). The HL-332-PIPAAm group also showed the highest cellular confluency (98.6 ± 0.5%). Islet cells cultured on the HL-332-PIPAAm surfaces showed a positive response in the glucose-stimulated insulin secretion test. LN511 also showed good results.


Regulated laminin-332 expression in human islets of Langerhans

Armanet M., Wojtusciszyn A., Morel P., Parnaud G., Rousselle P., Sinigaglia C., Berney T., Bosco D.
The FASEB Journal, 2009

In this article, the authors show that LN-332 is expressed in isolated human islets with labeling confined to endocrine cells. Labeling in isolated islets is granular but don’t colocalize with hormone secretory granules. LN- 332 is most abundant in cultured islets compared to freshly isolated islets and is found in culture medium, which suggests that it is secreted by islets. When islets are exposed to interleukin (IL)-1B, expression and secretion of LN-332 increase compared to control. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited culture and IL-1B-induced LN-332 expression in islets. These results show that LN-332, known to have some beneficial effect on B-cells in vitro, is produced and secreted by endocrine islet cells and is up-regulated by stressing conditions such as culture and IL-1-exposure. Altogether, their observations can be considered as prerequisites necessary to hypothesize that the in vitro effect of LN-332 on B-cell secretion, viability, and replication are of physiological relevance as well.


Blockade of 1 Integrin–Laminin-5 Interaction Affects Spreading and Insulin Secretion of Rat Beta-Cells Attached on Extracellular Matrix

Parnaud G., Hammar E., Rouiller D.G., Armanet M., Halban P.A., Bosco D.
Diabetes, 2006

In this study, the authors show that outside-in signaling via engagement of B1 integrins by laminin-332 is an important component of normal B-cell function. By using specific blocking antibodies, the authors demonstrated that laminin-332 is the component present in 804G matrix (rat bladder carcinoma cell line), responsible for the effect of 804G matrix on B-cell function and spreading. Only the B1 integrin was detected in B-cells, a well-known laminin-332 ligand. Anti–B1 integrin antibody reduced the spreading of B-cells on 804G matrix. Blockade of the interaction between B1 integrins and laminin-332 resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti–B1 integrin antibody also inhibited focal adhesion kinase phosphorylation induced by 804G matrix. In conclusion, anti–B1 integrin and –laminin-332 antibodies interfere with spreading of B-cells, resulting in decreased insulin secretion in response to glucose.


Extracellular Matrix Protects Pancreatic Beta-Cells Against Apoptosis. Role of Short- and Long-Term Signaling Pathways

Hammar E., Parnaud G., Bosco D., Perriraz N., Maedler K., Donath M., Rouiller D.G., Halban P. A.
Diabetes, 2004

B-cells cultured on laminin-332 rich surfaces improve their functions. This study provides evidence for activation of signaling pathways and gene expression by laminin-332 leading to improved B-cell survival. Laminin-332 protect sorted rat B-cells against apoptosis under standard conditions (11.2 mmol/l glucose, 10% serum), after serum deprivation (1% serum), and in response to interleukin-1 (IL-1; 2 ng/ml), compared with the poly-L-lysine control. Caspase-8 activity was reduced in cells cultured on laminin-332, whereas FAK, Akt and ERK phosphorylation was augmented. Treatment with an anti-1 integrin antibody, with the ERK pathway inhibitor PD98059, and/or with the phosphatidylinositol 3-kinase inhibitor LY294002 augmented cell death on the laminin-332 rich matrix but not on pLL.


Importance of Cell-Matrix Interactions in Rat Islet Bets-Cell Secretion In Vitro. Role of a6B1 Integrin

Bosco D., Meda P., Halban P.A, Rouiller D.G.
Diabetes, 2000

The present work addresses the influence of short-term cell-matrix interactions on islet B-cell function and provides first insight into the molecular basis of these interactions. When primary rat B-cells were allowed to attach to a laminin-332 rich matrix (804G matrix), there was an increased insulin secretory response to secretagogues. This change was the result of an increase in the proportion of actively secreting B-cells and in the amount of insulin secreted per active cell. In turn, the spreading or flattening of B-cells on this matrix was enhanced by secretagogues, and flattened cells secreted more insulin than rounded cells. a6B1 integrins are present heterogeneous at the surface of islet cells in situ and is upregulated by insulin secretagogues. a6B1 expression is higher in spreading cells and anti-a6B1–specific antibodies decrease spreading. These observations demonstrate that islet cell–matrix interactions can improve the sensitivity of insulin cells to glucose and are mediated, at least in part, by a6B1 integrins, suggesting that outside-in signaling through a6B1 integrin plays a major role in the regulation of B-cell function.


Unique basement membrane structure of human pancreatic islets: implications for b-cell growth and differentiation

Otonkoski T., Banerjee M., Korsgren O., Thornell L.-E., Virtanen I.
Diabetes, Obesity and Metabolism, 2008

The authors shown that in the human islets, a double BM structure surrounding each blood vessel within the islet. In addition, a continuous peri-islet BM was surrounding the entire islet, invaginating into the islet tissue together with the arterioles. The capillaries are surrounded by a double BM both in foetal and adult tissues. The B-cells are facing a BM that is separate from the endothelia, unlike the situation in mouse where the B-cells interact directly with BMs of capillary endothelial cells. Here they show that (i) a1 is not expressed in the adult human pancreas; (ii) a2 is only expressed in the exocrine pancreas; (iii) a4 is expressed in the blood vessel BMs; (iv) a5 and b1 are expressed similarly, both in the endocrine and endothelial BMs in the islets. Taken together, this suggested that there is a double-layered BM organization around the vascular channels of human islets: the inner vascular leaflet of the duplex contains Lms-411/421 and -511/521 whereas the outer leaflet facing the parenchymal endocrine cells contains only Lm-511. In contrast to the adult pancreas, laminin a1 chain could be found in the acinar BMs of fetal pancreas and faintly also in the developing islets. Similarly, as in the adult pancreas, immunoreactivity for laminin a5 chain was distinctly surrounding the developing islets and also in BMs of intra-islet vessels. Strong expression of laminin B1 and g1 in fetal pancreas.  a3 and b1 integrin subunits were found both on the vascular channels and the endocrine cells. However, integrin a6 subunit was clearly absent from the endocrine cells and only expressed in the endothelial cells. The islet cells facing this BM have a strong and polarized expression of Lutheran glycoprotein, which is a well-known receptor for the laminin a5 chain. Dispersed human islet cells adhere to purified human laminin-511 and the binding is equally effectively blocked by a soluble form of Lutheran as by antibody against integrin B1. The results reveal that the BM structure of human islets, different from rodents.


Extracellular matrix microarrays to study inductive signaling for endoderm specification

Braga Malta D.F., Reticker-Flynn N.E., da Silva C.L.,. Cabral J.M.S, Fleming H.E., Zaret K.S., Bhatia S.N., Underhill G.H.
Acta Biomaterialia, 2016

The authors implemented an extracellular matrix (ECM) array platform that facilitates the study of 741 distinct combinations of 38 different ECM components in a systematic, unbiased and high throughput manner. Seeded definitive endoderm (DE) cells onto the arrays and evaluated cell adhesion and hepatic and pancreatic differentiation. When comparing the ECM conditions that best supported hepatic and pancreatic differentiation, we noted that two combinations (fibronectin + merosin (laminin α2) and laminin (α1) + superfibronectin) are among the most robust domains for both hepatic and pancreatic differentiation.


Blood vessels of human islets of Langerhans are surrounded by a double basement membrane

Virtanen I., Banerjee M., Palgi J., Korsgren O., Lukinius A., Thornell L.E., Kikkawa Y., Sekiguchi K., Hukkanen M., Konttinen Y.T., Otonkoski T.
Diabetologia. 2008

Immunohistochemistry revealed a unique organisation for human laminin-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained laminin-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the laminin alpha5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas alpha(3) and beta(1) integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in short-term culture of human islets. The adhesion of dispersed beta cells to laminin-511 was inhibited equally effectively by antibodies to integrin and alpha(3) and beta(1) subunits, and by soluble Lutheran peptide.


Localization of laminin alpha4-chain in developing and adult human tissues

Petäjäniemi N., Korhonen M., Kortesmaa J., Tryggvason K., Sekiguchi K., Fujiwara H., Sorokin L., Thornell L.E., Wondimu Z., Assefa D., Patarroyo M., Virtanen I.
J Histochem Cytochem., 2002

Recent studies suggest important functions for laminin-411 in vascular and blood cell biology. Western blotting techniques we show that it recognizes the human laminin alpha4-chain. Immunoreactivity for the laminin alpha4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the laminin alpha4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of laminin alpha4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Laminin alpha4- and laminin alpha2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced laminin alpha4-chain as M(r) 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum.