Fibrin supports human fetal islet-epithelial cell differentiation via p70s6k and promotes vascular formation during transplantation

Abstract

The human fetal pancreas expresses a variety of extracellular matrix (ECM) binding receptors known as integrins. A provisional ECM protein found in blood clots that can bind to integrin receptors and promote β cell function and survival is fibrin. However, its role in support of human fetal pancreatic cells is unknown. We investigated how fibrin promotes human fetal pancreatic cell differentiation in vitro and in vivo. Human fetal pancreata were collected from 15 to 21 weeks of gestation and collagenase digested. Cells were then plated on tissue-culture polystyrene, or with 2D or 3D fibrin gels up to 2 weeks, or subcutaneously transplanted in 3D fibrin gels. The human fetal pancreas contained rich ECM proteins and expressed integrin αVβ3. Fibrin-cultured human fetal pancreatic cells had significantly increased expression of PDX-1, glucagon, insulin, and VEGF-A, along with increased integrin αVβ3 and phosphorylated FAK and p70 s6k . Fibrin-cultured cells treated with rapamycin, the mTOR pathway inhibitor, had significantly decreased phospho-p70 s6k and PDX-1 expression. Transplanting fibrin-mixed cells into nude mice improved vascularization compared with collagen controls. These results suggest that fibrin supports islet cell differentiation via p70 s6k and promotes vascularization in human fetal islet-epithelial clusters in vivo. Islet transplantation for the treatment of diabetes is currently limited by a shortage of available donor pancreata. 1 To surpass this problem, generating β cells from progenitor cell sources is a viable option. 2 However, this procedure and others like it produce low numbers of effective, functional β cells. 2 A greater understanding of the factors, especially those in the microenvironment, that regulate pancreatic development and islet cell differentiation is required to significantly increase the yield of β cells that these procedures generate for islet transplantation.Human pancreatic development begins at day 26 post conception, with tubular structures protruding from the ventral and dorsal side of the foregut endoderm. Two transcription factors that are required for the initiation of pancreatic development are PTF-1A and PDX-1, 3 in which the latter also promotes β cell maturation and function. 4 By 8 weeks of age, SOX9 is expressed in the pool of pancreatic progenitors 5 and is essential to support their proliferation and survival as well as maintain a transcriptional network with other factors. 6 SOX9 is also an important regulator of pancreatic endocrine cell differentiation. 5 Insulin positivity emerges around 7.5 weeks post conception, followed by glucagon and somatostatin 1 week later. 7 Islet-like structures eventually form where cells commonly express more than one hormone. 7 Subsequently, clustering occurs leading to construction of the islet of Langerhans. 8,9 Cell receptors in the human fetal pancreas that allow interactions with the extracellular environment (ECM) are integrins. Integrins are a family of highly expressed heterodimeric α and β receptors that bind to mo...

Development of a Bioartificial Vascular Pancreas

“…Fibrin was selected as the hydrogel of choice due to the fact that it has shown success in islet transplantation studies, facilitates microvessel ingrowth, and has an easy-to-use crosslinking mechanism achieved by combining fibrinogen with thrombin. 51 – 55 …”

“…Fibrin was selected as the hydrogel of choice due to the fact that it has shown success in islet transplantation studies, facilitates microvessel ingrowth, and has an easyto-use crosslinking mechanism achieved by combining fibrinogen with thrombin. [51][52][53][54][55] Rat-sized BVPs were created using rat pancreatic islets coated around decellularized human umbilical arteries (DHUA) that were 1 mm in diameter and 1-1.5 cm in length, with a wall thickness of 300-500 μm. 56,57 Pancreatic islets were isolated from adult rat pancreata using a series of enzymatic digestion steps, and then were coated onto the outer surface of DHUA by combining islets, fibrinogen, and thrombin together into a mold containing the DHUA (Figure 1(b)).…”

Decellularized bladder as scaffold to support proliferation and functionality of insulin‐secreting pancreatic cells

“…In the present study, since no metabolically active cells were observed on SDS‐processed bladders, we decided it was not worth performing immunostaining and GSIS with INS‐1 cells seeded on those. Previously, studies have been conducted to characterize INS‐1 cell proliferation and functionality on fibronectin‐coated TCPS, RGD‐coated TCPS, and fibrin 68,71,72 . Here, TCPS was selected as positive control, as it is our established reference for INS‐1 cell attachment and functionality 73 .…”

“…Previously, studies have been conducted to characterize INS-1 cell proliferation and functionality on fibronectin-coated TCPS, RGDcoated TCPS, and fibrin. 68,71,72 Here, TCPS was selected as positive control, as it is our established reference for INS-1 cell attachment and functionality. 73 matrices such as MatriStem, Oasis or others could have been an option for instance, but it would be a study on its own, as no study of INS-1 cells on these matrices has been reported.…”

Economic 3D-printing approach for transplantation of human stem cell-derivedβ-like cells

“…Furthermore, much of the prior research has been focused on cellular encapsulation, which prevents vascularization of the transplanted graft, that causes cellular hypoxia, as oxygen is only delivered to the cells through diffusion, leading to either necrosis or greatly reduced function of transplanted islets [17,18]. A macroporous device would allow vascularization of the graft, improving survival and function and reducing delays in glucose sensing, and can be loaded with fibrin, which is biocompatible and degradable, to further promote β cell survival and function along with host integration and vascularization [19][20][21][22][23].…”