Enhanced structure and function of human pluripotent stem cell‐derived beta‐cells cultured on extracellular matrix

Abstract The differentiation of human stem cells into insulin secreting beta‐like cells holds great promise to treat diabetes. Current protocols drive stem cells through stages of directed differentiation and maturation and produce cells that secrete insulin in response to glucose. Further refinements are now needed to faithfully phenocopy the responses of normal beta cells. A critical factor in normal beta cell behavior is the islet microenvironment which plays a central role in beta cell survival, proliferation, gene expression and secretion. One important influence on native cell responses is the capillary basement membrane. In adult islets, each beta cell makes a point of contact with basement membrane protein secreted by vascular endothelial cells resulting in structural and functional polarization. Interaction with basement membrane proteins triggers local activation of focal adhesions, cell orientation, and targeting of insulin secretion. This study aims to identifying the role of basement membrane proteins on the structure and function of human embryonic stem cell and induced pluripotent stem cell‐derived beta cells. Here, we show that differentiated human stem cells‐derived spheroids do contain basement membrane proteins as a diffuse web‐like structure. However, the beta‐like cells within the spheroid do not polarize in response to this basement membrane. We demonstrate that 2D culture of the differentiated beta cells on to basement membrane proteins enforces cell polarity and favorably alters glucose dependent insulin secretion.


| INTRODUCTION
Type 1 diabetes is caused by the autoimmune destruction of insulinsecreting pancreatic beta cells. 1 Treatment requires lifelong insulin injections and repeated monitoring of blood glucose throughout the day. Even with the best control, blood glucose commonly fluctuates outside the normal range 2 and leads to long-term complications that can have serious acute consequences, such as coma. 3 Replacing the lost beta cells is in principle a cure as shown by the effectiveness of islet transplantation. 4 However, issues with the quality and quantity of donor islets, as well as the consequences of long-term immunosuppression, have driven the need for alternative approaches. Recent work on stem cells has made exciting progress and beta-like cells, derived from human pluripotent stem cells (embryonic stem cell [ESC] and induced pluripotent stem cell [iPSC] refereed hereinafter as hPSC), can respond to glucose and secrete insulin. 5,6 Over the last 5 years, the differentiation protocols have been further refined 7 and the cells produced are close to phenocopying the native beta cells. 8,9 However, current hPSC-derived beta cells still contain and secrete less insulin than native beta cells, and they do not respond as well to normal blood glucose concentrations and have a limited lifespan. 8,9 The current focus in stem cell research has largely been at the level of the single cell and differentiation protocols target developmental switches to drive toward beta cell maturity. 5,6,9 The results of these protocols are typically spheroids of a few hundred cells composed of beta-like cells as well as other cell types. Despite improvements in differentiation methods, these cells have compromised metabolic glucose sensing and glucose stimulated insulin secretion (GSIS). 10 Differentiated cells express comparable levels of metabolic enzymes 10 and other gene signatures, 5,7-9 however, the microenvironment around each individual cell, within a spheroid, is not deliberately controlled and may or may not be optimal for cell function. This could be profoundly important because the normal islet environment around individual native beta cells has a dramatic effect both on their structure 11,12 and function. [13][14][15][16] Identifying the key aspects of these native environmental influences and recapitulating them in stem cell approaches could open new avenues in the goal to more faithfully phenocopy the function of native beta cells.
One aspect of the native islet microenvironment, the extracellular matrix, specifically the capillary basement membrane has a major effect on beta cell function. In native islets, the basement membrane is secreted by endothelial cells 11,17 and has a crucial impact on beta cell structure 15 and function. [18][19][20] In terms of structure, the beta cells contact the basement membrane only at the capillary interface. 11,15 This discrete contact area locally activates integrin-dependent focal adhesions, drives beta cell polarization, 15 and has multiple functional effects, such as proliferation, 11,21 insulin gene expression, 11 and the control of insulin secretion. 19,22 The effect of basement membrane proteins on stem cell-derived beta cells has not been investigated until very recently. The inclusion of basement membrane protein at specific stages of differentiation play role in beta cell fate determination 23,24 and function. 24 However, these studies do not address the presence or the effect of basement membrane proteins on the function of fully differentiated cells.
Here, we employed modifications of the typical hPSC-derived beta cells stem cell maturation protocols 5 to produce spheroids of cells, including cells with a beta cell phenotype and other cell types, such as alpha-like cells. Two different human pluripotent stem cell lines were used for this study; human iPSC (ATCC-ACS-1030) and human ESC Ins GFP/W (MEL1). 25 We term both the ESC and iPSC pluripotent stem cell lines, hPSC. We demonstrate that differentiated hPSC-derived spheroids do contain basement membrane, but it is present as unorganized structures within the spheroids with some beta-like cells contacting the matrix and others not. We measure the subcellular organization of the cells within the spheroid and show that they have aberrant polarization and altered structural arrangement when compared to native beta cells. In further experiments, the differentiated cells were cultured as monolayers on basement membrane coated dishes. We show that the cells polarize with respect to the coated surface and the response to a glucose challenge is favorably altered when compared to cell spheroids with a decrease in basal insulin secretion and an enhanced secretory index.
We conclude that the addition of basement membrane to fully differentiated hPSC-derived beta cells does drive changes in cellular structure facilitating the recruitment of insulin secreting machinery which enhances insulin secretion. This provides a platform for future work that aims to provide a better microenvironment for hPSC-derived beta cells to enhance cell function and viability.

| Cell lines and culture conditions
Human iPSC obtained from American Type Culture Collection (ATCC-ACS-1030) and human ESC line Ins GFP/W (MEL1) was obtained from Murdoch Children's Research Institute, Victoria, Australia. 25 Both the

Significance statement
Current protocols are producing stem cell-derived beta cells that secrete insulin in response to a glucose challenge.
These cells are functionally close to normal beta cells, but refinements are needed in order to achieve full recapitulation of normal beta cell behavior. One promising avenue to enhance cell behavior is to look specifically at how beta cell structure and function is regulated by the environment of normal islets. In islets the basement membrane, secreted by vascular endothelial cells, has a direct effect on beta cell differentiation, survival, structure, and function. Each beta cell contacts the capillary basement membrane which then locally activates integrins and forms focal adhesions. The native beta cells then orientate with respect to the capillary to become polarized and to target insulin secretion to this capillary interface. This article reports on the effect of culturing human stem cells-derived beta cells on basement membrane proteins, showing that this enforces structural polarity in the cells and significantly enhances glucose stimulated insulin secretion. The findings from this study suggest that adding environmental cues from the islet, such as basement membrane proteins, could be a promising route to enhance the function of human stem cells-derived beta cells.    After fixation cells were washed with PBS followed by 1 hour's incubation in blocking solution on ice. Cells were further washed and incubated in blocking buffer containing primary antibodies for 2 hours on ice. Cells were washed three times using PBS followed by 1 hour's incubation in secondary antibodies on ice. Secondary antibodies were washed, and cells were resuspended in 1% fetal calf serum (FCS) in PBS for cell sorting/analysis. Cells were sorted using 10-laser BD influx (BD Biosciences) and analyzed using FlowJo software.

| Gene profiling using nanostring technology
For gene profiling human ESC, pooled samples from differentiation batches of human ESC-derived spheroids and human islets from two different preparations were sampled. Total RNA was extracted using Isolate II RNA Mini Kit (Bioline; BIO-52072). Concentration of RNA was analyzed using nanodrop. The precise quantity and quality of RNA were determined using Bioanalyzer RNA 6000 Nano assay (Agilent; 5067-1511). The RNA Integrity Number value for the RNA samples were between 9.9 and 10. For the gene profiling of 50 selected genes, nCounter GX Custom CodeSets (NanoString; 116000001) was used. For each sample, 50 ng of RNA was hybridized with Reporter and Capture ProbeSet in a volume of 15 μL. Hybridization was carried at 65 C for 18 hours followed by a ramp down to 4 C. The hybridization samples were then loaded on nCounter SPRINT Cartridge (nanoString; 100 078) and run on nCounter SPRINT profiler. Data were analyzed using nSolver. Recommended quality control was performed. Background was subtracted using spiked negative controls. Absolute count of RNA was further normalized using the POLR2A housekeeping gene and plotted as heat map using GraphPad prism.

| Human hPSC-derived spheroids express monohormonal cells and exhibit insulin secretion upon glucose challenge
We established a stem cell differentiation protocol to produce spheroid cell clusters that contain beta-like cells. Our protocol used human ESC and human iPSC and was based on published reports with a six-stage, 34-day-long protocol. 5,7 Differentiation was car- We conclude that our differentiation protocol leads to a major population of monohormonal beta-like cells that express many of the markers of beta cell identity and maturity. We show that the beta cells 3.2 | Expression of basement membrane proteins form a mesh-like structure in the differentiated spheroids The differentiation protocol outlined above specifically aims to produce adult beta cells and therefore the structure of the spheroid forms spontaneously. Given native endocrine cells do not secrete basement membrane, 17 we wanted to determine if any basement membrane proteins are present in the spheroids. Past work has shown some basement membrane proteins in the periphery of hESC-derived pancreatic progenitor spheroids. 31 Here, we wanted to determine if they are present in the mature differentiated hPSC-derived spheroids and their spatial relationship with insulin-expressing beta-like cells.
In mouse 12  protein. 34 We conclude that basement membrane proteins are secreted by nonendocrine fibroblast-like population of differentiated spheroids and contact some beta-like cells. We next set out to determine if these contacts with the basement membrane influence betalike cell structural polarity.

| Differentiated beta-like cells do not show polar domain organization
The characteristic response of adult beta cells to the capillary basement membrane is integrin mediated cell orientation. 15  something not seen in native islets. We did observe expression of integrin β1, overlapping with Liprin α1 (Figure 4F-G). This is what is seen in native cells but, unlike native beta cells, this domain was not within a discrete subportion of the cell membrane but instead spread across much of the cell surface. We conclude that although the hPSC-derived beta cells do express these proteins (also seen in the transcriptome data of Figure 2D), there is little evidence for polar organization and therefore it is unlikely that they have functional significance in the control of insulin secretion from the spheroids.  15 We specifically chose to use cells from fully differentiated spheroids because basement membrane is known to control the fate of pancreatic progenitors. 23,24 Fully differentiated hESC-derived spheroids were dispersed into single cells and plated on basement membrane protein coated dishes (see Supplementary Information). We observed cobblestone sheets of beta-like cells in close contact with each other (Figure 5A,B). After 3 days of culture on basement membrane proteins (laminin 511, or collagen IV or fibronectin), GSIS assays were performed, exposing the cells sequentially to 3 mM (basal) and 25 mM (stimulated) glucose ( Figure 5C,D). Compared to relatively high levels of basal insulin secretion in the differentiated spheroids ( Figures 1G, 2B, and 5C), cells on basement membrane proteins showed a significant reduction in basal insulin secretion ( Figure 5C).

| Interaction between beta-like cells and basement membrane proteins favorably alters GSIS
The stimulated insulin secretion (fold change from basal, stimulation index) was significantly upregulated on all the three basement membrane proteins ( Figure 5C,D) with a maximum of 3.9-fold increase with cells on laminin 511. We conclude that culture of cells onto basement membrane proteins significantly affects the insulin secretion and, by lowering the basal secretion, the fold increase is substantially improved when compared with spheroids.

| DISCUSSION
Remarkable progress has been made in generating insulin secreting beta-like cells from stem cells in last decade. 5,6 For the purpose of scalability and robust differentiation, cells are maintained as spheroids in 3D culture, which means they will have endocrine to endocrine cell interactions that are expected to be analogous to those found in a native islet. However, the maturation and insulin secretion stimulation index of these cells are inferior compared to the human islets. Enrichment of beta cells and resizing of the clusters results into some improvement in the insulin secretion capacity, 8 but they still have compromised glucose metabolism and insulin secretion. 10 The identity of transporters and ion channels in beta cells that regulate glucose dependent control of insulin secretion is well known. 35   In this study, we show that basement membrane proteins are present as a diffuse web-like structure in the differentiated spheroids.
The origin of these basement membrane protein is not known but they unlikely to be secreted by the beta cells. 17 The disorganized basement membrane web we observed in the spheroids ( Figure 3C-K) is therefore likely to come from nonendocrine cells that are a "by-