TY - JOUR
AU1 - Lewis, Phillip L.
AU2 - Wells, James M.
AB - Abstract Strategies to mitigate the pathologies from diabetes range from simply administering insulin to prescribing complex drug/biologic regimens combined with lifestyle changes. There is a substantial effort to better understand β-cell physiology during diabetes pathogenesis as a means to develop improved therapies. The convergence of multiple fields ranging from developmental biology to microfluidic engineering has led to the development of new experimental systems to better study complex aspects of diabetes and β-cell biology. Here we discuss the available insulin-secreting cell types used in research, ranging from primary human β-cells, to cell lines, to pluripotent stem cell-derived β-like cells. Each of these sources possess inherent strengths and weaknesses pertinent to specific applications, especially in the context of engineered platforms. We then outline how insulin-expressing cells have been used in engineered platforms and how recent advances allow for better mimicry of in vivo conditions. Chief among these conditions are β-cell interactions with other endocrine organs. This facet is beginning to be thoroughly addressed by the organ-on-a-chip community, but holds enormous potential in the development of novel diabetes therapeutics. Furthermore, high throughput strategies focused on studying β-cell biology, improving β-cell differentiation, or proliferation have led to enormous contributions in the field and will no doubt be instrumental in bringing new diabetes therapeutics to the clinic. Engineered in vitro systems have the potential to revolutionize our understanding metabolic diseases and the countless roles β-cells play in them. Many different sources of insulin-secreting cells are available for engineered systems each with their own strengths and weaknesses pertinent specific downstream applications. For example, high throughput studies require large numbers of cells allowing cell lines and stem cell-derived β-cells to be suitable sources. However, functional studies into β-cell physiology as well as organ-organ interactions require physiologically mature cell types that more closely mimic human biology. This review covers specific sources insulin-secreting cells as well as applications in fluidic culture devices and high throughput methodologies as they pertain to the study of β-cell physiology with a focus on diabetes. Open in new tabDownload slide Open in new tabDownload slide cell culture, diabetes, experimental models, pancreas, pancreatic differentiation, pluripotent stem cells, technology Significance statement While superficially a disease of insulin secretion and sugar metabolism, the deeper complexities of diabetes necessitate advanced engineered strategies to uncover new treatment approaches. In vitro models of β-cells and insulin secretion in a human-specific context have therefore emerged as powerful tools to study diabetes. This article reports several advances including different sources of insulin-secreting cells, organ-on-a-chip models, and high throughput strategies designed to deepen our understanding of β-cell biology and to better treat disease. INTRODUCTION Diabetes is a pervasive and growing problem across the world. With age-adjusted diabetes prevalence increasing from 4.3% in 1980 to nearly 9% in 2016 and over 415 million people worldwide living with diabetes,1,2 there is a clear need for the development of novel therapies to combat the loss and dysfunction of β-cells. In type 1 diabetes (T1D) where β-cells are absent, glycaemia can be effectively managed with exogenous insulin therapy. While managing glucose has been highly burdensome historically, new technologies such as continuous glucose monitoring (CGM) and insulin pumps have provided a much-improved quality of life. The majority of people with diabetes have type 2 diabetes (T2D) (90%-95%). T2D can be mitigated with lifestyle changes, or using pharmaceutical approaches that center around increasing peripheral tissue insulin sensitivity (eg, metformin), increasing insulin secretion from existing β-cells (eg, sulfonylureas), or by altering hormonal interaction with other endocrine organs (eg, incretin mimetics, DPP-IV inhibitors). By far the most successful treatment for T2D and its associated sequelae is gastric bypass surgery, which results in rapid restoration of β-cell function as well as improved liver and cardiovascular function before appreciable weight loss occurs.3,4 It is not known how gastrointestinal surgery results in a reversal of T2D but highlights the role that interorgan communication plays in healthy and diseased β-cell physiology. A deeper understanding of islet and β-cell physiology and how soluble factors from other organs feed into the restoration of normoglycemia is paramount to developing novel therapies for diabetes. Historically, animal models have provided the most insight into islet pathophysiology. Significant differences between humans and model organisms have led to drugs failing during clinical trials; for example, troglitazone was safe in animal testing but hepatotoxicity led to it being removed from American markets.5 The preponderance of examples of drugs passing preclinical and phase 1 trials but failing at later stages highlights the need for human cell and tissue model systems to study human physiology, diseases, toxicity and drug efficacy. The convergence of multiple fields ranging from developmental biology, endocrinology, chemistry, biomaterials science, and chemical engineering has brought forth numerous platforms to study many different aspects of islets and insulin-secreting cells. For example, physiologically responsive human β-like cells can now be derived from pluripotent stem cells (PSCs) and sophisticated engineered systems can precisely stimulate and measure islet and β-cell physiology. Moreover, it is now possible to generate other metabolically critical organ tissues in vitro and study their interactions with β-cells in efforts to model T2D. In this review, we will discuss recent progress in the development and use of human in vitro model systems to study β-cells, from cell sources to their applications in fluidic systems and in high throughput strategies. SOURCES OF INSULIN-PRODUCING CELLS The choice of a cell source for studying diabetes and islet or β-cell physiology ultimately depends on the application in question (Figure 1). Studying the role of a gene in islet development, for example, is better-suited for rodent or PSC-based approaches as opposed to cell lines or primary isolated human islets. However, human islets may be better-suited to study human-specific insulin secretion physiology given the relative immaturity of PSC-β-cells and differences between humans and model organisms. Each source has its own unique strengths and weaknesses in the context of in vitro studies, with some experimental questions necessitating more than one approach. FIGURE 1 Open in new tabDownload slide Sourcing of insulin producing cells for in vitro engineered systems. Primary isolated human islets are the closest approximation to what would be seen in vivo; however, drawbacks in variability are often a concern. Pseudoislets are an approach to mitigate some variability. PSC-derived sources offer a unique approach to study human physiology, while model organisms can place islet physiology in a whole-organism context. β-Cell lines are by far the easiest to use of all categories; however, they are perhaps the least physiologically representative of their in vivo counterparts FIGURE 1 Open in new tabDownload slide Sourcing of insulin producing cells for in vitro engineered systems. Primary isolated human islets are the closest approximation to what would be seen in vivo; however, drawbacks in variability are often a concern. Pseudoislets are an approach to mitigate some variability. PSC-derived sources offer a unique approach to study human physiology, while model organisms can place islet physiology in a whole-organism context. β-Cell lines are by far the easiest to use of all categories; however, they are perhaps the least physiologically representative of their in vivo counterparts Model organisms Model organisms have remained the gold standard for islet and diabetes research. Research findings around islet development and function have been remarkably reproducible due to the consistency of inbred animals. Furthermore, islet preparations for in vitro experiments are similar batch-to-batch and lab-to-lab. Cryopreserved rodent islets are also readily available for purchase from several sources. Nevertheless, there are structural and functional differences between human and rodent islets. Mouse and rat islets highly express the GLUT2 receptor, making them more susceptible to streptozotocin (STZ)-induced β-cell death and are widely used as in vivo model of islet destruction during diabetes.6 Mouse and human islets are structurally different, with mouse islets being half the size of human islets, with endocrine cells that are organized differently.7 Human islets also have a unique vasculature8 and undergo unique changes in response to disease compared with rodents.9 The functional differences in insulin secretion physiology between human and mouse β-cells have been thoroughly reviewed.10 In summary, human islets are sensitive to lower concentrations of glucose and the differences in oscillatory waves of calcium influx and insulin secretion may ultimately be due to architectural differences. However, due to the limited availability and batch to batch variability, rodents remain a powerful tool for mechanistic studies of islet development and disease both in vivo and in vitro. Aside from rodents, many other vertebrate models have been used for diabetes and islet research. Fish and frogs have been used to study pancreas development because of the ease of embryonic manipulation and in the case of zebrafish their genetic tractability and transparency.11,12 Larger animals such as nonhuman primates (NHP) and swine are not typically used in early-stage basic research because of ethical issues and cost. However, NHPs have been used as models for xenotransplantation and immunoisolation strategies13 in testing porcine islets as a possible therapeutic in humans.14 While there are functional differences in insulin secretion between porcine, NHP, and human islets,15 the availability of porcine islets is a considerable advantage of xenotransplantation over allotransplantation.16 Human Cadaveric human islets are a first choice for studying human physiology in vitro, and their availability in recent years has improved. However, these islets are often those rejected for transplantation and are of variable quality, resulting in significant differences in function between batches of islets.17 This variability can be mitigated with the formation of “pseudoislets,” which are enzymatically dissociated islets that are reaggregated into uniformly sized spheroids.17 While an aggregate of any insulin-producing cell, including cell lines can be termed a pseudoislet,18 for the purposes of this review, pseudoislets will refer to reaggregated primary islets. Pseudoislets are more uniform in size and in nutrient responsiveness, however, tend to have lower overall insulin content than their unmanipulated islet counterparts.17,19 The dissociation and reaggregation also allows for efficient viral transduction for manipulation of gene expression and introduction of live imaging reporters.20-22 Another configuration of islets is monolayer culture, which are more uniform and have been used for high throughput applications (see below). However, the dispersing of islet cells and growth on monolayers fundamentally changes islet architecture and thus is disruptive to insulin secretion physiology. The restored cell-cell contacts of pseudoislets rescues calcium physiology and allows for the study of interendocrine cell communication between β-cells and other islet cell types.23 Human islets have also been used therapeutically by transplantation into patients with difficult-to-control “brittle” T1D. Islet transplantation typically follows the Edmonton protocol where islets are enzymatically liberated from the rest of the pancreas and infused into the hepatic portal vein, allowing engraftment into hepatic lobules. During Total Pancreatectomy with Islet Autotransplantation (TPIAT) for patients with chronic pancreatitis, a patient's own islets are subjected to this procedure. While many institutions are developing methods and materials to improve survival and engraftment of transplanted islets, the insurmountable obstacle to the wide use of cadaveric islets for therapy is their limited availability. Furthermore, insulin dependence, along with other complications, may return in many patients receiving allogeneic islets due to many factors not limited to autoimmune destruction.24 For these reasons, the field of transplantation-based therapeutics has focused on a promising and unlimited source of insulin-expressing cells, human PSCs. PSC-derived The past decade has seen remarkable advances in the development of β-like cells and islets derived from PSCs as reviewed in.25,26 PSC-derived β-like cells can be generated in an unlimited and reproducible manner, are of consistent quality, and can be autologous for autotransplantation. Several companies (Sigilon/Eli Lilly,27 Evotec/Sanofi, Novo Nordisk, Semma,28 Sernova,29 Seraxis,30 and Viacyte31) have been developing therapeutics for the treatment of T1D that combine PSC-derived islets with encapsulation devices that afford some level of immunoprotection. As an alternative to immune protective devices some groups are developing genetically modified “universal donor” iPS lines that can evade the immune system and prevent the need for immunosuppressants. The goal is to develop an implantable and easily-retrievable biologic that will serve as a surrogate endocrine pancreas. While human PSCs have been transformative to study human β-cell development, most early protocols only were able to generate embryonic precursors that required transplantation and maturation in rodents to become nutrient responsive β-like cells.32 The more recent generation of protocols have been able to show nutrient responsiveness in static conditions28,33,34 and even dynamic glucose responses including first and second phase secretion profiles.35-37 While these cells are still not molecularly or functionally equivalent to human β-cells, there is every reason to believe that additional advances will continue to improve β-like cell function. For example, alternating 12 hours cycles of high and low glucose37 or cell sorting36 and assembly of β-cell-like aggregates35,38,39 enhances many aspects of β-like cell maturation such as mitochondria number/density and insulin secretion dynamics. Applications of PSC-based approaches to model diseases (including diabetes) have yielded a wealth of information on the roles genes play in islet differentiation and function.25 For example, increased susceptibility to gluco/lipotoxicity, both in vitro and in vivo was demonstrated using PSC-β-cells with mutated genes associated with T2D (CDKAL1, MT1E, KCNJ11, KCNQ1).40,41 However, T1D or T2D are rarely monogenic diseases, and in vitro modeling of T2D especially will require long-term culture of functionally mature β-like cells to adequately mimic disease progression. Cell lines The consistency and reproducibility of cell lines combined with their ease of use and maintenance make them indispensable tools for molecular biological studies. Many different insulin-secreting cell lines have been developed from human and rodent β-cells through SV40 immortalization or isolated insulinomas as reviewed in.42 Regardless of the source of immortalization, the primary disadvantage of β-cell lines is their lack of physiologic responsiveness to nutrients as evident in tests of glucose-stimulated insulin secretion (GSIS). β-Cell lines can be hypersensitive to glucose, with other lines insensitive to it, while some become progressively more insensitive as passage number increases. Although they differ from line to line, β-cell lines are generally less mature than human islets. Transciptomic and proteomic comparisons between β-cell lines and primary human islets confirm that there are many differences.43,44 The appropriate glucose responsiveness of the rat insulinoma cell line INS-1 and the mouse insulinoma cell line MIN6 (in the presence of nicotinamide) has made them a popular first choice in the development of engineered systems to study insulin secretion.45,46 Historically, few human β-cell lines have been available to study insulin secretion. The human β-cell EndoC-βH1 was developed by Ravassard et al by transplanting immortalized human fetal pancreatic bud tissue into immunocompromised mice.47 While more similar to human fetal β-cells, this line shows remarkable glucose responsiveness in vitro and upon transplantation in addition to not expressing markers of other pancreatic cell types. A similar cell line, EndoC-βH3, expresses CRE-ERT2, allowing its proliferation to be arrested upon exposure to tamoxifen.48 Cell lines whose proliferation is easily controllable (EndoC-βH3, R7T1) are excellent candidates for studies into β-cell proliferation. The insulin secretion efficacy of most cell lines can also be enhanced with cell aggregation.49-53 Therefore, β-cell line aggregates cultured in this manner serve as low-cost surrogates for islets/pseudoislets. ENGINEERED FLUIDIC PLATFORMS FOR STUDYING ISLETS AND INSULIN SECRETION PHYSIOLOGY The study of islet physiology and (dys)function with fluidic systems has emerged as an entire subfield of tissue-on-a-chip research due to the many advantages of flow systems in studying dynamic insulin secretion. By mimicking blood flow, β-(like)-cells are exposed to a nutrient concentration that is not impacted by the β-cells' own secretions and metabolism. Furthermore, secreted hormones are immediately swept away from β-cells, mitigating any autocrine effects of insulin that may not be physiologically relevant. Fluidic systems also have the advantage of constant sampling, allowing for a more detailed, time-resolved, resolution of an islet's secretion profile, and in some cases allowing for measurement of several different physiologic phenomena on a per-cell or per-islet basis. Fluidic devices have been well-suited to investigate the effects of fluid flow on islet function. Fluid flow enhances the viability, insulin secretion, and response to secretagogues of rat islets when compared with a static system54 and can improve GSIS of pseudoislets.55 Flow was also shown to lead to other morphological changes such as more abundant microvilli and increased amounts of tight junctions. The impact of flow on may impart beneficial effects on resident islet endothelial cells, indirectly improving islet function and viability.55,56 Numerous labs have devised platforms for analysis of islet secretions, which has been thoroughly reviewed previously.57-59 Discussed below are recent advances and new directions the organ-on-a-chip field has recently taken with β-cell research. Using fluidic systems to study β-cell physiology Fluidic systems for analysis of β-cell secretion, regardless of endpoint application, have settled on several common approaches. These include an islet and cell aggregate trap system which gently immobilizes cells and enables fluid contact with and flow around the islet surface. Islets can be trapped using a mesh system,60 with narrowing of fluid channels,61,62 or with troughs into which islets and cell aggregates will settle.63 Alternatively, pseudoislets can be formed on the chip itself which can then be perfused during long-term culture.55,64 Fluidic systems have also incorporated on-chip analysis or sample collection for subsequent analysis, reducing workload and enhancing the number of samples able to be collected compared with traditional methods (Figure 2). Commercially available fluidic platforms such as those manufactured by Biorep65 allow for sample collection, while Seahorse by Agilent66 systems continuously measure metabolic activity in a well plate format and allow for fluid collection for off-platform analysis. On-chip analysis dramatically accelerates workflow with the compromise of increased cost and occasionally reduced versatility in terms of output measurement options. On-chip readouts in through live imaging or electrodes, for example, do not require fluid sampling, and therefore enable the same off-chip quantifications to be performed without sacrificing sample volume. FIGURE 2 Open in new tabDownload slide Quantitatively measuring islet and β-cell physiology. Measurements from whole islets typically focus on peptide hormone secretion. Any method developed to quantify secreted peptides can therefore be used, with some having more utility for certain proteins (eg, insulin). Interrogating individual β-cells focuses on specific aspects of GSIS physiology, listed adjacent to each output are examples of detection methods. Influx/efflux of several ions can be quantified using fluorescent dyes or indicator proteins. Patch clamping can also be geared toward specific ions. Indicator proteins have also been used to study protein/vesicle trafficking as well as mitochondrial redox state. Many different dyes and fluorescent proteins have been developed to measure these β-cell functions. APG, Asante potassium green; ELISA, enzyme-linked immunosorbent assay; GEPIIs, genetically-encoded potassium ion indicators; GLP-1, glucagon-like peptide 1; IAPP, islet amyloid polypeptide; R123, rhodamine-123 FIGURE 2 Open in new tabDownload slide Quantitatively measuring islet and β-cell physiology. Measurements from whole islets typically focus on peptide hormone secretion. Any method developed to quantify secreted peptides can therefore be used, with some having more utility for certain proteins (eg, insulin). Interrogating individual β-cells focuses on specific aspects of GSIS physiology, listed adjacent to each output are examples of detection methods. Influx/efflux of several ions can be quantified using fluorescent dyes or indicator proteins. Patch clamping can also be geared toward specific ions. Indicator proteins have also been used to study protein/vesicle trafficking as well as mitochondrial redox state. Many different dyes and fluorescent proteins have been developed to measure these β-cell functions. APG, Asante potassium green; ELISA, enzyme-linked immunosorbent assay; GEPIIs, genetically-encoded potassium ion indicators; GLP-1, glucagon-like peptide 1; IAPP, islet amyloid polypeptide; R123, rhodamine-123 Some immediate changes in islets that occur upon exposure to a nutrient stimulus can only be observed with on-chip analysis. For example, upon exposure to glucose, β-cells will depolarize leading to an immediate influx of calcium ions which leads to vesicle fusion and insulin secretion. Controlled fluid exchange enables more precise exposure of cells to nutrients/secretagogues compared with manual exchanging of media. Continuous imaging correlated with fluid flow dramatically increases data fidelity by reducing error that may occur with timing or manual pipetting. Calcium flux can be imaged using indicator dyes such as Fura-2/4 am63 or Fluo-264 or using calcium-responsive proteins such as GCaMP6.22,67 Imaging platforms also enable a large number of islets to be simultaneously interrogated for calcium flux in response to hypoxia and secretagogues.68 The precision of engineered systems can exacerbate the inherent variability in biological systems, and incorporating large numbers of islets within a perfusion system can help smooth over that variability. β-Cells are highly metabolically active, with perturbations in mitochondrial activity being exceptionally relevant to diabetes.69 Dynamic mitochondrial activity can be visualized with Rhodamine 123.70 Glutathione redox state and hydrogen peroxide concentration of rat islet mitochondria were monitored real time in a perfusion system employing mitochondria-targeted ratiometric fluorescent proteins.71 Zbinden et al devised a system using Raman microspectroscopy to study multiple aspects of EndoC-βH31 aggregate function simultaneously in real time.72 Multireadout systems have also been designed to merge immunoassays and imaging,73 with some incorporating optogenetics to further interrogate islet function.74 Insulin secretion is by far the most popular islet output and can be measured in real time or in downstream off-chip analysis. Insulin secretion itself can be indicative of islet health if isolated from patients with diabetes.75 Continuous real-time on-chip measurements of insulin output can be taken using fluorescence anisotropy from islet arrays62 or single islets.76 This approach offers a uniquely precise and uninterrupted measurement rarely seen in perfusion platforms. Insulin secretion from a single pseudoislet was able to be quantified (off chip) using a hanging drop perifusion system.77 In single islet scenarios, higher resolution aspects of insulin secretion physiology can be observed such as oscillations during second phase insulin secretion which has previously only been shown with live Ca2+ imaging.78,79 Features such as the brief dip in first phase insulin secretion have never been shown in human islets. Furthermore, finer details in insulin secretion are essentially impossible to observe in static traditional culture methods. Continuous live monitoring or rapid sampling of small numbers of islets (or single islets) is therefore the optimal choice if such features of insulin secretion need to be observed. The time-resolution and range of concentrations of a flow system are the two factors that ultimately determine which insulin secretion detection method is most appropriate. Technical descriptions of different islet secretion detection methods are reviewed in.58 An indirect way to measure insulin secretion is using dyes that detect the zinc that is incorporated into insulin secretory vesicles.80-83 Potassium efflux, although an intermediate step in the progression of GSIS, has been measured within islets using fluorescence dyes.84 Schulze et al devised a platform with immediate measurements of NAD(P)H using fluorescence imaging, and oxygen consumption rate with O2 sensors.63 This was combined with buffer collection and subsequent off-chip insulin quantification. Glucagon output from PSC-derived cells has been measured using BioRep platforms33,36,37,85; however, other protein output that could provide important information about islet function (islet amyloid polypeptide [IAPP], somatostatin, pancreatic polypeptide [PP], ghrelin) are rarely measured. In one case a more sensitive technique, surface plasmon resonance (SPR), was used to quantify islet output of insulin, glucagon, and somatostatin.86 These multiparametric techniques allow for complex physiological studies of islet hormone secretions in response to nutrients and therapeutics. While in-depth analysis of sampled media is possible with off-chip techniques such as proteomics, continuous monitoring with methods such as imaging or SPR afford a time-resolved view of β-cell physiology. While there have been progress in simplifying perfusion systems,87 most of these technologies require expensive specialized equipment and skills that are not easily transferrable to other laboratories. Organs on a chip: The emergence of microphysiologic systems to study organ function and interaction As described above, fluidic systems have lent themselves to the development of microphysiological systems (MPSs) that allow for analysis of β-cell and islet function. The body of literature surrounding static coculture studies with islets and/or β-cells is extensive and largely suggest that coculture with nearly any cell type enhances β-cell function and viability.88,89 Alternatively, β-cell death can be stimulated by coculturing with immune cells in experiments designed to model immunological aspects of T1D. However, modeling functional interorgan crosstalk can be difficult to quantify in traditional static culture configurations. Engineered fluidic systems can enhance both control over multitissue interactions, as well as quantification of those interactions with many methods like those described in the previous section. MPSs have now been developed for most organ systems, including many organs that have endocrine interactions with the pancreas such as intestine, liver, adipose, muscle, and brain (Figure 3).90,91 Due to its essential role in drug metabolism, liver MPSs have been developed by several groups.92 Several platforms have been used to study liver function in the context of diabetes. Nonalcoholic fatty liver disease (NAFLD) is a diabetes comorbidity that has seen many engineered in vitro models.93-96 MPSs can be configured to study liver-pancreas communication and have been used to model how β-cells communicate to hepatocytes to store glycogen or release glucose in response to insulin and glucagon, respectively. Treating a fatty liver disease model with drugs like metformin, for example, would increase hepatocyte sensitivity to β-cell secreted insulin. In one example, pseudoislets impacted the activity of aggregates of the immortalized hepatocyte cell line HepaRG cells to monitor and take up glucose from the media.97 Moreover, coculture enhanced the function of both constructs after long-term (15 days) culture, indicating that hepatic secretions maintain pseudoislet function, and vice versa. This was also demonstrated with primary rat islets and hepatocytes.98 A β-cell/liver MPS could be used in the future to study pathologic triggers such as inflammatory cytokines and how they cause the β-cell and liver dysfunction that is associated with T2D.99 FIGURE 3 Open in new tabDownload slide Interorgan crosstalk with the endocrine pancreas. Summarized are influences each organ (system) has on pancreatic endocrine secretin. Listed beside each organ are the effects caused by hormones secreted by islets. While not an endocrine interaction, direct autonomic nervous system contact with islets influence endocrine secretion. The GI tract secretes ~20 separate hormones in addition to being responsible for nutrient absorption, shown here are hormones known to influence islet secretion. Three major energy storage/metabolism organs (liver, muscle, adipose) primarily influence pancreatic function by modulating blood glucose directly. However, they also modulate islet activity through endocrine interactions. ACh, acetylcholine; ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone—vasopressin; ANS, autonomic nervous system; BG, blood glucose; CNS, central nervous system; DPP4, dipeptidyl peptidase 4; EEC, enteroendocrine cell; FABP4, fatty acid-binding protein 4; FFA, free fatty acids; FGF21, fibroblast growth factor 21; GI, gastrointestinal; GIP, glucose insulinotropic polypeptide; GLP1/2, glucagon-like peptide 1, 2; NMU, neuromedin U; NPY, neuropeptide Y; PYY, peptide YY; SCFA, short chain fatty acids; SeP, selenoprotein P; SST, somatostatin; TG, triglycerides; TNF-α, tissue necrosis factor α; VIP, vasoactive inhibitory peptide FIGURE 3 Open in new tabDownload slide Interorgan crosstalk with the endocrine pancreas. Summarized are influences each organ (system) has on pancreatic endocrine secretin. Listed beside each organ are the effects caused by hormones secreted by islets. While not an endocrine interaction, direct autonomic nervous system contact with islets influence endocrine secretion. The GI tract secretes ~20 separate hormones in addition to being responsible for nutrient absorption, shown here are hormones known to influence islet secretion. Three major energy storage/metabolism organs (liver, muscle, adipose) primarily influence pancreatic function by modulating blood glucose directly. However, they also modulate islet activity through endocrine interactions. ACh, acetylcholine; ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone—vasopressin; ANS, autonomic nervous system; BG, blood glucose; CNS, central nervous system; DPP4, dipeptidyl peptidase 4; EEC, enteroendocrine cell; FABP4, fatty acid-binding protein 4; FFA, free fatty acids; FGF21, fibroblast growth factor 21; GI, gastrointestinal; GIP, glucose insulinotropic polypeptide; GLP1/2, glucagon-like peptide 1, 2; NMU, neuromedin U; NPY, neuropeptide Y; PYY, peptide YY; SCFA, short chain fatty acids; SeP, selenoprotein P; SST, somatostatin; TG, triglycerides; TNF-α, tissue necrosis factor α; VIP, vasoactive inhibitory peptide Other MPSs have been developed for tissues directly involved in the normal and pathologic handling of blood glucose including adipose tissue100,101 and skeletal muscle.102,103 A similar approach was used to study adipose tissue in engineered systems104 and combined islet/adipose systems.105 Insulin secretion was shown to be augmented in islets placed downstream of adipose tissue in an MPS.106 Studying interactions between β-cells and the major energy storage/metabolism organs (liver, adipose, muscle) is technically challenging in MPS. Culture media is typically used in volumes where any storage/release of glucose from these organs in response to insulin/glucagon would not be detectible. Other surrogate readouts for insulin stimulation (or lack thereof) such as presence of glycogen granules or AKT signaling can be used.107 Adipose-immune crosstalk has also been studied in the context of T2D in the absence of insulin-producing cells.108 The GI tract is collectively the largest endocrine organ in the human body and plays a central role in regulating the endocrine pancreas via its secreted hormones and absorbed macronutrients. Bariatric surgery itself is largely considered the only immediate “cure” for T2D, with an immediate cessation of symptoms observed before any loss of weight.109 This metabolic shift is largely thought to be due to the alterations in nutrient exposure to different regions of the gut, and the resulting changes in the secreted hormones.3 Hormones in the GI tract are secreted by enteroendocrine cells (EECs) which are exceedingly rare (1%-3% of the intestinal epithelium) making their study notoriously difficult. Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), the “incretins,” are secreted by the EEC subtypes K and L cells, respectively, and are well-known to enhance glucose-stimulated insulin secretion. Several drugs are clinically available that harness the incretin effect.110,111 The L-cell line GLUTag was combined with INS-1 cells in an MPS to demonstrate an incretin effect, with dynamic secretion of both GLP-1 and insulin.112 However, as discussed above, the use of murine cells and transformed lines are not ideal models of human physiology. Moreover the intestine secretes >20 hormones whose influence on pancreatic endocrine hormone secretion is understudied,110 with other nonincretins such as PYY thought to also play a role in reversal of T2D postbariatric surgery.113 Therefore, developing primary human EEC MPSs to investigate the physiologic function and therapeutic potential of intestinal hormones in restoring β-cell function is paramount. Researchers have engineered platforms to explore vascularizing hydrogels containing encapsulated islets either in preparation for transplantation114-116 or for in vitro perfusion.56,117 Many of these in vitro systems are used in the study of T1D, often incorporating lymphocytes and vasculature into an engineered system to model autoimmune attack of islets.118 The destruction of vasculature due to diabetes was modeled independently of islets using PSC-derived endothelial cells.119 However, diabetes as a disease involves many tissues, necessitating design of more complex systems. The feasibility of a platform containing 4, 7, or 10 interconnected tissues (including islets) was demonstrated either driven by a pump120 or pumpless.121 These studies highlighted the compounding technical challenges that arise as each new MPS was added. Therefore, combining more than two tissues to effectively model endocrine disfunction in diabetes is a challenge, requiring a coordinated cross-disciplinary effort. Incorporating readouts in such complex systems will take the form of organ or cell-specific readouts (eg, Ca2+ indicator proteins) that are pertinent to a specific MPS design, or highly general approaches (eg, off-chip proteomics) to quantify tissue-tissue interactions. The widespread physiologic dysfunction of diabetes and the importance of insulin in everyday metabolism implies there is room for discovery in virtually any multitissue MPS involving islets.122-124 However, a pervasive shortcoming in the field is that new fluidic systems are not broadly adopted by other labs after the initial publication. It is common that there are no follow up publications, possibly due proprietary concerns because of attempts at commercialization, or the technical challenges of using these new complicated MPS systems. Also, the need to continue to develop new and better systems to publish papers is what pushes bioengineers to continuously design new platforms in lieu of using tools already at their disposal to probe deeper into β-cell physiology. These issues are being addressed by funding agencies, such as the NIH's National Center for Advancing Translational Sciences (NCATS) Tissue Chip program, which focuses on better sharing new MPS technologies with the broader research community. HIGH THROUGHPUT APPROACHES TO STUDY Β-(LIKE)-CELL BIOLOGY AND PHYSIOLOGY In order to be considered high throughput, a system must employ experimental replicates and dependent variables in quantities order(s) of magnitude higher than traditional approaches. This can be achieved through miniaturization, scale up, and automation of experimental techniques, data acquisition, and analysis. High throughput strategies involving islets and insulin-secreting cells focus primarily on identifying novel targets or drugs for the long-term goal of mitigating T1D or T2D. Historically, β cell lines have been the starting material of choice for HT screens due to the required scale up, minimal sample to sample variation, and two-dimensional (2D) configuration for imaging.125 However, technological advances such as robust protocols for PSC-derived β-like cells, rapid three-dimensional (3D) imaging, automated liquid handling, and automated image analysis have expanded researchers' capabilities to employ more physiologically mimetic systems in vitro.126 As discussed below many high-throughput screening (HTS) approaches focus on measuring changes in gene expression in 2D or 3D culture schemes, with detection of functional changes in HT occasionally requiring more nuance. 2D plate-based approaches 2D plate-based HT formats have been the gold standard of HT methods for decades. Superficially, cells are plated in a multiwell plate (96, 384, 1536-well plates), subjected to some treatment, and evaluated using automated image acquisition and analysis. These approaches have been applied to a wide variety of biological questions and are easily adaptable to evaluating islet and β-cell biology thanks to the availability of β-cell lines. One of the most evaluated aspects of β-cell biology in HT approaches is proliferation. Patients suffering from both T1D and T2D would benefit from enhanced β-cell proliferation; however, there are no clinically available drugs that are marketed for this purpose. Patients with both T1D and T2D may still have residual β-cells, which has led to a large research effort do identify compounds that can stimulate β-cell renewal.127 The R7T1 β-cell line contains a tet-on construct, only permitting its proliferation in the presence of a tetracycline. It is therefore an easy model to study β-cell proliferation in multiwell plates using viability-based assays (eg, CellTiter Glo).128,129 Images based assessment of cell proliferation via EDU incorporation and Ki67 staining has been used to identify novel genes involved in β-cell proliferation130 as well as compounds that enhance proliferation.131 Care should be taken using R7T1, as chemicals similar to tetracyclines may also induce proliferation.129 When dispersed primary islets are used, extra steps need to be taken to ensure proper physiological function and data integrity, such as pre-treating plates with extracellular matrix and omitting data from non-β-cells using image analysis, respectively.131 In that regard, an HT approach has also been developed to screen polymer surfaces for optimal attachment of dispersed islet cells, quantified with automated image analysis of insulin-expressing cells.132 2D plate-based HTS make excellent use of reporter-driven live cell automated imaging. Quantifying live changes in gene expression has unique utility in designing PSC differentiation protocols. Adapting PSCs to HT approaches has been thoroughly reviewed elsewhere.133 These approaches have helped define protocols that use small molecules that are now commonly used in many PSC differentiation protocols,134 including those designed to produce β-(like)-cells.135 The gradual replacement of recombinant proteins with small molecules is greatly reducing cost and encouraging more labs to explore stem cell differentiation protocols, with HTS playing an essential role. Reporter-driven HT approaches for β-cell differentiation center around increasing expression of genes critical to β-cell development, such as PDX1, NKX6.1, NEUROG3, MAFA and INS1, among others.136 Assaying for compounds that induce expression of certain transcription factors appropriate later in pancreatic differentiations, especially insulin, requires starting with cells already geared toward the pancreatic endoderm or endocrine precursors. For example HTS identified novel compounds that induce or enhance PDX1 expression during PSCs differentiation137,138 and in pancreatic ductal adenocarcinoma cell lines.139 Some approaches incorporate multiple gene reporters to screen for compounds that may enhance both PDX1 and INS1, for example.140 This same reporter system was used to identify a novel role of Na+ channels in modulating insulin expression.141 HTS was used to screen for the prohormone VGF,142 which has been shown to be essential in normal β-cell function and development.143 While cell lines or dispersed islets are well-suited in 2D HTS approaches, results are often validated using intact primary islets and animal models. The next generation of HTS could use physiological readouts such as insulin secretion or membrane depolarization, although adapting these to high throughput pose significant challenges, as described below. 3D approaches The frequent usage of imaging/image analysis in HT models poses unique challenges when employing more physiologically relevant 3D approaches. Advances in recent years in such as tissue clearing (eg, CLARITY144) and rapid 3D imaging platforms (eg, spinning disk, light sheet microscopy) have accelerated the merging of 3D models and HT technologies. A fluidic device combining on-chip culture, fixation, clearing, and imaging was demonstrated in a proof-of-concept study describing mouse islet vasculature in HT.145 Current costs of adopting these approaches has precluded widespread adoption, however. In that regard, simple epifluorescence plate-based imaging approaches can be applied to cell aggregates, such as dual INS1-NEUROG3 reporter transfected PSC aggregates.146 Higher resolution images of entire aggregates are not necessary in this specific application, as the presence of and ratios between two fluorescent reporters are the primary output. Automated image analysis can also be used for morphological analysis of islets encapsulated within biomaterials.147 This approach has been applied to encapsulation strategies148 and can be further applied to widely adopted immunoisolation strategies employed by several labs seeking to optimize islet health and engraftment upon transplantation,13,149,150 as well as stem cell differentiation.151 3D models have been applied where the primary output is not imaging-based, such as experiments studying secreted factors or lytic end point analyses. Aggregates of INS-1832/13 cells or human pseudoislets can be assayed for compounds that modulate static GSIS when placed in a 384-well plate.152 Aggregate-based approaches are often used in fluidic platforms to a certain HT degree, and may have several outputs quantified as discussed in the next section. Imaging aggregates in 3D has several technical challenges, such as movement of aggregates within the culture vessel because of plate scanning during image acquisition. Inspiration can be taken from approaches developed for whole organism HT studies using zebrafish. A workflow for HTS using transgenic insulin-YFP zebrafish that incorporates z-depth autofocusing and regional scanning/reporting in HT formats (termed automated reporter quantification in vivo [ARQiv]).153 This workflow was later applied to identify compounds that boost β-cell development154 or proliferation,155 taking advantage of the zebrafish's unique pancreas architecture. A separate group employed a fluorescent ubiquitylation-based cell cycle indicator (FUCCI) to identify compounds that induce proliferation in β-cells.156 Compounds that affect surrounding cells (islet vasculature, stroma) and indirectly enhance β-cell proliferation can be evaluated in whole-organisms HT scenarios. Other adverse effects, such as off-target carcinogenesis, can also be evaluated in this setting. HT functional studies High throughput approaches have enormous utility in probing β-cell biology. Understanding of the primary mechanisms behind β-cell apoptosis, metabolism, insulin secretion, and morphology can illuminate new opportunities for which therapeutics can be designed. However, designing HT studies based upon β-cell's physiological output can be technically challenging. Many of the studies above assay for changes in gene or protein expression; however, most of their follow-up validation studies that involve β-cell physiology do not qualify as HT. Indeed, reserving complex functional studies for low throughput validation is the primary reasoning for simplified, first-pass HT approaches. However, studying the biological end goal (eg, enhancing insulin secretion) in HT would potentially yield better results by streamlining workflow and cutting out intermediate steps. Several studies have measured static GSIS in HT in 2D arrays using cell lines,130,157-159 occasionally employing the use of automated liquid handling. Fluorescent dyes can also enable indirect HT static GSIS measurements via Ca2+ imaging using plate formats and automated liquid handling.129 However, PSC-based approaches have shown, Ca2+ imaging is not necessarily indicative of insulin secretion.160 As a method to employ fast and easy-to-use plate reader-based approaches, Min6 cells have been modified to cosecrete an insulin-luciferase fusion protein, and assayed for GSIS-enhancing compounds in HT.161 A similar technique was also used with INS1E cells157 to monitor GSIS. Other aspects of INS1E secretion physiology were quantified in scalable plate reader-based format using the membrane potential indicator dye DIBAC4(3), the Ca2+ dye Fura 2-AM, mitochondrial activity with rhodamine 123, and membrane lipid tension using FliptR.157 Several fluidic platforms can be used in HT; however, detecting secreted insulin in large volumes of fluid requires that several islets/aggregates be grouped together.64,65 The device designed by Lee et al involves 110 miniature bioreactors to study 1.1B4 cell line physiology under glucotoxic or lipotoxic conditions.64 This platform enables the formation of aggregates, live imaging, perfusion media sampling for GSIS, and cell lysis for genomic analysis all on the same culture device all in HT numbers. HT analysis of multiple biological processes of single islets using Raman microspectroscopy in a nondestructive manner was demonstrated in.72 Nondestructive techniques have potential applications in screening islets prior to transplantation without having to sacrifice tissue as scarce as islets. β-Cell death is involved in both T1D and T2D, and may be caused by immune-mediated destruction or exhaustion and gluco/lipotoxic conditions. Preventing β-cell destruction by these mechanisms is therefore a goal of several HT approaches that screen compounds that mitigate endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as well apoptosis. As these processes are not exclusive to β-cells, several commercially available kits are for a variety of cellular processes can be easily scaled to HT levels. Some studies described in this section scaled popular MTT/XTT viability kits to HT.157,158,162 Dispersed human islets were used in a 384-plate-based siRNA screen, using automated liquid handling, to screen for novel antiapoptotic genes. Apoptosis as well as cellular reducing power were quantified using commercially available kits. Plate-based caspase activity assays (eg, Caspase-Glo) facilitate apoptosis quantification in 2D formats, while ER stress can be measured with CHOP-luciferase reporters.159 ER stress can also be measured indirectly by quantifying the ratio of XBP1 spliced/unspliced, in addition to quantifying phosphorylated IRE1-α protein, both of which can be done in HT.158 CONCLUSION Engineered human β-cells, β-cell functional readouts and engineered systems that allow for high throughput drug screening are all examples engineered-inspired approaches to study β-cells. Many of these systems have been used to uncover aspects of islet biology and insulin secretion physiology impossible to observe in traditional static or low-throughput systems. Features of dynamic insulin secretion profiles have been discovered as a result of single islet engineered systems. Novel roles of support cells within islets, such as endothelial cells, have been uncovered thanks to fluidic systems. Scientists and engineers are only beginning to scratch the surface of modeling interorgan crosstalk using MPS. MPS studies hold promise in the development of drugs that harness interorgan crosstalk, such as incretin-modifying drugs that modulate intestine-pancreas communication for the treatment of T2D. Many researchers are even using MPS to study ways to mitigate immune system attack of β-cells through islet vasculature. With the massive scale up underway, progressively more engineered strategies qualify as HT. HT strategies are already causing massive changes in the ways researchers evaluate efficacy of small molecule-induced changes in gene expression. New approaches to quantify β-cell function in HT can lead to the development of novel drugs, or drug classes, that can directly influence β-cell physiology. Moreover, researchers are developing novel ways to evaluate and enhance the physiology if engineered β-cells. Researchers have even created fully synthetic insulin secreting cells capable of responding to glucose.163 Furthermore, only briefly touched upon in this review are the remarkable advances achieved in engineered extracellular matrix platforms for islet culture and transplantation.164,165 The increased sensitivity, sophistication, and versatility of the engineered models described above have achieved profound impact in their ability further dissect islet biology and insulin secretion physiology. Future approaches combining many of the approaches outlined above will no doubt lead to significant discoveries eventually leading to vast improvements in the treatment of diabetes. ACKNOWLEDGMENTS J.M.W. and P.L.L. are supported by the NIH grants P01 HD093363 and UG3 DK119982 (J.M.W.), a Shipley Foundation award (J.M.W.), and an award from the Allen Foundation (J.M.W.). We would also like to thank Dr Heather A. McCauley for critical reading of this manuscript. AUTHOR CONTRIBUTIONS P.L.L.: manuscript conception, writing, editing, final approval; J.M.W.: manuscript conception, editing, final approval. CONFLICT OF INTEREST J.M.W. declared research funding from the NIH. P.L.L. declared no potential conflicts of interest. DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article as no new data were created or analyzed in this study. REFERENCES 1 Collaboration NRF . 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Funding information Allen Foundation; Eunice Kennedy Shriver National Institute of Child Health and Human Development, Grant/Award Number: P01 HD093363; National Institute of Diabetes and Digestive and Kidney Diseases, Grant/Award Number: UG3 DK119982; Shipley Foundation © AlphaMed Press 2021 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Engineering-inspired approaches to study β-cell function and diabetes
JF - Stem Cells
DO - 10.1002/stem.3340
DA - 2021-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/engineering-inspired-approaches-to-study-cell-function-and-diabetes-kQzaz5djbK
SP - 522
EP - 535
VL - 39
IS - 5
DP - DeepDyve
ER -