Many cell types within the body, such as those in muscle and fat, require insulin to intake glucose from their surrounding environment so that they can use it as energy. This insulin is produced and secreted by a type of endocrine cell called the β cell within the pancreas. These β cells are able to sense changes in the level of glucose within the blood and secrete insulin to keep this blood glucose level within a narrow range. When a person eats, for example, the carbohydrates in the meal are quickly broken down into glucose and absorbed into the bloodstream. The β cells rapidly sense this spike in blood sugar and secrete enough insulin to allow this glucose to enter cells throughout the body. As glucose leaves the bloodstream and enters cells, the β cells shut off their insulin secretion so that the blood glucose concentration will not fall below a crucial lower threshold that is required for proper neurological function. Because glucose is a main source of energy for our bodies, the regulation of blood glucose levels is constantly adapting to changes in our energy intake and expenditure throughout the day. Additional factors, such as the release of stress hormones, can also alter the sensitivity of cells to insulin.
For most people, this dynamic regulation of blood glucose concentration takes care of itself. However, patients with diabetes mellitus have lost their ability to secrete an adequate amount of insulin either because their β cells were destroyed through an autoimmune process (type 1) or due to a deficiency in either insulin secretion by β cells or in the way insulin is used (type 2). Without insulin, glucose is not able to enter cells and instead remains in the blood. Over the short term, cells are essentially starving for energy. Over the long term, persistent elevated blood glucose levels can lead to a variety of complications due to the way glucose interacts with various tissues at these high concentrations. Thus, it is critical that patients with diabetes try to maintain their blood sugar level as close to normal as possible. All patients with type 1 diabetes, as well as many with type 2, must inject insulin throughout the day in order to survive. As you can imagine, injecting an estimated amount of insulin does not control blood sugar levels nearly as well as the fine-tuned regulation performed by a β cell. Injecting too much insulin can cause blood sugar levels to dip dangerously low in the short term, while injecting too little insulin will result in elevated blood sugars that can cause complications overtime. Due to changing insulin demands based on factors such as food intake, physical activity, stress levels, and sleep, it can be difficult for patients to maintain their blood glucose levels in a normal range all the time. While improvements in technology such as insulin pumps and glucose sensors have undoubtedly helped make living with diabetes easier, it still is an imperfect and often burdensome therapy.
An alternative method to controlling blood glucose concentration would be to replace the destroyed or damaged β cells. Such a cell replacement therapy would be life-changing for many diabetic patients, freeing them from the stress of managing this chronic disease. To this end, much progress has been made in the generation of β cells from human pluripotent stem cells. This differentiation methodology tries to mimic the signaling cues a cell experiences during embryonic development through the use of growth factors and small molecules, driving the stem cells through a number of intermediate cell types on their way to becoming functional pancreatic β cells. The development of this differentiation process has been accomplished through the excellent work of various research groups over the past two decades, resulting in sequential differentiation stages that have pushed the cells closer toward a β cell fate. Eventually, a breakthrough occurred in 2014 when several research groups published protocols that demonstrated robust generation of glucose-responsive, insulin-secreting stem cell-derived β (SC-β) cells [1-2], which have been further improved since then [3-4].
While these protocols successfully utilized the timed application of soluble molecules to direct differentiation, one potentially overlooked source of signaling can arise from the insoluble microenvironment surrounding a cell, such as through the extracellular matrix and its physical properties. As I explored incorporating some of these microenvironmental cues into various stages of the differentiation protocol, it became apparent that culturing pancreatic progenitors on a rigid polystyrene surface promoted the desired PDX1+/NKX6-1+ pancreatic progenitor phenotype but blocked their subsequent conversion to endocrine cells. This observation was interesting, as previous protocols that successfully generated SC-β cells were either done completely with suspension culture or required clustering of the cells during endocrine induction. A well-known effect of culturing cells on a stiff substrate is the polymerization of their actin cytoskeleton, which they use to pull against the rigid surface and which can influence multiple signaling pathways. I thus screened various compounds known to influence the cytoskeleton and found that depolymerizing F-actin with the compound latrunculin A during the first 24 hours of endocrine induction was enough to overcome this signaling bottleneck and promote robust formation of endocrine cells using traditional planar culture techniques, as described in our recent Nature Biotechnology paper .
In our new Nature Protocols paper , we provide a step-by-step guide for generating and characterizing SC-β cells using this planar differentiation methodology. In our experience, there are several unique advantages for doing the protocol this way. First, it simplifies the procedure by requiring only basic stem cell culture techniques rather than relying on more complicated suspension or clustering methods. In fact, we now reliably have new lab members that have little cell culture experience quickly learn how to successfully generate functional SC-β cells, often on their first attempt. Secondly, in our hands, we are able to generate more SC-β cells per volume of media used throughout the differentiation than with our previous suspension method. Furthermore, the cells we generate with the planar protocol are often more functional than those generated in suspension culture. Importantly, we are now able to generate SC-β cells from cell lines that we could not get to work with the previous suspension protocol. In particular, we provide data for the successful generation of SC-β cells from 10 human pluripotent stem cell lines from a wide variety of genetic backgrounds, 5 of which we have not published on previously. This list includes induced pluripotent stem cell lines derived from patients with type 1 diabetes, type 2 diabetes, Maturity-Onset Diabetes of the Young (MODY), and Wolfram Syndrome, providing the opportunity to better study different diabetic phenotypes in the lab.
Despite the excellent work that has been accomplished in developing a methodology for generating SC-β cells over the past two decades, there has been a lack of a user-friendly guide for making these cells. We hope that combined with the improved simplicity of the procedure and increased reproducibility across cell lines, this protocol will be a helpful guide that will decrease the barrier of entry for researchers to generate their own SC-β cells and use them in their diabetes research. And ultimately, we sincerely hope that increased accessibility to these cells in diabetes research will eventually help lead to the successful development of a β cell replacement therapy, providing a functional cure for people with diabetes.
Figure 1: Protocol for making SC-β cells. The generation of insulin-producing SC-β cells is accomplished through the sequential addition of growth factors and small molecules, driving stem cells through several intermediate cell types over the course of 5 weeks. The quality of a differentiation can be assessed at select stages along the way by measuring protein markers with flow cytometry (FC) and immunocytochemistry (ICC), and the functional properties of the final SC-β cells can be assessed by a glucose-stimulated insulin secretion (GSIS) assay. The SC-β cells are generated in planar culture using standard cell culture plasticware, but these cells can be aggregated into clusters using a simple procedure for use in other assays, such as transplantation into diabetic mice.
 Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. Generation of functional human pancreatic β cells in vitro. Cell. 2014 Oct 9;159(2):428-39. doi: 10.1016/j.cell.2014.09.040.
 Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, Asadi A, O'Dwyer S, Quiskamp N, Mojibian M, Albrecht T, Yang YH, Johnson JD, Kieffer TJ. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nature Biotechnology. 2014 Nov;32(11):1121-33. doi: 10.1038/nbt.3033.
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 Hogrebe NJ, Augsornworawat P, Maxwell KG, Velazco-Cruz L, Millman JR. Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells. Nature Biotechnology. 2020 Apr;38(4):460-470. doi: 10.1038/s41587-020-0430-6.
 Hogrebe NJ, Maxwell KG, Augsornworawat P, Millman JR. Generation of insulin-producing pancreatic β cells from multiple human stem cell lines. Nature Protocols. 2021 Aug 4. doi: 10.1038/s41596-021-00560-y.