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A method for the generation of human stem cell-derived alpha cells

biological techniques pancreas stem-cell differentiation

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#1 Engadin

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Posted 08 May 2020 - 01:00 PM


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O P E N   A C C E S S   S O U R C E :   nature

 

 

 

 

 

 

Abstract
The generation of pancreatic cell types from renewable cell sources holds promise for cell replacement therapies for diabetes. Although most effort has focused on generating pancreatic beta cells, considerable evidence indicates that glucagon secreting alpha cells are critically involved in disease progression and proper glucose control. Here we report on the generation of stem cell-derived human pancreatic alpha (SC-alpha) cells from pluripotent stem cells via a transient pre-alpha cell intermediate. These pre-alpha cells exhibit a transcriptional profile similar to mature alpha cells and although they produce proinsulin protein, they do not secrete significant amounts of processed insulin. Compound screening identified a protein kinase c activator that promotes maturation of pre-alpha cells into SC-alpha cells. The resulting SC-alpha cells do not express insulin, share an ultrastructure similar to cadaveric alpha cells, express and secrete glucagon in response to glucose and some glucagon secretagogues, and elevate blood glucose upon transplantation in mice.
 
 
Introduction
 
Although diabetes primarily involves beta cell dysfunction, there is mounting evidence that alpha cell defects play a role in disease etiology1,2. Patients with type 1 diabetes must cope with significant fluctuation in blood glucose levels including acute hypoglycemia3.
 
In the healthy pancreas, hormone-expressing endocrine cells function within the islets of Langerhans to precisely regulate blood glucose and energy metabolism. During hypoglycemia (low blood glucose), islet alpha cells secrete glucagon4 which raises blood glucose levels by increasing glycogenolysis and gluconeogenesis in the liver5,6. Although alpha cells persist in diabetic islets, these alpha cells are often incapable of mounting an appropriate glucagon response, perhaps due to the absence of alpha cell−beta cell interactions7. Recent studies implicate dysfunction of alpha cells as a contributing factor in the elevated blood glucose levels observed in diabetic patients4,7.
 
Published methods to make pancreatic beta cells8,9,10,11 all report a minor portion of alpha and delta (somatostatin secreting) cells. Additionally, there are several reports on the conversion of various cell types into alpha cells via transdifferentiation12,13,14. In 2011, Rezania et al.15 reported a protocol for generating glucagon-positive cells that exhibited some glucagon secretion in vitro; however, upon transplantation of 1.9 million cells into mice, these cells had limited physiological effects. Despite these early efforts to generate glucagon-positive cells, the production of alpha cells has not been reproduced nor widely adopted by the field.
 
We previously reported the generation of functional pancreatic beta cells from human pluripotent stem cells using a six-step directed differentiation protocol8. That protocol generates beta cells as well as side populations including polyhormonal cells and nonendocrine cells11. The presence of polyhormonal cells in pancreatic differentiations have also been observed in other reported protocols9,16,17. Several reports described polyhormonal cells as having features of immature beta cells9,17,18,19; however, beyond the expression of insulin protein, there has been little evidence to support the similarity of polyhormonal cells to beta cells, let alone their capacity to differentiate into beta cells. In contrast, others have reported that polyhormonal cells are present during development, contribute to alpha cells later in development, express several markers of alpha cells, and give rise to glucagon-expressing cells when transplanted11,15,20.
 
Reports from the literature have referred to INS+/GCG+ cells in turn as “bi-hormonal”21, “polyhormonal”9,22,23,24, “alpha-like cells”17, or “alpha cells”25. The nomenclature of the INS+/GCG+ cells produced in directed differentiation protocols has become equally confusing. In Veres et al.11, we performed extensive scRNA-seq studies on beta cell differentiations and observed a population of cells expressing many markers of alpha cells which we labeled as “alpha-like” cells irrespective of insulin expression. In this manuscript we refine this definition of “alpha-like” cells to distinguish between cells that have detectable insulin protein which we define as “pre-alpha” cells and those that do not express insulin, which we define as “SC-alpha cells”. We favor the term “pre-alpha” rather than “bi-hormonal” or “polyhormonal” because it more accurately depicts the similarity of these cells to alpha cells yet distinguishes them from more mature alpha cells that do not express insulin (SC-alpha).
 
Here we build upon our previous reports to develop a protocol for the generation of SC-alpha cells. The protocol robustly produces a transient pre-alpha cell intermediate, which has a transcriptional signature similar to alpha cells except that they express insulin in addition to glucagon. We identify a small molecule capable of driving pre-alpha cells to an alpha cell identity and demonstrate the ability to produce approximately 30% SC-alpha cells in vitro. Our results show that these SC-alpha cells have a transcriptional signature similar to human cadaveric alpha cells, are responsive to some glucagon secretagogues, and elicit a robust physiological response within 4 weeks of transplantation in mice. The SC-alpha cells generated can recapitulate central aspects of alpha cell biology and represent an efficient and scalable avenue to produce alpha cells for use in islet organoids for cell replacement therapy, drug screening, disease modeling, and may accelerate exploration of alpha cell biology.
 
 
Results
 
Optimization of differentiation to generate pre-alpha cells
 
Our previously published protocol for generating stem cell-derived beta cells (SC-beta cells) produces a small but significant population of pre-alpha cells8,11. Figure 1a shows a typical result wherein 27% of the cells are glucose-responsive SC-beta cells and 9% are pre-alpha cells expressing both insulin and glucagon. Kelly et al.16 suggested that these pre-alpha cells (referred to as polyhormonal in ref. 16) come from progenitors that fail to express NKX6.1. Thus, we sought to modify our beta cell protocol to prevent or reduce induction of NKX6.1 at stage 4. Using the HUES8 embryonic stem cell line, we observed that removal of KGF, SANT-1 and treatment with LDN only on day 2 of stage 3 results in a decrease of NKX6.1-positive cells11. In addition, we observed that treatment with LDN on day 1 of stage 4 and incubation with no factors for the remainder of stage 4 resulted in a significant population of Chromogranin A+/NKX6.1− cells. Together these protocol modifications (Fig. 1b) resulted in a large fraction of pre-alpha cells as marked by the coexpression of insulin and glucagon proteins (Fig. 1c). In the HUES8 cell line, this pre-alpha cell optimized protocol produces an average of 62.6 ± 2.3% insulin+ and glucagon+ coexpressing pre-alpha cells and a small percentage (<10%) of monohormonal glucagon-expressing SC-alpha cells (Supplementary Fig. 1). Immunofluorescent staining confirms a high proportion of cells that express both insulin and glucagon protein (Fig. 1d) distributed throughout the cell clusters.
 
 
41467_2020_16049_Fig1_HTML.png
 
Fig. 1: The development of protocols for generating pre-alpha cells.
a Embryonic stem cell differentiation to SC-beta cells results in a heterogeneous population. In this differentiation, 8.9% of cells coexpress markers for insulin and glucagon. b Schematic of directed differentiation protocol from hPSC into pre-alpha cells. The factors added at each step are noted below, catalog numbers are provided in Supplementary Table 1. c In the resulting pre-alpha cell differentiation, 63% of the cells stain for glucagon and insulin proteins. d Immunofluorescent cross-sectional staining of a cluster for insulin and glucagon. Scale bar = 50 μm. e Single-cell RNAseq analysis of pre-alpha cells from the SC-alpha protocol. Expression data are visualized with a tSNE plot where cells are clustered based on the similarity of transcript expression patterns. The expression profile for cells in the pre-alpha cell cluster is similar to the expression profile of human islet alpha cells (Supplementary Fig. 17b). Population ratios by this analysis are consistent with the flow cytometry plot in c. f Histograms of expression levels (as violin plots) for pancreatic hormones of SC-alpha cells and cadaveric human islets27. g Pre-alpha cells secrete glucagon, but do not secret insulin. Representative ELISA measurements of secreted glucagon (left panel) from HUES8 differentiated pre-alpha cells or human islets challenged sequentially with 2.8 or 20 mM glucose with a 60-min incubation for each concentration. Representative ELISA measurement of secreted full processed human insulin (right panel) from HUES8 differentiated pre-alpha cells or human islets challenged sequentially with 2.8 or 20 mM glucose with a 60-min incubation for each concentration. Data presented as mean glucagon or insulin secreted per 1000 total cells ± SEM (n = 3 biologically independent samples) Significance calculated using a two-tailed paired Student’s t test. ESC: embryonic stem cell, DE: definitive endoderm, GTE: gut tube endoderm, PP: pancreatic progenitor, EP: endocrine progenitor, PA: pre-alpha cell, KGF: keratinocyte growth factor, LDN: LDN193189, Alk5i: Alk5 inhibitor II, Repl.: replicating cells.
 
 
 
Pre-alpha cell transcriptional profile
 
We investigated the transcriptional signature of the pre-alpha populations produced at the end of stage 5 by single-cell RNAseq. Using single-cell sequencing (inDrops)26, we profiled 2043 cells from a pre-alpha cell differentiation revealing four distinct cell populations (Fig. 1e). Confirming the immunostaining and flow cytometry analysis, we observed a population of cells that express both insulin and glucagon transcripts, although expression of insulin transcripts was significantly lower than glucagon transcripts (mean tpm of 649 vs. 214,320; Fig. 1f and Supplementary Fig. 2a), indicating that these cells have downregulated insulin expression. This pre-alpha cell population (pink in Fig. 1e) expresses a transcriptional signature more similar to alpha cells than to beta cells (Supplementary Figs. 2b and 3). In addition to expressing insulin and glucagon transcripts, the pre-alpha cells also express transcripts for several markers of alpha cells and lack several key markers for beta cells. For example, pre-alpha cells express transcripts for ARX, IRX1, and IRX2, transcription factors that are expressed in endogenous alpha cells27 (Supplementary Fig. 2b), but do not express transcripts for beta cell markers such as NKX6-1, PDX1, and PAX4 (Supplementary Fig. 3). Figure 1f shows the relative transcript expression levels of pancreatic hormones in the pre-alpha cell population compared to the major endocrine cell types from human islets.
 
In addition to the pre-alpha cell population, two minor cell populations are present including a TPH1-positive enterochromaffin population11 and a nonendocrine population. The presence of these minor populations compared to the large number of pre-alpha cells confirms at the transcriptional level our finding that the majority of cells produced with this protocol are pre-alpha cells. Since the transcriptional profile of pre-alpha cells closely resembles alpha cells, we hypothesized that the pre-alpha cell population may be useful in generating stem-cell-derived alpha (SC-alpha) cells.
 
 
Pre-alpha cells secrete glucagon but not insulin
 
Due to the transcriptional similarity of pre-alpha cells to primary (cadaveric) alpha cells, we evaluated the functional response of pre-alpha cells to glucose. Pre-alpha cell clusters secrete glucagon under low-glucose (2.8 mM) conditions and suppress glucagon secretion at high glucose (20 mM) concentrations (Fig. 1g). Membrane depolarization induced by high (30 mM) extracellular K+ stimulated glucagon secretion in both pre-alpha cells and mature human islet alpha cells. When assessing insulin secretion from these cells with a human specific processed-insulin ELISA, pre-alpha cells do not secrete significant quantities of processed insulin compared to human islets. Although these cells express insulin protein (Fig. 1c), the c-peptide antibody used in flow cytometry and immunofluorescence experiments does not distinguish fully processed insulin from its precursor, indicating that pre-alpha cells do not process proinsulin into insulin.
 
To analyze the insulin processing in pre-alpha cells, we evaluated proinsulin processing by Western blot (Supplementary Fig. 2c). In human islets, the majority of proinsulin protein is converted to insulin, while in pre-alpha cells, the majority remains as proinsulin. We assessed transcriptional expression of the prohormone converting enzymes PC1 and PC2 (PCSK1 and PCSK2 genes) and found that pre-alpha cells expressed PCSK2 to a much higher degree than they express PCSK1 (Supplementary Fig. 2b). Thus, pre-alpha cells transcribe the insulin gene and produce proinsulin protein, but do not cleave proinsulin nor secrete mature insulin in significant quantities.
 
 
The pre-alpha cell is a transient state in vitro and in vivo
 
Previous reports demonstrated the presence of a small population of alpha cells in grafts from transplanted SC-beta cell differentiations8. We postulated that these alpha cells were derived from the pre-alpha cell side populations present in these SC-beta cell differentiations. As such, we tested the ability of pre-alpha cells generated in our protocol to convert into SC-alpha cells post transplant. We transplanted 5 million pre-alpha cells under the kidney capsule of (n = 10) immunocompromised (SCID-beige) mice and observed a reduced expression of insulin protein over the course of 4 weeks. Grafts were retrieved 14, 28, or 56 days after transplantation and assessed for hormone expression using antibodies that react with insulin and glucagon proteins. At 14 days after transplantation, pre-alpha cells continued to express insulin and glucagon proteins (Fig. 2a left, Pearson’s R value = 0.57). When grafts were evaluated at 28 days, few insulin protein-expressing cells were observed, whereas glucagon protein-expressing cells persisted (Fig. 2a middle, Pearson’s R value = 0.15). This population of monohormonal glucagon-expressing cells were observed for up to 56 days post transplant (Fig. 2a right, Pearson’s R value = 0.06). These results suggest that insulin protein expression is reduced in pre-alpha cells and glucagon protein expression is maintained with extended time in vivo. This result is consistent with previous studies which concluded that cells expressing both insulin and glucagon can resolve into alpha cells20,25,28,29. To exclude the possibility that the increase in SC-alpha cells observed after transplantation was due to selective replication of an SC-alpha subpopulation and/or concomitant death of pre-alpha cells, we evaluated cell replication and apoptosis during this in vivo maturation (Supplementary Fig. 4). Rarely were TUNEL+/glucagon+ cells observed. Although low levels of Ki67-positive replicating cells were observed, they occurred equally in cells expressing both insulin and glucagon and glucagon-only (Supplementary Fig. 4).
 
 
41467_2020_16049_Fig2_HTML.png
 
 
Fig. 2: Insulin expression is reduced following transplantation and extended culture in vitro.
Expression of insulin and glucagon in grafts after transplantation of pre-alpha cells under the kidney capsule of mice (n = 10 animals) at 14 days (left), 28 days (middle) and 56 days (right) post transplant. Inlay shows zoomed-in view of cells at each time point. Coexpression of insulin and glucagon is observed at 14 days. At 28 and 56 days, insulin expression is absent from the majority of cells. Images are representative of n = 2 grafts harvested at each time point. Grafts harvested at 4 and 7 days are not shown. Scale bar = 50 μm. b Extended culture in vitro results in a fraction of pre-alpha cells reducing insulin expression and continuing to express glucagon (highlighted in red). After 28 days extended culture, about 23% of cells express glucagon but not insulin.
 
 
 
Given the ability of pre-alpha cells to reduce insulin protein expression after transplantation, we tested whether this conversion can occur in vitro. After generation of pre-alpha cells, we continued culturing the cells for an additional 4 weeks in media without growth factors. During this extended culture period, cells were sampled at 7-day intervals and assessed for insulin and glucagon protein expression by flow cytometry. As shown in Fig. 2b, the vast majority of these cells (>80%) expressed both insulin and glucagon proteins at the beginning of the culture period. After 14 days, little change had occurred as >80% of the cells still expressed insulin and glucagon protein. After 21 days, a population of insulin-, glucagon+ cells appears with ~20% of the cells expressing only glucagon protein. This population of monohormonal SC-alpha cells that appeared at day 21, persists at day 28 of extended culture. These results indicate that upon extended culture in vitro, a fraction of pre-alpha cells reduce insulin protein expression and become SC-alpha cells.
 
 
PKC activation promotes maturation of SC-alpha cells
 
Given that pre-alpha cells can convert to SC-alpha cells in vivo and in vitro, we sought to identify signals that promote this conversion by performing a small-molecule screen. HUES8 embryonic stem cells were differentiated using the pre-alpha cell optimized protocol to the end of stage 5 (stage 6 day 1), arrayed into 384-well plates and incubated with a custom 43-member small-molecule library (Supplementary Table 3) where compounds were chosen for their activity in targeting signaling pathways. After 96 h of treatment (stage 6 day 5), cells were fixed and stained for insulin and glucagon (Fig. 3a). High-content imaging was used to quantify the dispersed (2D) cell populations for the percentage of cells expressing each hormone individually and the percentage of pre-alpha cells marked by expression of both hormones (Fig. 3b, c). The effect of each compound was evaluated in quintuplicate assays. The protein kinase c (PKC) activator phorbol 12,13-dibutyrate (PDBu) decreased the percentage of insulin protein-expressing cells and increased the percentage of glucagon-expressing cells compared to vehicle controls. To confirm that the effects of this compound were not unique to the planar assay format, PDBu was evaluated in the final stage of the 3D directed differentiation protocol. Over the course of a 28-day treatment, PDBu induced a significant population of pre-alpha cell clusters to reduce insulin protein expression compared to control (33 ± 6% vs. 16 ± 5%; n = 5; Fig. 3d and Supplementary Fig. 5). The resulting cell population contained significantly more alpha cells that did not coexpress the insulin protein (Fig. 3e).
 
 
41467_2020_16049_Fig3_HTML.png
 
 
Fig. 3: Screen to identify compounds that promote alpha cell identity.
Small molecules targeting known pathways (43 compounds) were incubated with pre-alpha cells in quintuplicate for 96 h. a Schematic of screening approach. Primary screening results showing the percentage of cells expressing both insulin and glucagon and c the percentage of cells expressing only glucagon. The data are presented as mean ± SEM (n = 5 biologically independent samples). The PKC activator PDBu significantly reduced the percentage of pre-alpha cells and increased the percentage of cells expressing glucagon. d Representative flow cytometry results of pre-alpha cells at stage 6 day 1, and stage 6 day 28 control (no treatment), or 28 days treatment with the PKC activator PDBu. e Immunofluorescence of clusters treated with and without PDBu for 28 days. Scale bar = 50 μm. f Additional PKC activators evaluated for their effect on pre-alpha cells (stage 6 day 1). All PKC activators increased the percentage of SC-alpha cells. The data are presented as mean ± SEM, significance calculated using an ordinary one-way ANOVA with Dunnett multiple comparison test (n = 3 biologically independent samples). g Stage 6 of protocol converts pre-alpha cells into SC-alpha cells.
 
 
 
To further explore the specificity of PKC activation, a small library of known PKC activators was evaluated. This structurally diverse set of compounds have all been reported to have PKC activating abilities. We added pre-alpha cell clusters (stage 6 day 1) to six-well plates and treated with either vehicle or each PKC activator for 28 days and assessed the percentage of monohormonal, glucagon-expressing alpha cells (Fig. 3f and Supplementary Fig. 6). While the control condition resulted in only 13.7% SC-alpha cells, treatment with each PKC activator resulted in a significant increase in the percentage of SC-alpha cells, though none more so than PDBu. To evaluate the stability of the shift from pre-alpha cells to SC-alpha cells, we withdrew PDBu from cells for 7 days and evaluated the percentage of monohormonal glucagon-expressing SC-alpha cells (Supplementary Fig. 7). Withdrawal of PDBu did not significantly affect the percentage of SC-alpha cells in our population. These results demonstrate that activation of PKC stably reduces the expression of insulin in the INS+/GCG+ population thereby accelerating the conversion process of pre-alpha cells to SC-alpha cells in vitro (Fig. 3g).
 
 
 
 
 
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