Continuous inhibition of Sonic hedgehog signaling effectively leads to differentiation of human-induced pluripotent stem cells into functional insulin-producing β cells

Background: Human-induced pluripotent stem cell (iPSC)-derived insulin-producing cells (IPCs) can be used for islet cell transplantation in type 1 diabetic patients and as patient-specic cells for the development of novel anti-diabetic drugs. Therefore, it is necessary develop a method for generating functional IPCs from iPSCs and simplifying the stepwise protocol. Methods: We compared combinations of small molecules that could eciently induce the differentiation of cells into a denitive endoderm, and preferentially into islet precursor cells. IPCs, generated using the optimal combination of small molecules, were conrmed to demonstrate insulin secretion in response to glucose stimulation. Finally, we re-constructed spheroid IPCs and veried the optimized culture and maturation conditions. Results: It was conrmed by quantitative polymerase chain reaction that denitive endoderm-specic markers were expressed differently depending on the combination of the small molecules used. Small molecule SANT-1 induced the differentiation of cells into functional IPCs by acting as an inhibitor of Sonic hedgehog signaling. Images of 2D culture showed that IPCs were spheroid-shaped from day 5 and demonstrated sustained insulin secretion. We developed a simple differentiation method using small molecules that produced functional IPCs that responded eciently to glucose stimulation in a relatively short time. Conclusions: We posit that this method along with a method that renes the process of differentiation can be used for growing IPCs that can be employed in clinical trials.


Background
Human embryonic stem cell (ESC)-or human-induced pluripotent stem cell (iPSC)-derived insulinproducing cells (IPCs) can be used not only for transplantation of islet cells, which are destroyed due to autoimmunity in patients with type 1 diabetes mellitus (DM), but also for identifying novel targets for the development of anti-diabetic drugs in vitro. Several reports on the generation of IPCs from ESCs or iPSCs are available [1][2][3][4][5]. In particular, a number of studies have successfully differentiated ESC or iPSC into IPCs by mimicking pancreatic developmental and established a stepwise protocol for the same [3,4,[6][7][8]. This protocol involves the addition of various differentiation-inducing factors to the culture media at each differentiation stage, because of which the expression of transcription factors that are speci c to that particular pancreatic developmental stage is induced. Small molecules are also being widely used as differentiation-inducing factors, and IPCs generated using small molecules have shown robust function in vivo and in vitro [9][10][11][12].
A growing number of small molecules that can be used both in vitro and in vivo to grow stem cells [13][14][15], direct their differentiation [10,11,[16][17][18], and reprogram somatic cells into a more naïve state [19] have been identi ed. These molecules also provide useful information on the signaling and differentiation mechanisms that regulate stem cell biology. Small molecules with unique biological activities enable the establishment of new biological studies and the development of new treatments by signi cantly lowering the cost of production. Therefore, we leveraged the advantages of these small molecules for IPC differentiation. We screened for small molecules that induced and regulated the differentiation of stem cells into pancreatic progenitor cells. These include dorsomorphin (Dor, a selective inhibitor of bone morphogenetic protein (BMP) signaling) [9,20], SB431542 (an inhibitor of the transforming growth factor-β (TGF-β) type I receptor/ALK5) [9,21,22], SANT-1 (an inhibitor of Sonic hedgehog signaling) [23][24][25][26], FR180204 (an ATP-competitive inhibitor of ERK1 and ERK2) [20,27], and retinoic acid (RA) [28][29][30]. Using this screen, we developed functional IPCs that responded to glucose in response to combinatorial treatment with small molecules.
Many researchers generated ESC-or iPSC-derived IPCs that had functions similar to those of natural islet cells. However, they were reported to lack the ability of functional insulin secretion in response to glucose stimulation [5,20,24] because the differentiation and culture conditions generated in vitro did not accurately mimic the natural developmental stages of the human pancreas. Therefore, although the combination of the differentiation-inducing factors is important, the method of producing these cells in culture must also be investigated.
In this study, we developed a simple and e cient stepwise protocol for generating IPCs from iPSCs using SANT-1 and FR180204. Combined treatment with small molecules induced the e cient differentiation of iPSCs into a de nitive endoderm, pancreatic progenitor cells, and nally IPCs. In addition, the functional IPCs were continuously cultured and matured to obtain a spheroid morphology similar to that of natural islet cells.

Small molecules
Several small molecular compounds were used to differentiate iPSCs into IPCs. In most cases, the small molecules inhibited the signaling pathways in cells. The differentiation process was divided into three steps to mimic the process of pancreatic development in the embryonic stage. The rst step was the formation of a de nitive endoderm, the second step was its differentiation into the pancreatic endoderm and pancreatic progenitor cells, and the third step was the induction of speci c differentiation of the cells into IPCs. Therefore, the differentiation-inducing small molecules were processed at each step, and the results were con rmed by comparing and analyzing the combinations of small molecules (Table 1).

Teratoma analysis
The iPSCs were harvested and dissociated into a single cell suspension using TrypLE TM Express (Gibco, NY, USA). The cells (2 × 10 6 cells) were subcutaneously injected dorsally into 8-week-old mice with severe combined immunode ciency (SCID). They were maintained under non-speci c pathogen-free (SFP) conditions at an experimental animal facility in Asan Institute. They were euthanized after the development of tumors >1 cm 3 , or following an observation period of 40 d. The tumor-containing tissue was xed with 4% paraformaldehyde (PFA), embedded in para n, and serially sectioned (4 µm sections). The tissue sections were stained with hematoxylin and eosin and subjected to histological analysis using a microscope. Total RNA was isolated using TRIzol reagent (Invitrogen), and used to synthesize rst strand cDNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen). The SYBR pre-mixed system SsoAdvancedTM Universal SYBR Green Supermix (BIO-RAD, CA), and speci c primers were used to determine the level of each mRNA transcript to relative to that of 18sRNA. Primer sequences used were the following: Sox17, forward 5'-CGCTTTCATGGTGTGGGCTAAGGACG-3', reverse 5'-TAGTTGGGGTGGTCCTGCATGTGCTG-3'; FoxA2, forward 5'-ACTGGAGCAGCTACTTAGCAGAGC-3', reverse

Flow cytometry
Differentiated cells were dissociated to single cells using Accutase, xed with 4% PFA, and permeabilized with Perm buffer III (BD Biosciences). The cells were incubated with normal horse serum for 10 min and then with mouse anti-insulin, rabbit anti-glucagon, and rabbit anti-Ngn3 antibody for 30 min at RT. The cells were then stained with Alexa Fluor 488-conjugated donkey antibodies directed against mouse or rabbit IgG for 30 min at RT, and ow cytometry was performed using FACSAria II (Becton Dickinson).

Statistics and reproducibility
Statistical tests performed for speci c data sets are described in the corresponding gure legends. Twotailed unpaired t-tests (Student's t-test) were used to measure standard deviation (SD). Two-way ANOVA test for multiple comparisons was used to calculate the signi cance, including P values. All statistical tests were performed using GraphPad Prism Software v8.

Characterization of iPSCs
We cultured the iPSCs on a vitronectin-coated culture dish without feeder cells. It was con rmed that colonies of iPSCs could be maintained stably in vitro for a long period of time (Fig. 1A). The speci c markers of iPSCs, Oct4 (Fig. 1B), Nanog (Fig. 1C), and Sox2 (Fig. 1D), which are expressed in the nucleus, could be identi ed by immunostaining of the undifferentiated cells. In addition, we con rmed that SSEA4, one of the representative cell surface markers of iPSCs, was expressed (Fig. 1E). We rst performed a teratoma analysis to observe the pluripotency of the iPSCs. The iPSCs that were transplanted into the mice grew into a solid mass of tissue for 40 d, and the surface of the tumor showed various types of tissue clusters (Fig. 1F). Sections of the solid masses were observed to show histological variation. As a result, representative tissues of the endoderm, ectoderm, and mesoderm were identi ed. Based on the identi ed characteristics of the iPSCs, we con rmed that the iPSCs were pluripotent and potentially capable of differentiating into IPCs.
E cient induction of differentiation of iPSCs into a de nitive endoderm using combinations of small molecules We investigated the small molecules capable of e ciently inducing differentiation of iPSCs into a de nitive endoderm ( Fig. 2A). Differentiation e ciencies of treatment combinations of CHIR99021 and LY294002 small molecules were compared based on high concentration of activin A (Fig. 2B) only on day 1 of the total induction period of 3 d. On immunostaining, the group treated with CHIR99021 and LY294002 together showed higher expression of FoxA2, a typical marker of the endoderm, than the groups treated with CHIR99021 and LY294002 separately (Fig. 2C). More CXCR4-expressing cells were observed in the group treated with both compounds than in the groups treated with the compounds separately (Fig. 2D). Based on the quantitative analysis using qPCR, FoxA2 expression was found to be slightly lower in the group treated with both compounds than in the CHIR99021-treated group. However, the expression of Sox17 and CXCR4 was higher in the group treated with both compounds (Fig. 2E).
Comprehensively, we concluded that treatment with CHIR99021 and LY294002 simultaneously was more effective in inducing the differentiation of iPSCs into a de nitive endoderm than treatment of these compounds separately.
The induction of differentiation of iPSCs into IPCs using combinations of small molecules Although iPSCs have differentiated into a de nitive endoderm, their internal ability to differentiate into mesodermal and ectodermal cells remains. Therefore, we used small molecules that are capable of inhibiting the signaling of cell differentiation pathways, excluding the pancreatic progenitor cell pathway ( Fig. 2A). In particular, we compared the effects of small molecules, which can inhibit the differentiation of cells into hepatic progenitor cells, based on the differentiation-inducing media containing small molecules such as Dor and SB431542 that inhibit differentiation of cells into ectodermal and mesodermal cells, respectively. In addition, we compared groups that were treated with growth factor FGF2, ERK signaling inhibitor FR180204, and hedgehog signaling inhibitor SANT-1 to cause differentiation of cells into mature pancreatic progenitor cells. It was con rmed that the morphologies of 6-day cultured cells were different under different differentiation-inducing conditions. In general, all conditions induced vigorous cell growth, resulting in high cell density. However, no signi cant difference in the morphology and growth frequency of cells of different groups could be observed under the microscope (Fig. 3A). Therefore, the expression of characteristic transcription factors of pancreatic progenitor cells, including Pdx1, Ngn3, Nkx6.1, Sox9, and NeuroD, was analyzed under all conditions. The overall expression of transcription factors was higher than that of basic differentiation factors in group 1. However, the expression of each transcription factor in SANT-treated groups 4 and 6 was signi cantly increased compared to that of each transcription factor in the other groups (Fig. 3B). These results demonstrated that inhibiting hedgehog signaling of a particular differentiation pathway in the differentiation of pancreatic progenitor cells was more effective for the differentiation of cells into pancreatic progenitor cells.
After differentiation of cells into pancreatic progenitor cells, induced by different conditions in step 2, the differentiation into IPCs was induced in a similar manner using the culture medium that contained Dexa, Nico, and fors (Fig. 4A). After 8 d of treatment with differentiation factors, gene expression of representative hormones of mature pancreatic islet cells, such as insulin and glucagon, and major transcription factors, such as Pdx1 and Nkx6.1, was con rmed by qPCR. The expression of each marker in all groups showed a relatively immaterial increased mean value compared to that in group 1, but no statistical signi cance was observed. However, SANT-1-treated groups 4 and 6 showed a marked increase in the gene expression compared to group 1, and due to this, statistical signi cance of all groups, including group 1, was con rmed (Fig. 4B). Immunostaining and ow cytometry analysis were performed for group 6, which showed a relatively successful differentiation compared to other groups. Insulin and glucagon-expressing cells were found scattered between the cells. In addition, it was con rmed that differentiation occurred in groups of small numbers than in large clusters (Fig. 4C). The Pdx1-expressing cells were identi ed in most of the groups, but were rarely observed along with insulin-expressing cells (Fig. 4D). Based on ow cytometry analysis, about 38% of all differentiated cells expressed insulin and 23% expressed glucagon. Expression of Ngn3, the most important transcription factor of pancreatic islet cells, was observed in about 66% of all differentiated cells (Fig. 4E). These results indicated that even in the presence of a large number of Pdx1-and Ngn3-expressing islet progenitor cells, relatively few cells could directly differentiate into islet cells, suggesting that differentiation conditions could be improved to obtain higher differentiation e ciency.

Comparison of functional insulin secretion ability of differentiated IPCs
We analyzed the quantitative differentiation of IPCs based on the different combinations of differentiation-inducing factors and determined whether there was a change in the glucose-regulated quality improvement of functional IPCs. First, the concentration of the spontaneously secreted insulin in the culture medium was analyzed on day 5 of differentiation of cells into mature IPCs. It was con rmed that all groups secreted insulin, but signi cance among the groups could not be observed (Fig. 5A). On day 8, signi cantly higher insulin concentrations, approximately 15 times higher than those on day 5, were observed in all groups. In particular, SANT-1-treated groups 4 and 6 showed higher insulin concentrations than the other groups, and there was no difference in the increase in concentrations among the other groups (Fig. 5B). fors is an adenylate cyclase activator that is involved in the vitality and growth of cells during IPC differentiation, but it also causes mature pancreatic cells to release more insulin. Therefore, we cultured the cells for another 4 d after removing fors from the differentiation culture media and then checked the concentration of insulin in the media. The group without SANT-1 showed reduced insulin concentration, more than twice less than that with fors, whereas the group treated with SANT-1 showed only a slight decrease in the insulin concentration (Fig. 3C).
We also determined whether the differentiated IPCs could secrete insulin by glucose stimulation. Group 1 without SANT showed increased concentration of insulin in the medium when KCL was added during differentiation, but no signi cant secretion based on glucose concentration was observed (Fig. 5D). However, the group with SANT showed an increase in insulin secretion in response to a high concentration of glucose (Fig. 5E, F). Overall, high levels of natural insulin and the ability to control insulin secretion based on glucose concentration were observed only in the group with SANT-1 that inhibited hedgehog signaling mechanisms. These results suggested that inhibiting hedgehog signaling played a major role in the differentiation of iPSCs into mature pancreatic islet cells.

IPC culture to manufacture cells similar to islet cells
The IPCs, differentiated using small molecules, were cultured in a 2D culture that was adhered to the culture dish. Thus, we attempted to cultivate spheroid clusters of IPCs similar to natural islet cells. After 10 d of step 3 of differentiation, the cells were separated, and a high density of cells was cultured. As a result, small aggregations of cells were observed on day 1, and smaller spheroid clusters of constant size were formed on day 5 (Fig. 6A). The spheroid IPCs were well-coordinated and coherent clusters of insulinand glucagon-expressing cells (Fig. 4B). To con rm the in vitro conditions that allowed long-term culture of differentiated cells before in vivo transplantation, it was con rmed that the culture conditions could maintain the spheroid morphology and basic insulin secretion ability of the IPCs.
The spheroid IPCs were cultured for a considerable period under any culture condition. It was con rmed that the insulin secretion ability of IPCs gradually decreased when the cells were cultured with differentiation-induction medium without FBS.
When fors was removed from the differentiation-induction medium, the basal c-peptide secretion was low but decreased signi cantly on day 11. Similarly, when the CMRL 1066 medium was used for islet cell culture, the c-peptide secretion was lower than that when fors was added, but remained stable for 7 d (Fig. 4C). We suggest that culturing spheroid IPCs in CMRL 1066 medium for 7 d represents the best culture condition because it is di cult to stably perform in vitro culture of normal islet cells for a long time.

Discussion
In this study, we demonstrated the differentiation of iPSCs into IPCs by a simple method using small molecules. As most small molecules are target inhibitors of biological pathways, we used small molecules to inhibit the differentiation pathways except that of IPCs. In other words, a combination of small molecules was developed to ensure the natural differentiation of iPSCs in only one direction towards IPCs.
To induce the differentiation of iPSCs into a de nitive endoderm, the GSK3β-speci c inhibitor CHIR99021 [20] and the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 were used in combination with activin A. As a result, the expression of FoxA2 was slightly higher in the group treated with the combination of CHIR99021 and activin A. However, based on the results of the expression of other speci c markers, we found that addition of LY294002 would have a better effect. Differentiation of cells into pancreatic progenitor cells was induced by the treatment of the de nite endoderm with FGF family members, RA, and Dor, as reported in previous studies. Therefore, we investigated whether IPC differentiation was better after treating with FR180204, an ATP-competitive inhibitor of ERK1 and ERK2 [20,27], and SANT-1, a Sonic hedgehog pathway antagonist [23,24], to inhibit differentiation into hepatocyte precursor cells instead [23][24][25][26]. In conclusion, the present study demonstrated that treatment with SANT-1 and FR180204 resulted in increased induction of differentiation into IPCs, increased insulin levels in the culture medium, and in particular, production of functional IPCs that were responsive to glucose.
In this study, IPCs were made more similar to natural islet cells by culturing them to obtain spheroid morphology because 2D IPCs were less capable of secreting insulin in response to glucose stimulation. Although we used combinations of small molecules to induce differentiation into 2D functional IPCs that respond to glucose, spheroid IPCs were developed by further maturing and growing the cells because it is easy to transplant them and they survive well even after transplantation.
We suggest that inducing the differentiation of iPSCs into IPCs using this method was suitable for use during human transplantation. The iPSCs were maintained and differentiated on xeno-free matrix vitronectin that supported the growth and differentiation of human iPSCs under serum-free feeder-free conditions. In addition, small molecules, which are more stable and safer than protein growth factors and are economical for mass production, were used to induce differentiation. Therefore, we will develop a method to ensure that the matured IPCs are functionally more similar to natural pancreatic islet cells and are suitable for use in clinical trials in further studies.

Declarations
Ethical approval and consent to participate    is relatively lower in this group than that in groups treated with activin A and CHIR99021. Each bar represents the mean ± standard deviation. *P <0.05, **P <0.005.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Table1.tif