Efficient Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells

Embryonic stem (ES) cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs) by a two-step differentiation protocol comprising of (i) the formation of definitive endoderm in monolayer culture by activin A, and (ii) this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.


Introduction
Human and mouse embryonic stem (ES) cells are capable of spontaneous differentiation into insulin-producing cells, among many other cell types. ES cells can be induced to preferentially differentiate into insulin-producing cells (IPCs) by changing the composition of the culture medium and causing expression of dominant transcription factor genes which are involved in pancreas development [1,2]. In previous studies, there are two main strategies for the differentiation of ES cells into IPCs: (i) embryoid body formation and (ii) definitive endoderm formation [3][4][5]. Because after spontaneous differentiation the number of specifically differentiated cell types is relatively low, the application of defined differentiation factors and selection of lineage-specific progenitor cells seems to be necessary for directed differentiation of ES cells into the desired cell types [6,7]. Differentiated cells can be obtained within approximately 33 days.
Until now, there is no report to directly induce definitive endoderm and pancreatic cells in monolayer cells at the same time. Here, we present a strategy for the differentiation of ES cells into IPCs by a two-step differentiation protocol comprising of (i) the formation of definitive endoderm in monolayer culture by activin A, and (ii) this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. The small bioorganic molecules can control cellular processes by modulation of metabolism, signal transduction pathways and gene regulation [8][9][10][11][12][13][14][15][16][17][18]. In our study, we demonstrated that bioorganic molecules provide key information to modulation of stem cell proliferation and differentiation at 7 days. We also combined application of the three analytical methods presented here-RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties.  Figure 1: Schematic representations of the differentiation protocol from ES cells into insulin-producing cells. Undifferentiated feeder-free ES-D3 cells were cultured in collagen-I-coated plates and incubated in differentiation DMEM/F-12 medium supplemented with 2 mM Lglutamine, 100 μM nonessential amino acids, 10 ng/mL activin A, 10 mM nicotinamide, and 1 μg/mL laminin with 10% FBS overnight. ES-D3 cells were next exposed to DMEM/F-12 medium supplemented with 2 mM L-glutamine, 100 μM nonessential amino acids, 10 ng/mL activin A, 10 mM nicotinamide, 25 μg/mL insulin, and 1 μg/mL laminin with 2% FBS for 6 days. Differentiated ES-D3 cells formation of islet-like clusters at 7 days. Bar = 50 μm.

Materials and Methods
fetal bovine serum (FBS) were purchased from Hyclone Laboratories Inc. Activin A was purchased from R&D system. Other chemicals were purchased from Sigma-Aldrich.

Statistical Analysis.
All data were performed in triplicate, and all experiments were repeated at least three times. Data were presented as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA, SAS 9.1.3, USA), followed by a Tukey's test to determine any significant differences. P values of less than 0.05 were considered statistically significant.

Gene Expression and Immunofluorescence Analysis of Insulin-Producing Cells.
In preliminary experiments, we found that 10 ng/mL activin A, 10 mM nicotinamide, 25 μg/mL insulin, and 1 μg/mL laminin under low serum are an ideal condition for differentiation of monolayer endoderm cells into IPCs at 7 days (shown in Figure 1). To assess IPCs developmental changes resulting from specific modifications of culture conditions, we evaluated the expression of various genes by a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. As shown in Figure 2, the results indicated that differentiated mouse ES cells expressed pancreatic transcription factors (Pax4 and Pax6), endocrine progenitor marker (Ngn3), definitive endoderm (Sox7), exocrine pancreas marker (Amylase), pancreatic β-cell markers (Insulin 1 and Insulin 2), and Langerhans αand δ-cells (Glucagon and Somatostatin). Fluorescence micrographs also demonstrated pancreatic hormone C-peptide expressing in IPCs (shown in Figure 3(a)). After 7-day treatment with 10 ng/mL activin A, 10 mM nicotinamide, 25 μg/mL insulin, and 1 μg/mL laminin, the percentage of C-peptide expressing cells increased to 67.3 ± 2.9% (Figure 3(b)). In the time course of the next 6 days, the efficiency of C-peptide expressing does not significantly change.

Insulin Content and In Vitro Glucose-Stimulated Insulin
Secretion. ES-3D cells were treated with glucose to evaluate whether IPCs released insulin in a manner that was dosedependent upon the amount of glucose added. As shown in Figure 4(a), it can be seen that insulin is released in a manner that is dependent directly upon the amount of glucose added. At 12.5 mM glucose, the insulin released was double the amount released at .5 mM glucose, and the amount of insulin released in cells treated with 12.5 mM glucose was approximately twice that in cells treated with 2.5 mM glucose (Figure 4(a)).
In the previous studies insulin content was increased following mouse ES cells differentiation in the presence of the phosphatidylinositol 3-kinase inhibitor LY-294002. The results showed that insulin content was increased approximately 1.3-fold compared with untreated control (Figure 4(b)).

Conclusions
Embryonic stem (ES) cells are a potential source for insulinproducing cells (IPCs), but existing differentiation protocols are of limited efficiency. The aim has been to develop an efficient differentiation protocol, in which we could induce differentiation by small specific molecules, including activin A, laminin, nicotinamide, and insulin. Permeable small molecules can control cellular processes by modulating signal transduction pathways, gene expression, or metabolism and have been effectively used in ESC differentiation protocols. These molecules, alone or in combination with specific growth factors and hormones, will likely provide key information to design specific culture media in order to obtain IPCs [8][9][10][11][12][13][14][15][16][17][18]. Until now, there is no report to directly induce definitive endoderm and pancreatic cells at the same time by small specific molecules. We here present a strategy for the differentiation of mouse ES cells into IPCs by a two-step differentiation protocol comprising of (i) the formation of definitive endoderm in monolayer culture by activin A, and (ii) this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Activin A, a member of the transforming growth factor-β (TGF-β) superfamily, has been shown to induce endodermal differentiation of the cells of this endodermal monolayer under low serum conditions. In most of the recent studies, activin A starting from the beginning of in vitro differentiation monolayer can be added to cause human ES cells to differentiate into definitive endodermal cells [6]. Nicotinamide (also known as niacinamide) is a form of vitamin B3, which enhances the in vitro differentiation of cultured human pancreatic cells, favoring the expression of insulin, glucagon, and somatostatin [14][15][16]. Laminin enhances IPCs differentiation with increases in insulin and Glut2 gene expressions, proinsulin, and Experimental Diabetes Research 5 insulin release in response to elevated glucose concentration [17,18].
In this study, pancreatic β-cells (Insulin 1 and Insulin 2) and Langerhans cells' markers (Glucagon and Somatostatin) could be identified in differentiated ES cells. Fluorescence micrographs also demonstrated that pancreatic hormone Cpeptide was expressed in IPCs, the percentage of C-peptide expressing cells about 67.3 ± 2.9%. Insulin content was increased in a glucose-dependent manner. After treatment with phosphatidylinositol 3-kinase inhibitor LY-294002, insulin was increased to 1.3-fold of that in untreated cells.
In summary, these studies show that small molecules induce definitive endoderm and pancreatic cells in monolayer cells at the same time in the differentiation process. Our differentiation system represents an efficient protocol to direct mouse ES cells into the pancreatic lineage by generating IPCs and is applicable to further strategies for the improvement of in vitro differentiation into functional insulin-producing cells.