## 3 Result
### Dynamic Glucose-Stimulated C-Peptide Secretion Profiles of Human Pls and HLSC-ILS  
After validation of the perifusion protocols in terms of reproducibility of the glucose concentration determined in each stimulation step, we evaluated whether the MPS could be suitable to assess the C-peptide secretion profiles of both human PI and stem-cell-derived islets (HLSC-ILS). For this purpose, we used human PIs derived from clinically different donors (healthy, obese, and type 2 diabetic) (Table 2), and HLSC-ILS recently described in our lab [3]. First, we performed the SPGP protocol (Figure 2A) to evaluate C-peptide secretion in response to an HG concentration (28 mM), and the complete degranulation in response to a high-potassium concentration (50 mM). As shown in Figure 3A-left, the healthy and HLSC-ILS secretion profiles were characterized by two main peaks corresponding to the secretion induced by HG (peak 1) and high potassium (peak 2). In addition, when low glucose (LG; 3 mM) was present (LG\({}_{1}\)-LG\({}_{2}\)-LG\({}_{3}\)), C-peptide concentrations returned to the basal state detected during pre-stimulation, therefore indicating that C-peptide secretion was dependent on glucose concentration. Interestingly, the C-peptide secretion profiles of obese and T2 DM groups were clearly dysregulated (Figure 3A-left). The amplitude of C-peptide secretion in response to HG stimulus in the obese group was twofold compared to the healthy group and failed to go back to the basal concentration (remained twofold) after KCl stimulation. On the other hand, the T2 DM profile displayed a significantly lower (\(p\leq 0.05\)) amplitude of response compared to the healthy group, and a lower stimulation index (SI) after HG (\(2.3\pm 1.1\) vs. \(3.5\pm 1.3\)) and KCl (\(3.4\pm 2.1\) vs. \(5.1\pm 0.9\)) stimulation (Figure S1A Complementary Materials). \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Healthy (_n = 3_)** & **Obese (_n = 2_)** & **T2 DM (_n = 3_)** \\ \hline Age (years) & \(36.7\pm 17.7\) & \(51\pm 5.6\) & \(61.6\pm 7.5\) \\ BMI (kg/m\({}^{2}\)) & \(24.4\pm 1.5\) & \(30.4\pm 3.2\) & \(29.9\pm 9.5\) \\ HbA1C (\%) & \(5.7\pm 0.2\) & \(5.1\pm 0.2\) & \(6.8\pm 1.3\) \\ \hline Groups comparison & Parameter & \(p\)-values \\ \hline Healthy vs. obese & & & \(p<0.01\) \\ Healthy vs. T2 DM & BMI (kg/m\({}^{2}\)) & & \(p<0.01\) \\ Obese vs. T2 DM & & & n.s \\ \hline Healthy vs. obese & & & n.s \\ Healthy vs. T2 DM & HBA1C (\%) & & \(p<0.001\) \\ Obese vs. T2 DM & & & \(p<0.001\) \\ \hline Healthy vs. obese & & & n.s \\ Healthy vs. T2 DM & Age (years) & & n.s \\ Obese vs. T2 DM & & & n.s \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Islet donor characteristics and comparison between groups. ANOVA with Turkey’s multi-comparison test was performed. Data are expressed as mean \(\pm\) SD. The DPG protocol (Figure 2B) consists of a narrow low-to-high glucose stimulus (3-17 mM) in order to investigate the possibility that stem -cell-derived islet-like structures might only respond to significant  
Figure 3: Comparison of dynamic C-peptide secretion profiles (left panels) and areas under the curve (AUCs, right panels) of human PIs derived from different donors and HLSC-ILS. (**A**) Single pulse of glucose (HG; 28 mM) and potassium (KCl; 50 mM) protocol; (**B**) two consecutive pulses of glucose (HG; 17 mM); (**C**) ramp of glucose concentration (3-30 mM); (**D**) constant high-glucose concentration (HG = 17 mM) protocol. Basal concentration of glucose (LG) for all protocols was 3 mM. Results are presented as mean \(\pm\) SD of PIs from healthy (\(n=3\)), obese (\(n=2\)), T2 DM (\(n=3\)) and HLSC-ILS (\(n=3\)–5). ANOVA with Turkey’s multi-comparison test was performed; \({}^{\star}\)\(p<0.05\); \({}^{\star\star}\)\(p<0.01\); \({}^{\star\star}\)\(p<0.001\); \({}^{\star\star}\)\(p<0.0001\); \({}^{\star\star}\)\(p<0.0001\). changes in glucose concentrations (3-28 mM).