AG73-GelMA/AlgMA hydrogels present a steady microenvironment for the era of pancreatic progenitor organoids | Journal of Nanobiotechnology

February 27, 2025

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SDC4 impacts pancreatic improvement and the differentiation of PPs

To analyze the involvement of the DEPs throughout varied levels of pancreatic improvement in mice, we utilized hierarchical clustering evaluation to look at a set of 1753 DEPs that had been constantly differentially expressed throughout three developmental levels (for detailed info, please check with our earlier examine). By using a cluster collection check, we recognized eight distinct protein expression profiles. Notably, SDC4 was robustly and constantly expressed, notably within the pancreases of the mice within the TW and EW teams (Fig. 1a). IF evaluation confirmed that the expression of SDC4 within the islet tissues of the EW mice was considerably higher than that within the islet tissues of the FW and TW mice, which correlates with the situation of insulin expression (Fig. 1b, c). In the meantime, in human pancreatic specimens, SDC4 expression was detected and confirmed an analogous spatial overlap with insulin expression (Fig. 1d). In keeping with our earlier methods for SC-β differentiation, we downregulated mobile SDC4 on the PG and PFG levels and induce their differentiation into PPs (Fig. 1e). The FCM outcomes revealed a considerably decrease share of NKX6.1⁺/PDX1⁺ PPs within the SDC4-shRNA group (15.35 ± 2.659%) than within the NC group (53.26 ± 2.738%) (P < 0.0001) (Fig. 1g and h). This discovering strongly suggests a considerable decline within the differentiation effectivity of PPs. IF and qRT‒PCR revealed a big lower within the expression of vital TFs, together with PDX1, SOX9, NKX6.1, GATA4, and PAX4, in SDC4-shRNA PPs (Fig. 1h, i). mRNA evaluation upon remedy with SDC4-shRNA and NC-shRNA validated the numerous downregulation of those key TFs in PPs upon the lack of SDC4 (Fig. 1j). KEGG evaluation revealed alterations in essential signaling pathways concerned in pancreatic endocrine cell improvement, together with insulin secretion and pancreatic secretion, following SDC4 downregulation (Fig. 1okay).

Preparation and cytocompatibility of the AG73 hydrogel

In our earlier examine, we efficiently developed a biomimetic hydrogel composed of 5% GelMA and a pair of% AlgMA that possesses physiological stiffness just like that of pancreatic tissue [11]. To reconstruct the microenvironment throughout pancreatic improvement and facilitate the mobile response to mechanical alerts from the ECM, we designed the AG73 hydrogel as a platform to judge how AG73-SDC4-mediated signaling impacts pancreatic differentiation (Fig. 2a). By conducting molecular docking simulations, we found that residue GLN-8 of AG73 kinds hydrogen bonds with HE-13 of SDC4, indicating a binding interplay between them (Fig. 2b). We formulated AG73 hydrogels at remaining concentrations of 0.1 mg/mL and 1 mg/mL (Fig. 2c). The presence of the attribute maleimide peak within the AG73 hydrogel was confirmed through nuclear magnetic resonance (NMR) evaluation, indicating the profitable binding of AG73 to fivepercentGelMA/2percentAlgMA (Fig. 2d). The rheological properties of the hydrogel, together with its storage modulus (G’, representing elasticity) and loss modulus (G”, indicating viscosity), had been analyzed. The outcomes revealed that upon publicity to UV radiation, the hybrid hydrogel exhibited improved elastic conduct and distinctive mechanical sturdiness, with G’ surpassing G’’ (Fig. 2e). After immersion in PBS for 48 hours, the hydrogel, low-AG73 hydrogel, and high-AG73 hydrogel exhibited swelling charges of 38.5 ± 3.4%, 40.6 ± 5.4%, and 35.5 ± 2.2%, respectively, with no vital distinction between them (Fig. 2f) (P > 0.05). On the idea of the stress‒pressure curves, the compressive moduli of the hydrogel, low-AG73 hydrogel, and high-AG73 hydrogel had been 10.9 ± 1.9 kPa, 10.1 ± 1.6 kPa, and 9.0 ± 1.4 kPa on day 1 and 9.6 ± 1.6 kPa, 9.1 ± 0.9 kPa, and eight.5 ± 1.2 kPa on day 14, respectively (Fig. 2g and h) (P > 0.05). There was no vital distinction within the compressive moduli among the many three hydrogels on both the primary or fourteenth day. These outcomes demonstrated that the incorporation of AG73 had no impact on the compression modulus of the hydrogel. Moreover, no vital lower within the compression modulus was noticed on day 14, indicating the superb stability of the ready hydrogels. The SEM outcomes revealed that the pore sizes of the three hydrogel teams had been 180.0 ± 15.8 µm, 145.2 ± 9.8 µm, and 136.4 ± 9.3 µm, respectively (P < 0.001) (Fig. 2i and okay). AG73 crosslinking led to a discount within the pore measurement; nevertheless, the ensuing pore measurement of the AG73 hydrogel remained inside an applicable vary can supplied satisfactory area for mobile development. The outcomes of calcein-AM/PI staining revealed that the hESCs’ survival fee within the three hydrogel teams was passable in day 3, and never many lifeless cells had been noticed (Fig. 2j). Moreover, the CCK-8 outcomes of hESCs proliferation within the hydrogels after 1 and three days weren’t considerably totally different among the many three teams of hydrogels (Fig. 2l) (P > 0.05). To evaluate the potential of the hydrogel and AG73 hydrogel to boost the differentiation of hESCs into PPs, we encapsulated hESCs and subjected them to a four-stage differentiation protocol. Upon analysis of the expression ranges of the important thing TFs related to PPs (PDX1, NKX6.1, SOX9, GATA4, GATA6, and PTF1A), our findings revealed that, in contrast with the hydrogel alone, the high-AG73 hydrogel considerably improved the differentiation effectivity of PPs (Fig. 2m).

HiPSCs era and differentiation into PP organoids

To reinforce the medical significance of this examine, we elected to make the most of hiPSCs in subsequent experiments. ADSCs from P3 to P5 had been chosen to induce hiPSCs differentiation (Fig. 3a). After roughly 28 days of differentiation, the hiPSCs clones turned seen (Fig. 3b). The FCM outcomes revealed {that a} exceptional 95% of those hiPSCs had been optimistic for NANOG/OCT4 and NANOG/SOX2 expression (Fig. S1a). Furthermore, IF evaluation revealed sturdy expression of OCT4, NANOG, SOX2 and SSEA4 (Fig. 3c). Concurrently, the hiPSCs offered optimistic ki67 expression, which is indicative of a strong proliferative capability (Fig. S1b). Karyotype evaluation of the chromosomes didn’t reveal any discernible mutations (Fig. S1c). The outcomes of the tumor formation experiment demonstrated that subcutaneous injection of hiPSCs into nude mice induced teratoma formation, which was characterised by well-differentiated buildings representing all three germ layers (the ectoderm, mesoderm, and entoderm), as revealed by histological staining (Fig. S1d).

After encapsulation into the hydrogels and differentiation into PPs, the hiPSCs shaped early mobile clusters that continued to proliferate till they reached the PP stage, as noticed through mild microscopy (Fig. S2a). IF evaluation was used to evaluate the differentiation effectivity of the PP organoids and revealed optimistic expression of the main TFs at totally different levels of cell differentiation, together with the DE stage (FOXA2/SOX17), PFG stage (FOXA2/PDX1), and PP stage (PDX1/NKX6.1), and consider the impact of the AG73 hydrogel on PP organoid differentiation (Fig. 3e). The hydrogel group offered a considerably decrease fee of optimistic TF expression. The FCM outcomes additional revealed considerably higher optimistic expression of PDX1/NKX6.1 within the PP organoids within the AG73 hydrogel group than in each the Matrigel group and the hydrogel group (Fig. 3f and g). Furthermore, DEG evaluation revealed considerably increased expression ranges of key TFs (NKX6.2, PDX1, GCG, GATA6, and many others.) within the PP organoids of the AG73 group than in these of the Matrigel group. Conversely, the hydrogel group offered decrease expression ranges of PDX1, GATA4 and NKXK6.2 than did the Matrigel group (Fig. 3i and j). KEGG pathway evaluation revealed that there was enrichment of DEGs primarily within the ECM-receptor pathway, focal adhesion, the PI3K-AKT signaling pathway and the MAPK signaling pathway. GO time period enrichment evaluation revealed that the main enriched organic processes and molecular capabilities had been primarily system improvement, tissue improvement, the ECM, and the event of multicellular organisms (Fig. S2c, S2d). Curiously, we discovered that the PP organoids inside the hydrogels exhibited a transparent lumen-like construction, particularly these within the AG73 group (Fig. 3e, S2b). IF revealed that epithelial markers, reminiscent of EPCAM, had been considerably upregulated, indicating that the AG73 hydrogel facilitated epithelial–mesenchymal transition (EMT) within the PP organoids (Fig. 3h).

To analyze the differentiation potential of PP organoids, we prolonged their directed differentiation into EPs and SC-β cells (Fig. 4a). Within the Matrigel group, EPs had been generated through self-organization in AggreWell 400 plates, and the EPs within the AG73 group had been launched from the hydrogel by collagenase remedy. The EPs of the Matrigel group gave the impression to be extra tightly packed than these of the AG73 group (Fig. 4c). Nevertheless, each teams of SC-β cells ultimately exhibited a tightly packed construction after 21 days tradition. There was no statistically vital distinction within the common diameters of EP and SC-islets between the 2 teams. The compact construction of AG73 hydrogel-derived SC-β was additional confirmed by H&E staining (Fig. 4e). IF was used to judge the expression of NGN3 and YAP1 within the EPs within the Matrigel group and the AG73 hydrogel group. Our outcomes indicated that YAP1 expression within the launched EP organoids was partially translocated to the cytoplasm, suggesting a lower in YAP exercise (Fig. 4b). The findings of earlier research have demonstrated that downregulation of YAP1 expression throughout the late stage of PPs improvement promotes the differentiation of those cells into EPs [21]. Each teams of SC-β cells expressed NKX6.1, INSULIN, and MAFA (Fig. 4f). The variations in TF expression between the 2 teams was additional analyzed through qRT‒PCR and FCM. The qRT‒PCR outcomes revealed that the SC-β cells within the AG73 hydrogel group offered considerably increased expression ranges of PDX1, NKX6.1, INS, MAFA, GCG and NEUROD1 than that within the Matrigel group (Fig. 4d). FCM revealed that the NKX6.1/INS and NKX6.1/MAFA optimistic charges within the AG73 hydrogel group had been 65.5 ± 3.2% and 41.3 ± 2.1%, respectively, which had been considerably higher than these within the Matrigel group (52.8 ± 3.0%, P = 0.0011 and 31.6 ± 2.9%, P = 0.0015, respectively) (Fig. 4g and h). The TEM outcomes revealed the presence of insulin granules within the SC-β cells of each teams, suggesting that the cells had matured (Fig. 4i). SC-β cell proliferation was additionally in contrast by evaluating ki67 expression, and there was no vital distinction between the SC-β cells derived from the AG73 hydrogel group and the Matrigel group. These findings recommend that each teams of cells didn’t excessively proliferate (Fig. 4j). As well as, glucose, KCl, and exenatide-4 had been utilized to the SC-β cells stimulate insulin, and considerably extra insulin secretion was noticed within the AG73 hydrogel group than that within the Matrigel group (Fig. 4okay, l and m).

In vivo transplantation into diabetic mice

The flexibility of SC-β cells to control blood glucose ranges in diabetic mice was confirmed after a 12-week transplantation experiment carried out in vivo. IF staining revealed that the differentiated SC-β cells from each teams continued to precise insulin and NKX6.1 after transplantation in vivo (Fig. 5g). The optimistic fee of INS+/NKX6.1 + cells derived from AG73 was considerably increased in comparison with that within the Matrigel group (P = 0.0011) (Fig. 5h). Furthermore, serum ranges of human C-peptide didn’t considerably differ at 1 week posttransplantation (P > 0.05). Nevertheless, there was a considerably higher disparity within the serum ranges of human C-peptide between the AG73 group and the Matrigel group at 6 weeks after transplantation (P = 0.0027) (Fig. 5b). Subsequent, we evaluated the rise in physique weight, blood glucose ranges, and glucose tolerance and clearance. The noticed adjustments in blood glucose ranges inside 120 min after intraperitoneal glucose injection indicated that diabetic mice handled with SC-β cells derived from the AG73 hydrogel exhibited considerably higher glucose tolerance and clearance than did mice within the Matrigel-treated group (Fig. 5c and d). Moreover, the physique weights of the mice transplanted with SC-β cells derived from the AG73 hydrogel had been notably higher than these of the mice transplanted with Matrigel at 12 weeks (Fig. 5f).

A major distinction in blood glucose ranges was noticed between the AG73 hydrogel group and the Matrigel group at 6 weeks after transplantation (P < 0.05). The blood glucose ranges of the AG73 hydrogel group had been 6.5 ± 1.1 mmol/mL, whereas these of the Matrigel group had been 11.3 ± 1.5 mmol/mL. By week 11 posttransplantation, each teams demonstrated enhancements of their blood glucose ranges, with measurements of 6.2 ± 0.4 mmol/mL for the AG73 hydrogel group and 6.6 ± 0.4 mmol/mL for the Matrigel group, indicating efficient restoration of regular blood glucose ranges. In contrast with these transplanted with Matrigel, diabetic mice that acquired SC-β cells derived from the AG73 hydrogel offered a extra speedy lower in blood glucose ranges, and this discount capacity appeared roughly 6 weeks after transplantation, versus the 9 weeks required by the Matrigel group (Fig. 5e). Furthermore, shortly after graft elimination, each teams of recipients skilled hyperglycemia, indicating that glucose homeostasis was altered as a result of transplanted SC-β cells. No vital tumor formation was noticed within the kidneys 12 weeks after transplantation of SC-β cells, indicating a positive security profile for SC-β cell transplantation. Moreover, IF staining of Ki67 within the transplanted space revealed no proof of extreme proliferation among the many transplanted SC-β cells (Ki67%<1%) (Fig. 5i).

Single-cell transcriptome evaluation of the PP organoids

To analyze how the AG73 hydrogel impacts PP organoid improvement on the mobile cluster stage, scRNA-seq was carried out on day 2 of the PFG transformation into PPs. All cells had been categorised into 15 clusters through t-SNE evaluation. The predominant clusters within the AG73 hydrogel group had been clusters 1, 3, 7, 8 and 9, whereas clusters 0, 1, 4 and 6 had been primarily noticed within the management hydrogel group (Fig. 6a and b).

Subsequent, we recognized development-related genes expressed in PPs from each the hydrogel group and the AG73 hydrogel group. Notably, the important thing TFs PDX1, SOX9, NKX6.2, GATA4, GATA6 and HES1 had been considerably upregulated in clusters 1, 2, and seven (Fig. 6c). t-SNE evaluation was subsequently carried out to look at the expression patterns of SOX9, GATA4, PDX1, and SDC4 in addition to their distributions throughout totally different clusters. Curiously, whereas PDX1, SOX9, and GATA4 had been upregulated within the AG73 hydrogel group in contrast with the hydrogel group, the expression of SDC4 didn’t considerably differ between these two teams (Fig. 6d). Evaluation of all the TF subpopulation revealed that cluster 7 offered excessive expression ranges of PDX1, SOX9, and YAP1 (Fig. 6f). The cells expressing PDX1, SOX9, and YAP1 had been recognized as bipotent PPs on the idea of earlier research (Fig. 6e) [22]. Subsequently, IF confirmed that PDX1 and SOX9 had been expressed within the PP organoids, as each had been detected in each experimental teams however they had been current at increased ranges within the AG73 hydrogel group (Fig. 6g, h). Cell cycle evaluation revealed that there was a rise within the proportion of cells in G1 part among the many AG73 hydrogel-derived PP organoids in contrast with these within the hydrogel group, indicating the suppression of cell cycle development (Fig. 6i and j). DEG evaluation additionally revealed vital upregulation of the pancreatic endocrine components FOXA2, SOX4, HES1, and different associated genes in clusters 1 and seven (Fig. 6okay). In the meantime, Determine S3 illustrates the marker genes for various clusters together with the KEGG analyses of cluster 0, 1, and seven. The additional refined pseudotime evaluation revealed a possible lineage relationship between the cells in clusters 1 and seven, suggesting that cluster 1 cells might function precursors for the event of cluster 7 cells (Fig. S4). Moreover, recognized vital genes that drive PP organoid differentiation through RNA profiling, the era of fee scatter plots and characterization of the distinct kinetic behaviors for every gene (Fig. S5). The cell–cell and cell–ECM interactions within the hydrogel had been additional analyzed. The AG73 hydrogel-derived PP organoids offered extra ligand‒receptor pairs between distinct clusters, indicating enhanced intercellular communication (Fig. 7a). The HSPG signaling household, which incorporates SDC4, was discovered to be upregulated in AG73 hydrogel-derived PP organoids (Fig. 7c) [23]. Subsequent evaluation revealed a big enhance within the abundance of SDC4-related receptors and ligands inside the AG73 hydrogel-derived PP organoids. Moreover, substantial activation of quite a few intently related receptors, reminiscent of FN and people within the integrin household, was noticed (Fig. 7b). Furthermore, STRING database evaluation advised intensive hyperlinks between SDC4, FN and integrins (Fig. 7d). Fibronectin comprises domains that may interact each SDC4 and integrins. The expression sample of ITGAV in each teams was additional validated through t-SNE evaluation, revealing that clusters 1 and seven had been considerably enriched inside the AG73 hydrogel group (Fig. 7e). To validate our speculation, we initially carried out IF to verify the coexpression of SDC4 and ITGAV within the PP organoids. We discovered that whereas SDC4 was expressed in each teams, the expression of ITGAV was notably higher within the AG73 hydrogel group (Fig. 7f). To additional validate how ITGAV impacts PP organoids improvement, an siRNA concentrating on ITGAV was launched to the AG73 hydrogel group throughout the PFG and PP levels, and the tradition was maintained till the PP stage was accomplished. IF evaluation revealed a concomitant discount in ITGAV expression, in addition to diminished ranges of the important thing TFs PDX1 and SOX9, inside the PP organoids (Fig. 7g and h). Furthermore, qRT‒PCR evaluation confirmed that the expression ranges of TFs, reminiscent of PDX1, NKX6.1, SOX9, GATA4, GATA6 and PAX4 in PP organoids additionally decreased together with ITGAV downregulation in PP organoids (Fig. S6a). The position of AKT signaling in PP organoids differentiation was subsequently investigated by incorporating inhibitors concentrating on the AKT signaling pathway throughout the PG and PFG levels. IF evaluation revealed a discount in SOX9 and GATA4 expression inside the PP organoids (Fig. 7i), supporting our findings. Moreover, qRT‒PCR evaluation confirmed the decreases within the expression ranges of PDX1, NKX6.1, SOX9 and GATA4, following inhibition of the AKT signaling pathway (Fig. S6b). Lastly, we examined the altered adhesion properties of PP organoids in hydrogels and located that SANIL2 and N-CAD had been upregulated within the AG73 hydrogel group, whereas E-CAD was downregulated. These findings recommend that the AG73 hydrogel promotes EMT in PP organoids (Fig. 7j, okay).

AG73-GelMA/AlgMA hydrogels present a steady microenvironment for the era of pancreatic progenitor organoids | Journal of Nanobiotechnology

SDC4 impacts pancreatic improvement and the differentiation of PPs

Preparation and cytocompatibility of the AG73 hydrogel

HiPSCs era and differentiation into PP organoids

In vivo transplantation into diabetic mice

Single-cell transcriptome evaluation of the PP organoids

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