Human pancreatic islet-derived stromal cells reveal combined features of mesenchymal stromal cells and pancreatic stellate cells

Abstract

Background: Mesenchymal stromal cells (MSCs) are recognized for their potential in regenerative medicine, attributed to their multipotent differentiation capabilities and immunomodulatory properties. Despite this potential, the classification and detailed characterization of MSCs, especially those derived from specific tissues like the pancreas, remains challenging leading to a proliferation of terminology in the literature. This study aims to address these challenges by providing a thorough characterization of human pancreatic islets-derived mesenchymal stromal cells (hPD-MSCs).

Methods: hPD-MSCs were isolated from donor islets using enzymatic digestion, immortalized through lentiviral transduction of human telomerase reverse transcriptase (hTERT). Cells were characterized by immunostaining, flow cytometry and multilineage differentiation potential into adipogenic and osteogenic lineages. Further a transcriptomic analysis was done to compare the gene expression profiles of hPD-MSCs with other mesenchymal cells.

Results: We show that hPD-MSCs express the classical MSC features, including morphological characteristics, surface markers expression (CD90, CD73, CD105, CD44, and CD106) and the ability to differentiate into both adipogenic and osteogenic lineages. Furthermore, transcriptomic analysis revealed distinct gene expression profiles, showing notable similarities between hPD-MSCs and pancreatic stellate cells (PSCs). The study also identified specific genes that distinguish hPD-MSCs from MSCs of other origins, including genes associated with pancreatic function (e.g., ISL1) and neural development (e.g., NPTX1, ZNF804A). A novel gene with an unknown function (ENSG00000286190) was also discovered.

Conclusions: This study enhances the understanding of hPD-MSCs, demonstrating their unique characteristics and potential applications in therapeutic strategies. The identification of specific gene expression profiles differentiates hPD-MSCs from other mesenchymal cells and opens new avenues for research into their role in pancreatic function and neural development.

Keywords: Pancreas; Pancreatic islets; Pancreatic mesenchymal stromal cells; Pancreatic stellate cells; Transcriptome analysis.

Fig. 1

Isolation and Immortalization of hPD-MSCs. (A) Phase-contrast micrograph depicting human pancreatic islets post-isolation, enveloped by adjacent exocrine cells. (B) Isolated islets adhere to the culture surface, with cellular growth evident on the outer layer. (C) The islet structure dissipated, giving way to cells attaching to the culture surface. (D) Subsequent cellular morphology was observed after several passages, illustrating the evolving characteristics during the cultivation process. (E) The proliferation rates of three averaged cell lines, comparing both immortalized and non-immortalized cell lines. The red vertical arrow indicates the moment of LV-hTERT transduction, while the red circle marks the point at which proliferation ceased. (F) RT-qPCR results for mRNA hTERT in PD-MSCs are depicted before and after immortalization, presented as the average of three independent repeats with standard deviations. Statistical significance was assessed using a paired student’s t-test, where ***P < 0.001

Fig. 2

Expression of mesenchymal stromal cell markers in PD-MSCs and their multilineage differentiation. (A, B) Representative flow cytometric analysis of immortalized and non-immortalized hPD-MSCs samples respectively, showing the specific expression of mesenchymal stromal cell markers CD106, CD105, CD90, CD73, and CD44. (C)Adipogenic differentiation of PD-MSCs compared to non-induced control, lipid droplet formation achieved through phase-contrast microscopy and staining with Oil Red O, enhanced with trypan blue. (D) Osteogenic differentiation of PD-MSCs compared to non-induced control, The deposition of calcified and mineralized extracellular matrix is visualized by phase-contrast microscopy and staining with Alizarin red S enhanced with trypan blue

Fig. 3

Expression of stemness and PSCs markers in PD-MSCs. (A) RT-qPCR results comparing the expression levels of stemness markers in PD-MSCs relative to iPD-MSCs, BM-MSCs, and AT-MSCs. (B) RT-qPCR results comparing the expression levels of LRAT enzyme in PD-MSCs compared to iPD-MSCs, BM-MSCs, AT-MSCs- human fibroblasts. Data presented as an average of at least 3 biological repeats with standard deviation, statistical significance was assessed using ANOVA analysis where *P < 0.033, **P < 0.0021, ***P < 0.0002, **** P < 0.0001. (C)immunocytochemistry staining results of PSCs markers in PD-MSCs including CXCL12, Desmin, Laminin, Nestin, α-SMA, GFAP, Fibronectin, Decorin, Collagen I

Fig. 4

Transcriptomic analysis of PD-MSCs. (A) Principal components plot for the variance stabilized counts of the obtained transcriptomes of PD-MSCs, iPD-MSCs, AT-MSCs, and BM-MSCs. (B) Heatmap of log-transformed transcripts per million (TPMs) of genes of various pancreatic markers for the studied MSCs samples. (C) Inferred proportions of pancreatic cells and correlation of the RNA-seq samples with the pancreatic Tabula Sapiens dataset by the CIBERSORTx. (D) Venn diagram of intersection of statistically meaningful (P.adj < 0.05) DEG with big effects (log2FC > 2) for PD-MSCs, original and immortalized (denoted as “iPD”) separately, vs BM-MSCs (blue) and AT-MSCs (green). (E) Heatmap of log-transformed transcripts per million (TPMs) of PD-MSCs-specific genes for the studied MSCs samples

Fig. 5

(A) Verification of transcriptome results by RT-qPCR in PD-MSCs compared to iPD-MSCs, BM-MSCs, and AT-MSCs. (B) expression of ISL1 in PD-MSCs and iPD-MSCs compared to BM-MSCs and AT-MSCs. Data presented as an average of at least 3 biological repeats with standard deviation, statistical significance was assessed using ANOVA analysis where **P < 0.0021, ***P < 0.0002, and ****P < 0.0001