Mesenchymal Stem Cells from Chronic Pancreatitis Patients Show Comparable Potency Compared to Cells from Healthy Donors

Jingjing Wang, Yong Zhang, Colleen Cloud, Tara Duke, Stefanie Owczarski, Shikhar Mehrotra, David B Adams, Katherine Morgan, Gary Gilkeson, Hongjun Wang, Jingjing Wang, Yong Zhang, Colleen Cloud, Tara Duke, Stefanie Owczarski, Shikhar Mehrotra, David B Adams, Katherine Morgan, Gary Gilkeson, Hongjun Wang

Abstract

Mesenchymal stem cells (MSCs) are proven to be beneficial in islet transplantation, suggesting a potential therapeutic role of them in total pancreatectomy with islet autotransplantation (TP-IAT) for chronic pancreatitis (CP) patients. We investigated whether MSCs derived from CP patients are suitable for use in autologous cell therapy. MSCs from healthy donors (H-MSCs) and CP patients (CP-MSCs) were studied for phenotype, colony formation potential, multilineage differentiation ability, proliferation, senescence, secretory characters, and immunosuppressive functions. The potential protective effect of CP-MSCs was evaluated on hypoxia-induced islet cell death. Cell surface markers were similar between H-MSCs and CP-MSCs, as well as the ability of colony formation, multilineage differentiation, secretion of vascular endothelial growth factor and transforming growth factor (TGF-β), senescence, and inhibition of T cells proliferation in vitro. We found that growth differentiation factor 6 and hepatocyte growth factor (HGF) were significantly downregulated, whereas TGFβ and matrix metalloproteinase-2 were significantly upregulated in CP-MSCs compared with H-MSCs, among 84 MSC-related genes investigated in this study. MSCs from CP patients secreted less HGF, compared with the H-MSCs. A higher interferon-γ-induced indoleamine 2,3-dioxygenase expression was observed in CP-MSCs compared to H-MSCs. Moreover, CP-MSCs prevented hypoxia-induced β cell deaths to a similar extent as H-MSCs. Regardless of moderate difference in gene expression, CP-MSCs possess similar immunomodulatory and prosurvival functions to H-MSCs, and may be suitable for autologous cell therapy in CP patients undergoing TP-IAT. Stem Cells Translational Medicine 2019;8:418-429.

Keywords: Chronic pancreatitis; Islet autotransplantation; Mesenchymal stem cells; Stem cell therapy.

Conflict of interest statement

The authors indicated no potential conflicts of interest.

© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Figures

Figure 1
Figure 1
Phenotype and colony forming ability of H‐MSCs and CP‐MSCs. (A): Images of representative cultures of H‐MSCs (upper panels) and CP‐MSCs (lower panels) that were taken using a phase contrast microscope. Light gray: isotype control; dark gray: expression of indicated molecules on H‐MSCs or CP‐MSCs. (B): Representative FACS analysis of MSC defining surface positive marker panel (CD44, CD90, CD105). (C): FACS analysis of CD31, CD45 and HLA‐DR on H‐MSCs and CP‐MSCs. (D): Total CFU‐F in H‐MSCs and CP‐MSCs with crystal violet at 14 days after culture. Abbreviations: CFU‐F, colony forming units‐fibroblast; CP‐MSC, mesenchymal stem cells from chronic pancreatitis patients; H‐MSC, mesenchymal stem cells from healthy donors.
Figure 2
Figure 2
Multilineage differentiation of H‐MSCs and CP‐MSCs: (A, B) adipogenic differentiation indicated by oil red staining; (C, D) osteogenic differentiation indicated by alizarin red staining; (E, F) chondrogenic differentiation indicated by alcian blue staining. Molecular markers of gene expression during each differentiation by real‐time PCR, (G) PPARγ, (H) LPL, (I) Runx2, (J) OCN, and (K) Col2a. Bars represent mean ± SD; data are representative of at least three independent experiments. Abbreviations: Col2a, collagen a1; CP‐MSC, mesenchymal stem cells from chronic pancreatitis patients; H‐MSC, mesenchymal stem cells from healthy donors; LPL, lipoprotein lipase; PPARγ, peroxisome proliferator‐activated receptor 2; OCN, osteocalcin.
Figure 3
Figure 3
Proliferation and senescence of H‐MSCs and CP‐MSCs. (A): Expression of cell cycle‐ and senescence‐related genes at mRNA level in H‐MSCs and CP‐MSCs at passage 6. (B): Representative images of SA‐β‐gal staining on H‐MSCs and CP‐MSCs at passage 9. Scale bar = 100 μM. (C): Percentages of senescent cells quantified by SA‐β‐gal + cells among all the cells. Bars represent mean ± SEM. Abbreviations: CP‐MSC, mesenchymal stem cells from chronic pancreatitis patients; H‐MSC, mesenchymal stem cells from healthy donors; SA‐β‐gal, senescence associated β‐galactosidase.
Figure 4
Figure 4
Expression profile of MSCs‐related genes in H‐MSCs and CP‐MSCs. (A): Volcano plot of 84 human MSCs‐related genes in CP‐MSCs versus H‐MSCs. Fold change of more than 1.5 was used as a threshold limit, with green circle indicating <1.5 and red circle indicating >1.5. The blue horizontal line indicates that the p value of the Student's t test threshold is .05. (B): Folds change of genes that are differentially expressed in H‐MSCs and CP‐MSCs. (C): Representative image on Western blot shows the expression level of MMP2 in H‐MSCs and CP‐MSCs at passage 6. (D): Quantification of band intensities of MMP2 expressions in HMSCs and CP‐MSCs. Bars represent mean ± SEM. n = 3. Abbreviations: CP‐MSC, mesenchymal stem cells from chronic pancreatitis patients; GDF6, growth differentiation factor 6; HGF, hepatocyte growth factor; H‐MSC, mesenchymal stem cells from healthy donors; MMP2, matrix metalloproteinase‐2.
Figure 5
Figure 5
Secretion levels of growth factors by H‐MSCs and CP‐MSCs. (A) VEGF, (B) TGF‐β, (C) HGF secretion in H‐MSCs and CP‐MSCs at passage 2 and passage 4 measured by ELISA (n = 3). Values represent mean ± SD, and n = 3 for both H‐MSCs and CP‐MSCs. * and ** indicates p < .05 and p < .01 versus H‐MSCs, respectively. Two‐tail Student's t test. Abbreviations: CP‐MSC, mesenchymal stem cells from chronic pancreatitis patients; HGF, hepatocyte growth factor; H‐MSC, mesenchymal stem cells from healthy donors; VEGF, vascular endothelial growth factor.
Figure 6
Figure 6
Immunosuppressive potential of H‐MSCs and CP‐MSCs. (A): Represent figure of cell proliferation measured by flow cytometry. CD4+ (middle panels) and CD8+ (right panels) T cell proliferation assay was performed using Cell Trace violet‐labeled human PBMCs. Cells were activated by anti‐CD3 and anti‐CD28 antibodies. MSC:PBMC ratio was 5:1. Neg: negative control. Pos: positive control. (B): mRNA expression of IDO after IFN‐γ stimulation in H‐MSCs and CP‐MSCs. Expression level of IDO mRNA relative to β‐actin was evaluated by quantitative SYBR green real‐time PCR. Delta CT method was applied to calculate the fold induction of IDO over the unstimulated control. Bars represent mean ± SD. n = 3–5, *** indicates p < .001 versus CP‐MSCs stimulated with IFNγ, † indicates p < .001 versus UC‐MSCs stimulated with IFNγ. One‐way ANOVA with Tukey's multiple comparison. Abbreviations: CP‐MSC, mesenchymal stem cells from chronic pancreatitis patients; H‐MSC, mesenchymal stem cells from healthy donors; IDO, indoleamine 2,3‐dioxygenase; IFN‐γ, interferon‐γ; PBMC, peripheral blood mononuclear cell; UC‐MSCs, umbilical cord‐derived MSCs.
Figure 7
Figure 7
CP‐patient islets coculture with or without CP‐MSCs 48 hours under normal or hypoxic conditions (1% oxygen). Representative pictures of immunofluorescent staining of islets cultured alone or with H‐MSCs or CP‐MSCs under normal (A–C) or hypoxia conditions (D–F) stained for insulin (red) and TUNEL+ (green) cells. At least 20 islets have been counted in each group. Scale bar = 100 μM. (G): Percentages of TUNEL+ cells among insulin+ cells in human islets cultured alone (HI), with H‐MSCs or with CP‐MSCs. ***, p < .001, One‐way ANOVA with Turkey's multiple comparison. Abbreviations: CP‐MSC, mesenchymal stem cells from chronic pancreatitis patients; H‐MSC, mesenchymal stem cells from healthy donors; TUNEL, TdT‐mediated dUTP nick end labeling.

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