Characterization of immortalized mesenchymal stem cells derived from foetal porcine pancreas

Abstract

Islet replacement therapy is limited by shortage of donor islet cells. Usage of islet cells derived from porcine pancreatic stem cells (PSCs) is currently viewed as the most promising alternative for human islet transplantation. However, PSCs are rare and have a finite proliferative lifespan. In this study, we isolated and established an immortalized mesenchymal stem cell (MSC) line derived from foetal porcine pancreas, by transfecting human telomerase reverse transcriptase (hTERT) and called these immortalized pancreatic mesenchymal stem cells (iPMSCs). The iPMSCs have been cultured for more than 80 passages and have capacity to differentiate into neurons, cardiomyocytes, germ cells and islet-like cells, analysed by morphology, RT-PCR, western blotting, immunofluorescence, immunocytochemistry and transplantation assay. Islets derived from iPMSCs reversed hyperglycaemia in streptozotocin-induced diabetic mice and secreted insulin and C-peptide in vitro. These results demonstrated that iPMSCs might provide unlimited resources for islet replacement therapy and models for functional cell differentiation.

Figure 1

Morphology of iPMSCs, hTERT expression and growth curves of iPMSCs. (a) typical primary PSC colonies; (b) morphology of untransfected iPMSCs; (c, d) typical spindle fibroblast‐like iPMSCs; (e) Giemsa staining of iPMSCs; Bar = 20 μm; (f), hTERT expression of untransfected cells, and hTERT was present in iPMSCs and stably expressed in iPMSCs at passages 35 and 75, but absent in untransfected cells; (g) growth curves of iPMSCs. ♦ (P75 iPMSCs); (P35 iPMSCs); (Untransfected P24 PSCs).

Figure 2

Characteristics of iPMSCs. Immunofluorescence analysis showed that iPMSCs were positive for PDX1, Vim, PC1/3, Glut2 and hTERT. Bar = 20 μm. RT‐PCR analysis showed that iPMSCs were positive for Vim, Ngn3, Mafa, NeuroD1, PC1/3, Glut2 and PDX1.

Figure 3

Detection of cell surface markers. Analysis of FACs showed that iPMSCs were positive for CD29 and CD44, and negative for CD34, CD45, CD71 and CD166.

Figure 4

Pluripotent ESC markers were expressed in iPMSCs. Immunocytochemistry analysis showed that iPMSCs were positive for pluripotent ESC markers (Oct4, Sox2, Nanog, C‐Myc and Klf4) and PCNA, Bar = 20 μm; RT‐PCR analysis showed that iPMSCs were positive for Oct4, Sox2, Nanog, C‐Myc, Klf4 and PCNA.

Figure 5

Neuron‐like cells were formed after induction. Nestin, NSE, GFAP staining were positive respectively after induction and analysed by immunohistology. Bar = 20 μm; and expression of Nestin and β III‐tubulin were up‐regulated in induced cells relative to untreated cells as analysed by RT‐PCR.

Figure 6

Cardiomyocyte‐like cells were formed after induction. Cardiac α‐actin, CT3, Islet1 staining were positive respectively after induction by 10 μm 5‐AZA or 10⁻⁷  m all trans‐retinoic acid in combination with 0.75% DMSO, Bar = 20 μm; cardiac specific markers analysed by RT‐PCR showed that cardiac α‐actin, Nkx2.5 were up‐regulated induced by 5‐AZA but there was no up‐regulation by or RA in combination with DMSO.

Figure 7

Germ cells were formed after induction. (a) EBs derived from iPMSCs; (b) Large round cells detached from the periphery of EBs; (c) EBs derived from iPMSCs expressed Nestin (ectoderm), Brachuary (mesdoderm) and PDX1 (endoderm) analysed by RT‐PCR. Follicle‐like cells were obtained from EBs derived iPMSCs and immunohistochemical analysis showed that large cells expressed Vasa, Scp3 and Oct4 (the markers of germ cells), bar = 20 μm; Germ cell‐specific markers showed that oocyte markers (Scp3, GDF9 and ZP3) were up‐regulated after induction as analysed by semi‐RT‐PCR, similar to that in porcine oocyte.

Figure 8

Potential differentiation of islet cells. (a) islet clusters derived from iPMSCs were formed at day 14; (b) islet clusters were positive for DTZ staining; islet clusters derived iPMSCs were positive for PDX1, C‐peptide, insulin and Nestin, Bar = 20 μm; (c) induced islets derived from iPMSCs and non‐induced iPMSCs expressed pancreatic stem cell markers including Vim, Ngn3, Mafa, NeuroD1, Glut2, PDX1 and insulin as analysed by RT‐PCR; (d) pancreatic islet‐specific markers (Ngn3, Mafa, NeuroD1, Glut2, PDX1 and insulin) in induced islets were up‐regulated relative to non‐induced iPMSCs after 1‐week induction, while expression levels of primary pancreatic specific marker (Vim) was down‐regulated relative to undifferentiated iPMSCs, and there was an upward trend expression of Mafa, NeuroD1, Glut2, PDX1 and insulin in the 2‐week induced group compared with 1‐week group as analysed by QRT‐PCR; (e) western blotting showed that expression of Vim, PDX1 and insulin in induced islets were up‐regulated relative to non‐induced iPMSCs.

Figure 9

Blood glucose levels of transplantation mice. Blood glucose level of transplantated mice gradually decreased after transplantation with induced islets derived from iPMSCs, and this was maintained for around 3 weeks at low levels (<16.65 mm), then increased up to >27 mm (♦). Blood glucose level of all diabetic model mice transplanted with culture medium was higher than 27 mm (). All diabetic model mice transplanted with non‐induced iPMSCs maintained a higher level of glucose than those in islets transplantation, but lower than in culture medium transplantation (). All normal control mice were at 3.54–6.5 mm(×).