Transdifferentiation molecular pathways of neonatal pig pancreatic duct cells into endocrine cell phenotypes

Transdifferentiation Molecular Pathways of Neonatal Pig Pancreatic Duct Cells Into Endocrine Cell Phenotypes G. Basta, L. Racanicchi, F. Mancuso, L. Guido, G. Luca, G. Macchiarulo, P. Brunetti, and R. Calafiore ABSTRACT Restrictions in availability of cadaveric human donor pancreata have intensified the search for alternate sources of pancreatic endocrine tissue. We have undertaken to assess whether nonendocrine pancreatic tissue, with special regard to ducts, including epithelial cells, and retrieved from neonatal pig pancreata that are used for islet isolation, may under special in vitro culture conditions generate endocrine cell phenotypes. Special care was taken to identify the time-related appearance of molecular and biochemical markers associated with ␤-cell specificity, in terms of glucose-sensing apparatus and insulin secretion. For this purpose, established ductal origin monolayer cell cultures were incubated with a battery of mono- or polyvalent growth factors. Morphological, immunocytochemical, molecular, and functional assays indicated that under special culture conditions ductal origin cells acquired an endocrine identity, based upon expression of key gene transcripts that govern the stimulus-coupled insulin secretory activity. Among factors eliciting transdifferentiation of ductal epithelial into endocrine cells, Sertoli cell (SC)-conditioned medium seemed to be the most powerful inducer of this process. In fact, the resulting cultures not only expressed ␤-cell– oriented metabolic markers but also were associated with insulin and C-peptide output at equimolar ratios. This finding indicates that SC coincubation, more than other conditions, caused originally ductal cell cultures to gradually differentiate and mature into ␤-cell–like elements. In vivo studies with this early cell differentiation product will test whether our approach may be suitable for correction of hyperglycemia in diabetic animal models.

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UCCESS OF HUMAN pancreatic islet cell allografts demonstrated by the Edmonton protocol,1 which is under trial by the Centers of the Immune tolerance Network,2 constitutes an important proof of principle, showing that under the best conditions islet transplant may induce a full, sustained remission of hyperglycemia among immunosuppressed patients with type 1 diabetes mellitus (TIDM). However, it is increasingly clear that the human procedure is limited by a series of problems, beginning with the restricted availability of cadaveric donor organs. Additionally, a single pancreas may not yield a sufficient islet mass to successfully engraft one diabetic recipient because of the persistent technical pitfalls in the digestion and cell purification of the harvest. Finally, recipients invariably require lifelong general, pharmacological immunosuppression to preserve islet graft survival and function, the long-term side effects largely remain unknown. These graft-related immunological problems might be avoided by recent advances in islet cell microencapsulation, as shown by our laboratory.3

This technique creates a physical, highly biocompatible, semipermeable immunoselective shield that surrounds the islets, allowing nutrients and oxygen but not antibodies on cellular mediators or the immune system, to pass through the membrane. Microcapsules basically include Na-alginate, a polysaccharide extracted from brown seaweeds, which we have uniquely complexed with an aminoacidic polycation, poly-L-ornithine.4 For this purpose, we recently have been granted permission by the Italian Institute of Health to begin a pilot, closed clinical trial of microencapFrom the Department of Internal Medicine, Section of Internal Medicine and Endocrine and Metabolic Sciences (Di.M.I.), University of Perugia, Perugia, Italy. Supported by the Consorzio Interuniversitario per i Trapianti d’Organo, Rome, Italy. Address reprint requests to Riccardo Calafiore, MD, Di.M.I., University of Perugia, Via E. Dal Pozzo, 06126 Perugia, Italy. E-mail: [email protected]

© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.10.026

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sulated human islet allografts in 10 patients with TIDM, following a multiple series of preclinical animal studies with encapsulated islet transplants. To circumvent the scarce availability of cadaveric human donor pancreatic tissue for islet ␤ cells, alternate sources should be considered with special regard to nonhuman (ie, porcine) islet tissue and/or pancreatic precursor cells that may under appropriate environmental conditions evolve toward endocrine, insulin-secreting cell phenotypes. As to the former, we and others have methods for separation and purification of neonatal pig islet cells (NPCCs).5–7 Problems related to incomplete functional maturation of these islets at the time of isolation are gradually being overcome in our laboratory,8 although the potential infectivity by porcine endogenous retroviruses in humans9 has so far limited, or anyway delayed, progress of this approach into clinical applicability. As far as the other possible research option is concerned, we had focused our initial attention on the use of neonatal pig pancreatic ducts as a potential source of insulin-producing cells.10 We have observed that pancreatic ducts under special culture conditions may indeed express insulin as well as other markers of precursor endocrine cells. At the end of this first attempt to identify endocrine phenotypes among epithelial ductal cells, we observed the presence of insulin, although no glucose-stimulated insulin release was elicited from this tissue. Possibly, an early maturational stage of these cells adversely affected the functional performance that is associated with well-differentiated islet ␤ cells. Neonatal pancreatic ducts have expressed some markers of ␤-cell maturation, such as the insulin transcriptional factor that is encoded by the pancreatic and duodenal home box gene 1 (PDX-1). In the early stage of development, all pancreatic precursor cells express PDX-1, while this expression is subsequently lost by the exocrine cells, which in fact begin to express p48 and finally amylase. On the contrary, endocrine-oriented cells maintain PDX-1 expression, adopting a Neurogenin 3–positive phenotype, followed by expression of Neuro-D/␤2. Expression of these gene products clearly indicates a switch of pancreatic precursor cells to endocrine-committed cells (␣, ␤, ␦, and PP) with the intervention of other regulatory genes. In our preliminary studies on the evolution of ductal origin cells toward endocrine precursors, we observed, under particular experimental conditions, that PD may form cell monolayers and subsequently cell aggregates or cysts that resemble islet-like structures, although they have no signs indicating the presence of a differentiated glucosesensing apparatus. To identify a stepwise path along which pancreatic ducts could generate functionally mature ␤ cells, in vitro, we have pursued the following research aims: (1) to promote in vitro differentiation of precursor pancreatic epithelial cell populations; (2) to identify and characterize morphological, molecular, and functional markers associated with ␤-cell functional competence within the pancreatic duct cell pop-

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ulations; and (3) to obtain stable ␤-cell–like cultures, eventually able to be expanded in vitro. MATERIALS AND METHODS Separation and Purification of NPCCs and Pancreatic Ducts We have isolated NPCCs and pancreatic ducts according to previously published methods.7,10 Briefly, the pancreata retrieved from 3- to 5-day old large white piglets were transported to the laboratory in cold UW-Belzer solution (Viaspan, BristolMyers Squibb, Latina, Italy). After mincing into small pieces (1 to 2 mm3), they were washed in Hank’s balanced salt solution (HBSS) (Celbio, Milano, Italy) prior to enzymatic digestion by gentle shaking in 2 mg/mL collagenase solution (Collagenase P, Roche Diagnostics SpA, Monza, Italy) for approximately 20 minutes at room temperature. After, washing in HBSS, NPCCs were plated in 100 ⫻ 15 mm. Petri dishes (Corning Inc, New York, NY, USA) in HAM F-12 medium (Celbio), supplemented with pen-strep (Sigma Aldrich, Milano, Italy) with 0.001% 3-isobutylmethylxanthine (Sigma), 10 mmol/L nicotinamide (Sigma), and 0.5% bovine serum albumin fraction V (Sigma). The tissue medium was changed at 1, 3, 5, and 7 of the 9 days of culture maintenance. Pancreatic ducts were then handpicked from the Petri dish containing NPCCs under a stereomicroscope. About 30 pancreatic duct per 25-cm2 flask (Corning) in Click’s medium (Celbio), supplemented with 10% fetal bovine serum (FBS) (Celbio), were seeded. At 7 to 10 days of culture, pancreatic ducts physically dissociated and started to form cell monolayers, which were not confluent. The cultures were thereafter expanded thrice by seeding pancreatic ducts from one to three flasks.

Sertoli Cell Isolation Sertoli cells (SC) were retrieved from the testes of the same neonatal piglets that donated the pancreas. After fine chopping and subsequent collagenase and trypsin digestion, we harvested bulk amounts of purified SC following a method previously published.11

Culture Conditions Primary. Isolated pancreatic ducts were culture maintained for 2 weeks in Click’s (Sigma-Aldrich) supplemented with 10% fetal calf serum (Celbio) in 25-cm2 culture flasks (Corning). Secondary. Confluence of pancreatic duct cell monolayers was obtained after treatment with trypsin/EDTA (Celbio) for 5 minutes. The cells were detached from one flask and replated into three new flasks in Click’s supplemented with FBS in 5% CO2 at 37°C. After 1 week of culture, the preparations underwent trypsin/ EDTA treatment as above and were replated in new flasks twice, so as to obtain expanded continuous monolayers. To this end, the following factors were added to obtain different culture conditions: high glucose (25 mmol/L); 10 ng/mL keratinocyte growth factor (KGF) (Pre-Protech, Tebu-Bio, Milano, Italy); 10 ng/mL hepatic growth factor (HGF) (Pre-Protech, Tebu-Bio); dialyzed/liophilized medium derived from primary homologous SC cultures with 1:64 dilution with Click’s (protein content ⫽ 82.5 mg/mL); Click’s supplemented with 10% FBS with no added growth factors (control).

Morphology and Immunocytochemistry Cell viability was assessed by staining with ethidium bromide and fluorescein diacetate under fluorescence microscopy (Leitz Ortho-

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Table 1. Primary Antibodies for Gene Product Immunodetection Antibody target

Host

Titer

Anti-human insulin* Anti-PDX-1** Anti-Glut-2**

Guinea pig Rabbit Rabbit

1:200 1:200 1:150

*Linco Research Immunoassay, St Charles, Mo, USA. **Chemicon.

the an ECL Western blotting detection kit (Amersham). Primary antibodies were diluted as follows: 1:1000 rabbit anti-PDX-1 (Chemicon, Prodotti Gianni, Milano, Italy); 1:2500 rabbit-antiGLUT2 (Chemicon); rabbit anti-glucokinase (GCK; Santa Cruz Biotechnology, DBA Italia, Milano, Italy). The secondary antibodies were 1:5000 goat anti-rabbit horseradish peroxidase– conjugated (Sigma). All antibodies were diluted in 0.1% Tris buffer saline Tween (Sigma) plus 5% nonfat dry milk (Sigma).

Radioimmunoassay mat IV, Wetzlar, Germany): viable cells appeared green, while damaged cells were red. Double fluorescence immunolabeling was sequentially performed on tissue preparations, using a battery of primary antibodies derived from different species and recognizing several gene products (Table 1). Antibodies were applied to prefixed cells (4% buffered formaldehyde). Secondary antibodies, conjugated with fluorescein for fluorescence microscopy examination, included: goat anti-rabbit IgG (Sigma), goat anti-mouse IgG, and rabbit anti-guinea pig IgG (Sigma). After appropriate rinsing, the slides were mounted with glycerol (90%) and phosphate buffer saline (Sigma). Cell nuclei were counterstained with propidium iodide (Sigma; 1 ␮g/mL) after treatment with 10 ␮g/mL RNA-ase A (Sigma) for 10 minutes at room temperature.

Molecular Studies Reverse transcription-polymerase chain reaction. Total cellular RNA from tissue seeded in 75-cm2 cell culture flasks was prepared by using the RNAeasy Mini Kit (QUIAGEN S.p.A., Milano, Italy) according to manufacturer’s instructions. RNA (0.5 to 1 ␮g total) was used to generate the first-strand cDNA by the Sensiscript Reverse Transcriptase kit (QUIAGEN). cDNA samples were subjected to polymerase chain reaction (PCR) amplification with specific primers under linear conditions. The cycling parameters were: denaturation at 94°C for 30 minutes, annealing at 58°C for 30 minutes (GAPDH, Nkx6.1), or 60.8°C for 30 minutes (PDX-1), or 62°C for 30 minutes (GK, GLUT-2, NeuroD), and elongation at 72°C for 30 minutes (35 cycles for all fragments). For the GK fragment ␣32P dNTP (Amersham, Newcastle, UK) was used. The PCR primers employed for amplification were: GAPDH (forward 5=-accacagtccatgccatcac-3= and the reverse 5=-tccaccaccctgttgctgta3=, 452 bp); Nkx6.1 (forward 5=-aggatccattttgttggaca-3= and the reverse 5=-cgccaagtatttcgtttgtt-3=, 111 bp); PDX-1 (forward 5=agagcccgaggagaacaag-3= and the reverse 5=-gcggcctagagatgtatttg-3=, 110 bp); GK (forward 5=-ggacctgaaaaaggtgatga-3= and the reverse 5=-catcttcacactggcctctt-3=, 99 bp); GLUT-2 (forward 5=-ccgagtttttcagtcaagga-3= and the reverse 5=-agtccgcaatgtactggaag-3=, 109 bp); Neuro-D (forward 5=-cctgtgcacccctactctta-3= and the reverse 5=tgcaggatagtgcatggtaa-3=, 272 bp). Amplification products were resolved on 2% agarose gels and the fragments yielded bands of expected molecular weight. Western blotting. For total protein extraction, the cells were suspended in lysis buffer (10 mmol/L Tris base, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 20 mmol/L NaF, 1% Triton X-100, 0.2 mmol/L Na vanadate, 0.5% Igepal CA-630, 0.2 mmol/L phenylmethanesulfonyl fluoride and kept on ice for 20 minutes. After centrifugation at 10,000 rpm for 5 minutes at 4°C, the supernatants were tested. Nuclear/cytoplasmic extraction was performed as previously described.12 Sample protein concentrations were assayed by the Bradford method (BIORAD assay, BIORAD Laboratories, Milano, Italy). Protein samples (100 ␮g) analyzed on 14% SDS-PAGE were transferred onto nitrocellulose membranes (BIORAD). Immunodetection was performed using

Insulin, proinsulin, and C-peptide contents in the media were assayed by radioimmunoassay (RIA) (Pantec Italia, Torino, Italy).

RESULTS Immunocitochemistry

After 30 days of culture under different conditions, the cells showed different immunopositivity for the examined antigens. In all the cases the cells were positive for PDX-1, GLUT-2, and insulin. PDX-1 was primarily in the cytoplasm of control cultures in a diffuse pattern (Fig 1, lane B). On the contrary, PDX-1 was associated with cytoplasmic staining with a punctate pattern in the following culture conditions: high glucose, SC-conditioned medium, HGF, and KGF. Moreover, in these conditions PDX-1 also showed a major perinuclear or nuclear staining. These data possibly reflect the formation of PDX-1-protein cytoplasmic clusters that precede nuclear translocation. GLUT-2 (Fig1A) showed a similar pattern in all culture conditions but was more intense in factorexposed than control cells. Insulin (Fig1C) showed a weak and diffuse staining in the control and in the HGF-treated conditions, while it appeared to be more intense with a punctate or granular pattern under other conditions, especially in the case of SC-conditioned medium. Reverse Transcription-PCR

RNA transcriptional levels of PDX-1, Nkx6.1, NeuroD, GLUT-2, and GK were assessed by reverse transcription (RT)-PCR in cells monolayers after 30 days in different culture conditions. Data concerning electrophoretic analysis of amplification products are reported in Fig 2A, with quantification of transcripts by densitometric analysis reported in Fig 2B. An increase in all transcripts was evident for all conditions compared with controls, with a major increase among KGF-treated cells (through 150% to 200% of increase). Interestingly, for GK and GLUT-2 transcripts, SCconditioned medium treatment induced a significant increase, of approximately 150%, while the other conditions seemed to be comparable. In the case of PDX-1 and NeuroD transcripts, HGF, high glucose, and SC-conditioned medium were associated with similar but small increases, while Nkx6.1 was greatly increased by KGF and high glucose. In this instance, HGF and Sertoli-conditioned medium treatments were associated with comparable effects. All of the fluorescence intensity of indicated amplifi-

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Fig 1.

BASTA, RACANICCHI, MANCUSO ET AL

Confocal detection of Glut-2 (A), Pdx-1 (B), and insulin (C), in cells monolayers after 30 days in different culture conditions.

cation products was normalized with the fluorescence intensity of the GAPDH amplification product. Western Blotting

Immunoblot analysis revealed the presence of PDX-1, GLUT-2, and GK proteins in cell monolayers after 30 days of different culture conditions (Fig 3). The analysis showed the presence of transductional products of apparent molecular weights consistent with previous observations. In fact, for all conditions we observed a spot of 52 KDa for GK, 50 KDa for GLUT-2, and 46 KDa for PDX-1, the last one probably being the phosphorylated isoform. To confirm the nuclear translocation of PDX-1, separate analysis was performed on the nuclear and cytoplasmic fractions (Fig 3A, bottom panel), demonstrating the presence of the 46 KDa form of PDX-1 also in the nuclear fraction. Interestingly, for GK we observed greater expression with SC treatment, while GLUT-2 expression seemed to be magnified by high glucose treatment. In contrast, PDX-1 did not show any major changes among all treatments although KGF was associated with an higher intensity. RIA Assay

Proinsulin, insulin, and C-peptide content in the culture media showed upon RIA assay (Fig 4) that regardless of treatment, the ductal origin cell monolayers were able to evolve to ␤-cell–like elements. Proinsulin levels under all

conditions were higher than the controls and also than the insulin levels, possibly reflecting low cell maturity (partially confirmed by the insulin/C-peptide ratio). On the contrary, SC-derived medium resulted in an increased proinsulin secretion with, even more insulin secretion as evidenced by an equimolar insulin/C-peptide secretory ratio. This result indicated a higher extent of maturation/differentiation of nonendocrine precursors into ␤-like cells. DISCUSSION

The search for an alternate tissue as a resource for donor islets for transplantation purposes in TIDM is today one of the major challenges for the academic community in this research field. Potential directions are use of either nonhuman (ie, porcine),13 genetically engineered artificial ␤ cells14 or pancreatic nonendocrine precursor cells as sources grafts in diabetic recipients. While previous studies15–17 have demonstrated that adult pancreatic human tissue may yield islet buds, the exact sequence of molecular events that underlie the transdifferention process from nonendocrine to functionally competent ␤-cell–like elements have so far remain unknown. Many of these studies have used ECM-like structures to favor the process.17,18 Our aim here was to add ad hoc selected growth factors, to epithelial cell monolayers derived from neonatal pig pancreatic ducts under specific culture conditions. We

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Fig 2. RT-PCR analysis of Pdx-1, Nkx6.1, NeuroD, Glut-2, and GK expression in cells monolayers after 30 days in different culture conditions. As internal control, GAPDH transcripts were amplified. (A) Electrophoretic analysis of amplification products. The reaction yielded the bands of the expected molecular weight (indicated in the figure). RT-PCR for GK was performed in the presence of ␣32 PdNTP. (B) Densitometric analysis of results reported in A. Bars represent the ratio between the fluorescence intensity of indicated amplification products and GAPDH amplification product.

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Fig 3. Western blot analysis in cells monolayers after 30 days in different culture conditions. (A) Top, glucokinase (GK, 52 KDa) and Glut-2 (50 Kda) immunodetection; (A) bottom, Pdx-1 (46 KDa). C indicates the cytoplasmic fraction, while N is the nuclear one. Cell extracts were sized by 14% SDS-PAGE and blotted as described. (B) Densitometric analysis of proteins showed in A, top.

sought to stepwise identify and characterize, the morphological, molecular, and functional markers of transdifferentiation of original cell elements into endocrine cell phenotypes. Special care was taken to detect the appearance of those components of the glucose-sensing apparatus that

Fig 4. Proinsulin, insulin, and C-peptide content in the media of cells monolayers after 30 days in different culture condictions.

represent specific hallmarks for ␤-cell– oriented differentiation. We have found that mono- or multifactorial promoters of cell growth facilitate differentiation/maturation to the point that cells originally devoid of any specific function are able to express insulin as well as other molecular elements as part of the complex of a glucose-sensing and stimuluscoupled insulin secretion apparatus. Our observations on gene transcriptional products associated with endocrine cell phenotypes deriving from epithelial ductal cells focused on the following items: (1) KGF-elicited increased mRNA for PDX-1, Nkx6.1, Neuro-D, as compared to controls19,20; HGF and high glucose also induced an increase in the above-mentioned transcriptional products, comparably, although the former is a growth factor and the latter, a nutrient21–24; SC-conditioned medium mainly raised GLUT-2 and GK mRNA levels, although to a lesser extent than KGF with no significant effects on other gene transcripts.25–29 Mature protein synthesis, as assessed by Western blot, indicated that SC was the most powerful inducer of

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GK production, as compared to other condions, which did not differ significantly between them; GLUT-2 was increased upon incubation with high glucose. From the obtained results it seems that SC-conditioned medium may drive ductal cell cultures toward thorough ␤-cell maturation as compared to other conditions. This finding was confirmed by insulin secretory patterns in the medium under basal conditions. In fact, the equimolar release of insulin and C-peptide, in SC-conditioned, but not other, culture conditions indicates that the former is associated with a greater extent of cell maturation, in terms of insulin release in response to exogenous glucose stimulation. In conclusion, ductal-derived cell cultures are potential candidates for ␤-like insulin-producing cells. It is possible to drive ␤-cell differentiation from these cultures without ECM-like support, just by using appropriate tissue culture conditions. Amongst these, we believe that SC-conditioned medium, containing a series of molecules and growth factors that modulate cell development, may represent the most powerful promoter of transdifferentiation of pancreatic neonatal tissue into ␤-cell–like insulin-secreting cell populations. REFERENCES 1. Ryan EA, Lakey JR, Paty BW, et al: Diabetes 51:2148, 2002 2. Berney T, Mathe Z, Bucher P, et al: Transplant Proc 36:1123, 2004 3. Calafiore R, Basta G, Luca G, et al: Biotechnol Appl Biochem 39:159, 2004 4. Calafiore R: Expert Opin Biol Ther 3:201, 2003 5. Binette TM, Dufour JM, Korbutt GS: Ann NY Acad Sci 944:47, 2001 6. Omer A, Duvivier-Kali VF, Trivedi N, et al: Diabetes 52:69, 2003

2863 7. Luca G, Nastruzzi C, Basta G, et al: Diabetes Nutr Metab 13:301, 2000 8. Luca G, Calvitti M, Baroni T, et al: Acta Diabetologica 4:207, 2003 9. Hunkeler D: Nat Biotechnology 17:1045, 1999 10. Basta G, Racanicchi L, Mancuso F, et al: Transplant Proc 36:609, 2004 11. Giannattasio AJ: Endocrinol Invest 25:RC23, 2003 12. Macfarlane W, McKinnon CM, Felton-Edkins ZA, et al: J Biol Chem 274:1011, 1999 13. Bonner-Weir S, Sharma A: J Pathol 197:519, 2002 14. Efrat S: Ann N Y Acad Sci 1014:88, 2004 15. Ramiya VK, Maraist M, Arfors KE, et al: Nat Med 6:278, 2000 16. Bonner-Weir S, Taneja M, Weir GC, et al: Acad Sci USA 97:7999, 2000 17. Gao R, Ustinov J, Pulkkinen MA, et al: Diabetes 52:2007, 2003 18. Bonner-Weir S: Endocrinology 141:1926, 2000 19. Movassat J, Beattie GM, Lopez AD, et al: Diabetologia, 46:822, 2003 20. Hardikar AA, Marcus-Samuels B, Geras-Raaka E, et al: Proc Natl Acad Sci USA 100:7117, 2003 21. He ZP, Tan WQ, Tang YF, et al: Differentiation 71:281, 2003 22. Mashima H, Shibata H, Mine T, et al: Endocrinology 137:3969, 1996 23. Rafiq I, da Silva Xavier G, Hooper S, et al: J Biol Chem 275:15977, 2000 24. Macfarlane WM, McKinnon CM, Felton-Edkins ZA, et al: J Biol Chem 274:1011, 1999 25. Li L, Seno M, Yamada H, et al: Am J Physiol Endocrinol Metab 285:E577, 2003 26. Luca G, Calvitti M, Neri LM, et al: J Invest Med 48:441, 2000 27. Lamb DJ, Spotts GS, Shubhada S, et al: Mol Cell Endocrinol 79:1, 1991 28. Mashima H, Ohnishi H, Wakabayashi K, et al: J Clin Invest 97:1647, 1996 29. Dufour JM, Gores P, Hemendinger R, et al: Cell Transplant 13:1, 2004