Journal of Stem Cells Research Development & Therapy Category: Medical Type: Research Article
Tissue Complex of Adult Pancreatic Duct and Vascular Endothelial Cells Promotes In Vitro Differentiation into Insulin-Producing Cells
- Jun Kanamune1*, Chongmun Kim2, Yasuhiro Iwanaga3, Jorge David Rivas-Carrillo4, Shoichiro Sumi1, Shinji Uemoto3, Kazuyuki Yokokawa2
- 1 Department Of Organ Reconstruction, Field Of Clinical Application, Institute For Frontier Medical Sciences, Kyoto University, Japan
- 2 Bioscience Department, IKKO-ZU Corporation, Kanagawa, Japan
- 3 Departmentof Surgery, Division Of Hepatobiliary Pancreatic And Transplant Surgery, Kyoto University, Japan
- 4 Department Of Physiology, Laboratory Of Immunology, Tissue Engineering And Transplant, University Of Guadalajara, University Center For Health Sciences, Mexico
*Corresponding Author:Jun Kanamune
Department Of Organ Reconstruction, Field Of Clinical Application, Institute For Frontier Medical Sciences, Kyoto University, Japan
Received Date: Mar 31, 2015 Accepted Date: Jun 03, 2015 Published Date: Jun 17, 2015
Vascular endothelial cells are known to be involved in pancreatic organogenesis. An important seminal work indicated a direct role of vascular endothelial cells in expressing Pdx1, a master transcription factor to engage in the expansion of endocrine precursor population . In organogenesis, contacts with endothelial cells allow endoderm to generate a pancreatic rudiment tissue expressing Pdx1 [6-10]. Moreover, biology of native islets indicates the importance of endothelial cells, as a paracrine source that secrete extracellular matrices  as well as soluble factors that promote islet survival and function [12,13].
Based on the view that adult pancreatic stem-like cells partially recapitulate the organogenesis in the course of differentiation into endocrinal cells [14,15], vascular endothelial cells possibly enhance their differentiation in the In vitro regeneration model . Using a pancreatic injury and regeneration model in adult mice, we previously found that doxorubicin-induced perturbation of vascular endothelial tissues significantly depleted of a population of CD133-positive pancreatic stem cells , indicating that vascular endothelial cells are involved in pancreatic regeneration.
In the light of these background data, we hypothesized that a direct contact between vascular endothelial cells and pancreatic stem duct cells would enhance the differentiation of the latter into endocrinal cells. In support of the hypothesis, the tissue complex of these two different cell populations that were isolated and expanded from a common tissue source of the pancreas showed increased proportions of insulin-producing cells and insulin-secreting abilities. The tissue complex when transplanted into the kidney normalized glucose levels in some individuals of diabetic mice.
In the course of this study, we investigated the role of another cell of mesodermal origin, stellate cell, in the In vitro proliferation of the pancreatic stem-like cells. Stellate cells and their role as a promoter of cell proliferation are well recognized particularly in the pathological setting of pancreatic inflammation. Stellate cells upon experimental pancreatic injuries are known to proliferate in the tissue regeneration . Because stellate cells produce mitogens such as cytokines and growth factors involved in precursory conditions leading to oncogenesis , they are thought to function as a paracrine source of similar mitogens in regenerating pancreas.
We tested if stellate cells would promote the murine adult pancreatic stem-like cells to proliferate in a co-culture model. The stellate cells kept in a transwell insert substantially increased the proliferation of an Aldeflour-positive pancreatic stem cells cultured in a common medium, which eventually established a monolayer of epithelial cell population with a potency to differentiate into endocrinal cells.
MATERIALS AND METHODS
Pancreatic tissue collection
Non-endocrinal epithelial cell culture
About 60% of the media was subsequently replenished in every 2-3 days. With this serum-free medium culture, scattered colonies of Non-Endocrinal Epithelial Cells (NEECs) were passaged into separate culture dish, so that they eventually formed dense cell clusters. After this stage, the cultures were occasionally treated with diluted dithizone solution to stain any remaining islet tissue contaminants to be manually removed. For passaging, 50 to 70%-confluent NEECs were washed twice with PBS and removed by incubating with Accutase (Innovative Cell Technologies, Inc.) at RT for less than 3min. The cells were gently removed by adding the culture medium, passed through a 40μm mesh to remove large tissue clumps, and pelleted cells were re-suspended with the same serum-free medium and seeded at or above 5.0 x 106 cells/ml on a collagen I-coated 10cm dish or a small fraction of these on 4-well slide chambers (Biocoat Cellware, BD Labware) for immunohistochemical analyses.
Vascular endothelial cell culture
Formation of tissue complex
RNA isolation and semi-quantitative RT-PCR
|Genes||GenBank no.||Forward primer||Reverse primer||Size|
Evaluation of insulin-secreting function
In vivo functional evaluation of tissue complex
Sorting of Aldefluor-positive cells by FACS
Promotion of Aldefluor-positive stem-like cells by co-culture with stellate cells
In vitro cultures of non-endocrinal epithelial cells
Figure 4: Relative expressions of genes: Ngn3, Pdx1, NeuroD and Ptf1a (A) and Notch1, Hes1, HNF6 and Nkx6.1 (B) based on semi-quantitative RT-PCR of the NEECs. The numbers 1-4 indicate the tissues or cell cultural samples used for the analyses: 1, pancreatic discards; 2, NEECs after 5 days of culture in serum-free media; 3, NEECs cultured with media containing all-trans retinoic acid A and cyclopamine KAAD for 24 hrs; and 4, NEECs’ culture control of 3. Arrows highlight that Ngn3 and Hes1 expressions seem synchronized in heterogeneous cell populations in the cultures.
Generation of tissue complex from NEECs and VECs
Tissues generated with and without VECs
Glucose stimulation tests
In vivo glucose-controlling ability of the generated tissues
Aldefluor-positive cells’ proliferation in co-culture with stellate cells
Both immunocytochemical and mRNA analyses indicated increased expressions of Ngn3 by NEECs when cultured with cyclopamine KAAD and all-trans retinoic acid, which suggests that the heterogeneous populations of NEECs indeed contained stem-like cells with endocrinal differentiation potentials. In synchronized differentiation of ES cells toward pancreatic endocrinal stem cells, Ngn3 expression is known to occur transiently following the down regulation of Notch1 and Hes1 [25,26]. Thus, the concomitant expression of Ngn3 along with that of Notch1 and Hes1 (Figure 4) characterizes the temporal heterogeneity of the NEECs whose subpopulations were undergoing different stages of endocrinal differentiation.
To test if the interaction with VECs would enhance endocrinal differentiation In Vitro, we co-cultured the VECs and NEECs to form a spherical tissue complex in a suspension culture, the method previously used for generating islet-like tissues and known to increase their insulin-secretion ability [27,28]. Cells dissociated from culture containers were swirl-incubated in a culture medium to promote direct homotypic and heterotypic cell-to-cell contacts. Furthermore, in the light of recent finding on ES cell’s differentiation into hepatocytes, swirling motion exerted a certain degree of sheer stress, which in combination with some soluble factors in the medium is known to enhance a differentiation process [29,30].
The spherical tissue complex had increased proportions of insulin-positive cells, amounts of secreted C-peptide, glucose responsiveness, and mRNA expressions of Ins1, Ins2 and glucagon. These data suggest that VECs enhanced the differentiation of progenitors residing in NEECs into functional endocrinal cells. Whether such enhancement was primarily of direct cell-to-cell contact or by means of some extracellular materials secreted by VECs is yet to be clarified. With respect to the contribution of the extracellular materials, it is worth noting that the co-culture increased the amount of tissues generated. Since the amount of VECs mixed with NEECs was less than 30% of the total cell number, the doubled tissue volume as a result of co-culture suggests that VECs had secreted and increased the amount of extracellular materials into the tissue complex.
In vivo evaluation of the tissue complex also showed its overall positive functional qualities in spite of the ambivalent outcomes regarding its In vivo glucose-controlling ability. Among the total of 9 recipient diabetic mice transplanted, only one individual recovered normoglycemia, but notably all of the recipients of the tissue complex survived throughout the experimental period, and a significant number of them had fluctuating blood glucose levels between 300 and 600mg/dl. In contrast, the control diabetic mice without the tissue transplants died during the experimental period.
Aldefluor-E-cadherin double positive pancreatic cells in adult mice have been isolated as a facultative endocrinal stem-like cell population, which required a spheroid culture for continuous clonetic proliferation . We found that these stem-like cells proliferated as a monolayer when co-cultured with stellate cells, suggesting that they require specific cellular niches but are somewhat flexible in choice of physical environments. Since the monolayer NEECs that we have established from pancreatic tissue debris contained some stellate cells, their continuous proliferation possibly entails similar interactions in the co-culture environments.
Our present data indicate that generation of tissue complex of NEECs and VECs both from pancreatic tissue debris can increase its potential of islet-like functions. In Vitro generation of islet-like tissues if performed efficiently will offset the shortage of transplantable donor islets. Once optimized, the tissue complex method will be a feasible application to human tissues for not only therapeutic but also in vitroscreening purposes.
- Puri S, Folias AE, Hebrok M (2015) Plasticity and dedifferentiation within the pancreas: development, homeostasis, and disease. Cell Stem Cell 16: 18-31.
- Carpino G, Cardinale V, Gentile R, Onori P, Semeraro R, et al. (2014) Evidence for multipotent endodermal stem/progenitor cell populations in human gallbladder. J Hepatol 60: 1194-1202.
- Wang Y, Lanzoni G, Carpino G, Cui CB, Dominguez-Bendala J, et al. (2013) Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis. Stem Cells 31: 1966-1979.
- Rovira M, Scott SG, Liss AS, Jensen J, Thayer SP, et al. (2010) Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas. Proc Natl Acad Sci USA 107: 75-80.
- Lammert E, Cleaver O, Melton D (2001) Induction of pancreatic differentiation by signals from blood vessels. Science 294: 564-567.
- Kim SK, Hebrok M (2001) Intercellular signals regulating pancreas development and function. Genes Dev 15: 111-127.
- Matsumoto K, Yoshitomi H, Rossant J, Zaret KS (2001) Liver organogenesis promoted by endothelial cells prior to vascular function. Science 294: 559-563.
- Cleaver O, Melton DA (2003) Endothelial signaling during development. Nat Med 9: 661-668.
- Crivellato E, Nico B, Ribatti D (2007) Contribution of endothelial cells to organogenesis: a modern reappraisal of an old Aristotelian concept. J Anat 211: 415-427.
- Zaret KS, Grompe M (2008) Generation and regeneration of cells of the liver and pancreas. Science 322: 1490-1494.
- Nikolova G, Jabs N, Konstantinova I, Domogatskaya A, Tryggvason K, et al. (2006) The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation. Dev Cell 10: 397-405.
- Bahary N, Zon LI (2001) Development. Endothelium--chicken soup for the endoderm. Science 294: 530-531.
- Johansson M, Mattsson G, Andersson A, Jansson L, Carlsson PO (2006) Islet endothelial cells and pancreatic beta-cell proliferation: studies In Vitro and during pregnancy in adult rats. Endocrinology 147: 2315-2324.
- Cano DA, Hebrok M, Zenker M (2007) Pancreatic development and disease. Gastroenterology 132: 745-762.
- Xu X, D'Hoker J, Stangé G, Bonné S, De Leu N, et al. (2008) Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell 132: 197-207.
- de Koning EJP, Nienaber C, Yatoh S, Patterson D, Rask-Madsen C, et al. (2006) Endothelial cell involvement in proliferation and differentiation of human pancreatic duct cells (Abstract). Diabetologia 49: 290.
- Rivas-Carrillo SD, Kanamune J, Iwanaga Y, Uemoto S, Daneri-Navarro A, et al. (2011) Endothelial cells promote pancreatic stem cell activation during islet regeneration in mice. Transplant Proc 43: 3209-3211.
- Erkan M, Adler G, Apte MV, Bachem MG, Buchholz M, et al. (2012) StellaTUM: current consensus and discussion on pancreatic stellate cell research. Gut 61: 172-178.
- Bachem MG, Schunemann M, Ramadani M, Siech M, Beger H, et al. (2005) Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology 128: 907-921.
- Nikolova G, Strilic B, Lammert E (2007) The vascular niche and its basement membrane. Trends Cell Biol 17: 19-25.
- Cirulli V, Beattie GM, Klier G, Ellisman M, Ricordi C, et al. (2000) Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells. J Cell Biol 150: 1445-1460.
- Menger MD, Vajkoczy P, Beger C, Messmer K (1994) Orientation of microvascular blood flow in pancreatic islet isografts. J Clin Invest 93: 2280-2285.
- Piper K, Brickwood S, Turnpenny LW, Cameron IT, Ball SG, et al. (2004) Beta cell differentiation during early human pancreas development. J Endocrinol 181: 11-23.
- Liew CG, Andrews PW (2008) Stem cell therapy to treat diabetes mellitus. Rev Diabet Stud 5: 203-219.
- Jensen J, Heller RS, Funder-Nielsen T, Pedersen EE, Lindsell C, et al. (2000) Independent development of pancreatic alpha- and beta-cells from neurogenin3-expressing precursors: a role for the notch pathway in repression of premature differentiation. Diabetes 49: 163-176.
- Jensen J, Pedersen EE, Galante P, Hald J, Heller RS, et al. (2000) Control of endodermal endocrine development by Hes-1. Nat Genet 24: 36-44.
- Ogata T, Park KY, Seno M, Kojima I (2004) Reversal of streptozotocin-induced hyperglycemia by transplantation of pseudoislets consisting of beta cells derived from ductal cells. Endocr J 51: 381-386.
- Todorov I, Omori K, Pascual M, Rawson J, Nair I, et al. (2006) Generation of human islets through expansion and differentiation of non-islet pancreatic cells discarded (pancreatic discard) after islet isolation. Pancreas 32: 130-138.
- Zhang S, Zhang Y, Chen L, Liu T, Li Y, et al. (2013) Efficient large-scale generation of functional hepatocytes from mouse embryonic stem cells grown in a rotating bioreactor with exogenous growth factors and hormones. Stem Cell Res Ther 4: 145.
- Sasaki K (2014) Large-scale generation of differentiated cells to achieve regenerative medicine. Stem Cell Res Ther 5: 10.
Citation:Kanamune J, Iwanaga Y, Rivas-Carrillo JD, Sumi S, Uemoto S (2015) Tissue Complex of Adult Pancreatic Duct and Vascular Endothelial Cells Promotes In Vitro Differentiation into Insulin-Producing Cells. J Stem Cell Res Dev Ther 2: 005.
Copyright: © 2015 Jun Kanamune, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.