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Published online November 16, 2006
Diabetes 56:3-7, 2007
DOI: 10.2337/db06-1165
© 2007 by the American Diabetes Association
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Islet-Derived Fibroblast-Like Cells Are Not Derived via Epithelial-Mesenchymal Transition From Pdx-1 or Insulin-Positive Cells

Lucas G. Chase, Fernando Ulloa-Montoya, Benjamin L. Kidder, and Catherine M. Verfaillie

From the Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota

Address correspondence and reprint requests to Catherine M. Verfaillie Stem Cell Institute, University of Minnesota, McGuire Translational Research Facility, 2001 6th St. SE, mail code 2873, Minneapolis, MN 55455. E-mail: verfa001{at}umn.edu

Abbreviations: BM-MSC, bone marrow mesenchymal stem cell; CAAGS, chicken ß-actin promoter/cytomegalovirus enhancer; EMT, epithelial-mesenchymal transition; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; MSC, mesenchymal stem cell; qPCR, quantitative PCR


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As recent studies suggest that newly formed pancreatic ß-cells are a result of self-duplication rather than stem cell differentiation, in vitro expansion of ß-cells presents a potential mechanism by which to increase available donor tissue for cell-based diabetes therapies. Although most studies have found that ß-cells are resilient to substantial in vitro expansion, recent studies have suggested that it is possible to expand these cells through a process referred to as epithelial-mesenchymal transition (EMT). To further substantiate such an expansion mechanism, we used recombination-based genetic lineage tracing to determine the origin of proliferating fibroblast-like cells from cultured pancreatic islets in vitro. We demonstrate, using two culture methods, that EMT does not underlie the appearance of fibroblast-like cells in mouse islet cultures but that fibroblast-like cells appear to represent mesenchymal stem cell (MSC)-like cells akin to MSCs isolated from bone marrow.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose many of their mature characteristics (i.e., tight junctions) and acquire properties commonly associated with mesenchymal cells (i.e., vimentin expression). While this process has been shown to be absolutely necessary for embryogenesis, it has also been associated with pathological conditions such as fibrosis and cancer (1). Two recent studies (2,3) have suggested that under certain culture conditions, pancreatic ß-cells can undergo EMT and dedifferentiate into fibroblast-like cells. In addition, these cells can be extensively propagated and subsequently redifferentiated into cells expressing insulin and glucagon. As harvested pancreatic islet fractions are never comprised of purely endocrine cells, these studies cannot prove that the pancreatic fibroblast-like cells are generated by EMT from ß-cells. Using rigorous genetic-based lineage-tracing models, we here investigate whether fibroblast-like cells observed in cultured islet fractions are derived from a pancreatic origin.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic animals.
For mouse experiments, the following strains were used: C57BL/6J, Ins2cre, Z/EG (The Jackson Laboratories, Bar Harbor, ME), Pdx1cre (Pedro Herrera, University of Geneva), and MIP-GFP (Manami Hara, University of Chicago). Transgenic offspring were genotyped by PCR, as described in the supplemental research methods (see online appendix, available at http://diabetes.diabetesjournals.org). All transgenic animals were bred and maintained under specific pathogen-free conditions. University of Minnesota housing and husbandry guidelines were adapted from requirements in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (4).

Isolation and culture of mouse islet–enriched pancreatic fractions.
Adult mice (10–24 weeks of age) were anesthetized with 250 mg/kg Avertin, the abdomen was opened, the pancreas was perfused, and the islets were purified as detailed in the supplemental research methods. Islet fractions were then seeded in a low-serum–, platelet-derived growth factor BB–, epidermal growth factor–, and leukemia inhibitory factor–supplemented culture media formulation (see supplemental research methods for detailed culture protocol) or as previously described (2,3). Bone marrow mesenchymal stem cells (BM-MSCs) were obtained from Dr. Darwin Prockop (Tulane University) and cultured as described (5). Methods for differentiation of Sca1+ fibroblast-like progenitors are described in the supplemental research methods.

Fluorescence-activated cell sorter analysis.
Cells were harvested using 0.25% trypsin (Mediatech, Herndon, VA), washed with PBS plus 2% fetal bovine serum and 1 mmol/l EDTA (Ambion, Austin, TX), and filtered through a 40-µm cell strainer (BD Biosciences, San Jose, CA) to remove remaining cell clusters. Cells from total islet cultures were stained with anti–CD45-APC (30-F11) and Sca1-PE (E13-161.7) (BD Biosciences), and CD45–/Sca1+ cells were selected using a FACSAria (BD Biosciences). Alternatively, 1 x 105 Sca1+–cultured cells were harvested and stained with anti–CD45-APC (30-F11), Sca1-PE (E13-161.7), and CD44-FITC [fluorescein isothiocyanate] (IM7) (BD Biosciences) and analyzed using the FACSCalibur flow cytometer (BD Biosciences). Anti-rat IgG2b{kappa}-APC (A-95-1), IgG2b{kappa}-FITC (A-95-1), and IgG2a{kappa}-PE (R35-95) antibodies (BD Biosciences) were used as isotype controls.

RNA isolation, cDNA synthesis, and quantitative real-time PCR.
Total RNA (1 x 105–1 x 106 cells) was isolated using an RNeasy Micro Kit (Qiagen, Valencia, CA). RNA was treated with Turbo DNA-free (Ambion) according to the manufacturer’s protocol and reverse transcribed into cDNA using Superscript III with random hexamers (Invitrogen, Carlsbad, CA). For real-time RT–quantitative PCR (RT-qPCR), cDNA was amplified using SYBR Green PCR Master mix with an ABI Prism 7700 (Applied Biosystems, Foster City, CA), as described in the supplemental research methods.

Genomic DNA isolation and excision PCR.
Genomic DNA was obtained from trypsinized and filtered day 10–14 Pdx1cre-Z/EG (ZPC) cultures using the QiaAmp DNA Micro Kit (Qiagen). PCR detection of excision was performed using excision (chicken ß-actin promoter/cytomegalovirus enhancer [CAAGS]-GFP)–and nonexcision (CAAGS-LacZ)–specific primers (Table 1 of online appendix). Standard PCR or real-time qPCR was performed using Platinum TaqDNA Polymerase (Invitrogen) or SYBR Green PCR Master mix, respectively, as described in the supplemental research methods. As a relative (100%) control, tail genomic DNA was obtained from a double-transgenic Actbcre-Z/EG animal. This animal provides expression of both Cre recombinase under control of the ß-actin promoter and the Z/EG reporter.

X-gal staining.
Mouse islet fractions from transgenic Ins2cre-Z/EG (ZI) mice were cultured in 24-well culture plates (BD Biosciences) in culture media as described. Once confluent, fibroblast-like cells were fixed in 0.2% glutaraldehyde/2% formaldehyde for 5 min and stained overnight at 37°C with X-gal stain as described (6).


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Consistent with previous reports on human islet cultures (2,3), we observed a population of fibroblast-like cells appearing when mouse islet–enriched fractions (Fig. 1A, a) were cultured in medium containing 2% fetal bovine serum, epidermal growth factor, platelet-derived growth factor BB, and leukemia inhibitory factor (Fig. 1A, b). Within this population, ~10–20% of the cells were CD45– and expressed the stem cell antigen Sca1 (Fig. 1B, a). The CD45–/Sca1+ cells, purified by fluorescence-activated cell sorter (FACS) analysis, could be maintained for >40 passages in the same media (Fig. 1A, c and d). The expanded Sca1+ cells coexpressed CD44 but not CD45 (Fig. 1B, b) and expressed characteristic epithelial and mesenchymal transcripts (Fig. 2A), a phenotype similar to that of islet-derived fibroblast-like cells described by others (2).


Figure 1
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  		FIG. 1. Morphology and FACS analysis of pancreas-derived fibroblast-like cells. A: After 5–7 days of culture, mouse islet–enriched fractions (a) revealed robust proliferation of fibroblast-like cells (b). B, a: Cells from 10- to 14-day cultures labeled with antibodies against CD45 and Sca1 were analyzed by flow cytometry. FACS-purified CD45–/Sca1+ cells were continually propagated (A, c and d) and analyzed by flow cytometry (B, b) with antibodies against Sca1, CD44, and CD45 (passage 18 shown). C: BM-MSCs were labeled with antibodies against Sca1 and CD44 and analyzed by flow cytometry. Original magnification: A, a and b: 10x; A, c: 20x; A, d: 40x.

 

Figure 2
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  		FIG. 2. Real-time RT-qPCR analysis and differentiation of Sca1+ pancreatic progenitor cells. A: RNA extracted from Sca1+ cells (passage 37) and from BM-MSCs were analyzed by real-time RT-qPCR for endodermal and mesodermal, transcripts. {Delta}Ct = Ct(gene of interest) –Ct(Actb). Expression levels: ++++, very high expression ({Delta}Ct <6); +++, high expression ({Delta}Ct 6–12); ++, moderate expression ({Delta}Ct 12–18); +, low expression ({Delta}Ct >18). B: Sca1+ progenitors (passages 16–37) were plated in differentiation medium containing hepatocyte growth factor and dexamethasone. Four-week cultures revealed the presence (10–20%) of adipocyte-like cells. a, unstained; b and c, Oil red O stained. a–c, original magnification 20x.

 
When switched to serum-free differentiation conditions (with or without individual or combinations of cytokines, including activin, bone morphogenetic protein-4, growth and differentiation factor-11, betacellulin, exendin-4, and/or hepatocyte growth factor), the mouse Sca1+ pancreas-derived cells did not acquire a more mature pancreatic phenotype (data not shown). We observed, however, a robust differentiation to lipid-filled adipocytes (Fig. 2B).

As the CD44+/Sca1+ phenotype and adipocyte differentiation potential of the Sca1+ pancreatic progenitors was reminiscent of BM-MSCs, we compared Sca1+ pancreatic progenitors with BM-MSCs. Like pancreas-derived Sca1+ cells, murine BM-MSCs were Sca1+ and CD44+ by flow cytometry (Fig. 1C). Real-time RT-qPCR demonstrated that both cell types expressed low levels of most epithelial markers (Fig. 2A), with pancreas-derived Sca1+ cells expressing high levels of CK18 and -19 and BM-MSCs expressing moderate levels of CK19. Likewise, undifferentiated human MSCs have been found to express both CK18 and -19 (7). Both mouse Sca1+ pancreas-derived cells and BM-MSCs expressed very low or undetectable levels of the endoderm/pancreas markers Afp, Sox17, Hnf1ß, Hnf3ß, and Pdx1 but expressed high levels of the mesenchymal markers Vim, Acta2, Cdh2, Csf1, Zeb, Snail1, and Snail2 (Fig. 2A).

To conclusively determine whether fibroblast-like cells in our pancreas cultures could be explained by EMT, we performed studies using two Cre-Lox recombination-based genetic lineage-tracing models. Islet-enriched fractions were cultured from ZI and ZPC murine pancreata using the expansion culture conditions described above. In these animals, the Pdx-1 or insulin-2 promoters, respectively, drive Cre recombinase expression, causing excision of the ßgeo gene in the Z/EG reporter. This event replaces ßgeo expression with permanent green fluorescent protein (GFP) expression (8) (Fig. 3A). In agreement with previously published reports (8,9,10), ZI animals displayed robust islet-specific GFP labeling (Fig. 3B, a), while ZPC animals have permanent GFP labeling of all endodermal pancreatic lineages (Fig. 3B, e). In cultures from ZI or ZPC islet-enriched fractions, few, if any, fibroblast-like cells expressed GFP, as evaluated by immunofluorescence microscopy (Fig. 3B, b–d, f, and g) or flow cytometry (Fig. 3C). Individual fibroblast-like GFP-positive cells were very rarely observed (Fig. 3B, g). Standard PCR (Fig. 3D, a) and real-time qPCR (Fig. 3D, b) on genomic DNA isolated from nonclustered cells within ZPC bulk cultures revealed minimal evidence of lineage tracing, as indicated by a lack of amplification of the CAAGS-GFP–excised Z/EG construct. The low level of excision detectable only by real-time qPCR was likely due to the persistence of a small number of individual mature pancreatic endocrine or exocrine cells remaining in the filtered, analyzed cell population. That the reporter construct was not silenced during culture is shown by persistent X-gal staining of fibroblast-like cells from ZI islet fractions (Fig. 3B, h). To rule out the possibility that the media formulation selectively expanded nonendodermal fibroblast-like cells, we also performed identical lineage-tracing studies using media as previously described (2,3). When islet-enriched fractions from ZI mice (Fig. 3E, a) were cultured for 14 days in this media, we again did not observe GFP-positive fibroblast-like cells (Fig. 3E, b–d).


Figure 3
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  		FIG. 3. Lineage tracing on proliferative fibroblast-like cell populations. A: PCR primers were designed to allow detection of excised and nonexcised Z/EG reporter constructs. A common CAAGS promoter forward primer (A, black arrow) was used along with a GFP-specific reverse primer (A, green arrow) or a ßgeo-specific reverse primer (A, blue arrow). B: Islet-enriched fractions were cultured from ZI (a) and ZPC (e) pancreata. Early (day 3) ZI cultures revealed GFP-negative fibroblast-like cell outgrowth (b). Late-stage (day 10–14) ZI (c and d) and ZPC (f) cultures displayed a confluent population of GFP-negative fibroblast-like cells. Very rare GFP-positive fibroblast-like cells were observed in ZI or ZPC cultures, as shown for a ZI culture (g, white arrow). Late-stage ZI cultures revealed active CAAGS promoter activity within the Z/EG reporter, as shown by X-gal staining (h). Original magnification: a, c, e, and f: 10x; b, d, g, and h: 20x. C: Flow cytometry of day 10–14 bulk ZI and ZPC cultures following cell cluster filtering revealed minimal evidence of GFP-positive cells. D: Genomic DNA from unfractionated, filtered day 10–14 ZPC cultures was analyzed by standard PCR (a) and real-time qPCR (b) using excision (CAAGS-GFP)–and nonexcision (CAAGS-LacZ)–specific primers. Pos, positive control. As a relative (100%) control for real-time qPCR, genomic DNA was obtained from a double-transgenic Actbcre-Z/EG animal. E: Islet-enriched fractions (a) were cultured from ZI pancreata using media as described (2,3). By day 14, abundant GFP-negative fibroblast-like cells could be detected surrounding GFP-positive islets (b–d). a–d, original magnification 10x.

 
These lineage-tracing studies provide evidence, under the tested conditions, that endodermal pancreatic cells or ß-cells do not undergo EMT and do not significantly contribute to the proliferating fibroblast-like cell population. When islet fractions from MIP-GFP mice (11) were cultured under our conditions, some GFP-positive cells were found to attain a fibroblast-like morphology (Fig. 1 of online appendix). As we did not stain these cultures for mesenchymal markers, we do not know whether these cells coexpressed ß-cell and mesenchymal markers, as has been previously observed (2,3). However, as revealed through the lineage-tracing studies, these insulin-expressing cells did not significantly contribute to the proliferative fibroblast-like population. This lack of expansion precluded testing whether these cells could redifferentiate into mature ß-cells. It is unknown whether differences between our results and those recently published (2,3) are due to undetermined differences in culture protocols or reflect the use of mouse pancreatic tissue rather than human tissue.


   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we demonstrate that in two different media formulations, fibroblast-like cells can be cultured from mouse islet–enriched pancreatic fractions. Lineage-tracing studies conclusively demonstrate that these fibroblast-like cells are not derived from a ß-cell or endoderm pancreatic origin and therefore do not result from ß-cells undergoing EMT. By contrast, the phenotype and differentiation potential of the proliferating fibroblast-like cells appears similar to that of BM-MSCs; hence, these cells likely represent mesenchymal cells from within the pancreas.


   ACKNOWLEDGMENTS
 
These studies were supported by National Institutes of Health award U19 DK61244, a grant from the Juvenile Diabetes Research Foundation, and a research grant from Athersys (Cleveland, OH).


   FOOTNOTES
 
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.

Posted on the World Wide Web at http://diabetes.diabetesjournals.org on 16 November 2006.

See accompanying commentary on p. 281.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication August 17, 2006 and accepted in revised form October 6, 2006


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Thiery JP, Sleeman JP: Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7:131–142, 2006[Medline]
  2. Gershengorn MC, Hardikar AA, Wei C, Geras-Raaka E, Marcus-Samuels B, Raaka BM: Epithelial-to-mesenchymal transition generates proliferative human islet precursor cells. Science 306:2261–2264, 2004[Abstract/Free Full Text]
  3. Ouziel-Yahalom L, Zalzman M, Anker-Kitai L, Knoller S, Bar Y, Glandt M, Herold K, Efrat S: Expansion and redifferentiation of adult human pancreatic islet cells. Biochem Biophys Res Commun 341:291–298, 2006[Medline]
  4. Grossblatt N: Guide for the Care and Use of Laboratory Animals. Washington, DC, National Academy Press, 1996
  5. Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ: Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103:1662–1668, 2004[Abstract/Free Full Text]
  6. Jadrich JL, O’Connor MB, Coucouvanis E: The TGF beta activated kinase TAK1 regulates vascular development in vivo. Development 133:1529–1541, 2006[Abstract/Free Full Text]
  7. Moriscot C, de Fraipont F, Richard MJ, Marchand M, Savatier P, Bosco D, Favrot M, Benhamou PY: Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro. Stem Cells 23:594–603, 2005[Abstract/Free Full Text]
  8. Novak A, Guo C, Yang W, Nagy A, Lobe CG: Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28:147–155, 2000[Medline]
  9. Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, Lindner J, Cherrington AD, Magnuson MA: Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 274:305–315, 1999[Abstract/Free Full Text]
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  11. Hara M, Wang X, Kawamura T, Bindokas VP, Dizon RF, Alcoser SY, Magnuson MA, Bell GI: Transgenic mice with green fluorescent protein-labeled pancreatic beta-cells. Am J Physiol Endocrinol Metab 284:E177–E183, 2003[Abstract/Free Full Text]

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