Islet-Derived Fibroblast-Like Cells Are Not Derived via Epithelial-Mesenchymal Transition From Pdx-1 or Insulin-Positive Cells
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Figures
- 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: 10×; A, c: 20×; A, d: 40×.
- 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. ΔCt = Ct(gene of interest) –Ct(Actb). Expression levels: ++++, very high expression (ΔCt <6); +++, high expression (ΔCt 6–12); ++, moderate expression (ΔCt 12–18); +, low expression (ΔCt >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 20×.
- 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 βgeo-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: 10×; b, d, g, and h: 20×. 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 10×.
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