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Bone morphogenetic proteins promote development of fetal pancreas epithelial colonies containing insulin-positive cells

Fang-Xu Jiang1,*, Edouard G. Stanley2, L. Jorge Gonez1 and Leonard C. Harrison1,*

1 Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute of Medical Research, PO The Royal Melbourne Hospital, Parkville 3050, Australia
2 Cancer Division, The Walter and Eliza Hall Institute of Medical Research, PO The Royal Melbourne Hospital, Parkville 3050, Australia



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  		Fig. 1. RT-PCR expression profile analysis of TGF-ß superfamily members. Total RNAs purified from developing pancreas between E13.5 and E17.5 are shown. mRNAs were incubated with (+) or without (-) reverse transcriptase and subjected to PCR with specific primers as described in Materials and Methods.

 


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  		Fig. 2. BMP-6 (10 ng/ml), in the presence of laminin-1 (160 µg/ml), promotes cystic colony formation from dissociated E15.5 mouse pancreas cells. Phase contrast images show cells cultured for 2 or 6 days in laminin-1 with and without BMP-6. In the presence of BMP-6, small colonies and some tubule-like structures (closed arrow, also inset) were observed by day 2; by day 6, colonies increased in number and were of variable size. Colonies <=30 µm (open arrow) were excluded from the quantitation of colony numbers in Fig. 3 below.

 


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  		Fig. 3. Frequency of pancreatic cell colonies (mean±s.d., n=3) as a function of increasing concentration of BMPs. Colonies were directly counted under a phase contrast microscope (x10) 6 days after culture in the presence of 160 µg/ml laminin-1. Colonies <=30 µm (see Fig. 2) were not counted.

 


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  		Fig. 4. Effects of TGF-ß superfamily members on pancreatic cell colony formation. Colonies (mean±s.d., n=3 separate experiments) were counted 6 days after culture with the indicated treatment in the presence of 160 µg/ml laminin-1. (A) Effect of BMP-6, activin A, TGF-ß1 (all 100 ng/ml) or vehicle alone. (B) Effect of BMP-5, activin A or TGF-ß1 (each 100 ng/ml) on BMP-6 (10 ng/ml)-induced colonies. (C) Dose-dependent inhibition by TGF-ß1 and activin A of BMP-6 (10 ng/ml)-induced colonies.

 


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  		Fig. 5. Fluorescence images of proliferating cells in fetal pancreas cell colonies. BrdU (100 µM) was added during the last 16 hours of 6 day culture of E15.5 pancreas cells in the presence of laminin-1 and BMP-6. BrdU incorporated into the DNAs of proliferating cells was stained with mouse monoclonal anti-BrdU (A) and isotype control IgG2a (B) antibody and visualized with fluorescein-isothiocyanate-conjugated anti-mouse immunoglobulins.

 


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  		Fig. 6. Characterisation of colony cells. H&E staining showing colonies lined by various types of epithelia (A). Under a higher magnification (oil lens) several types of epithelia are shown: (B) columnar, (C) cuboidal, (D) squamous or (E) a mixture. (F) periodic acid Schiff (PAS) reaction staining of polysaccharides (pink, arrow) in the basement membrane surrounding the colony cells. (G) E-cadherin (E-cad), a marker of epithelial cells, detected by rat anti-mouse E-cadherin antibody and visualised by peroxidase-conjugated rabbit anti-rat immunoglobulins (brown).

 


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  		Fig. 7. Immunocytochemistry for proinsulin, insulin, glucagon and amylase in colony cells. The colony cells were directly fixed (for proinsulin only) or harvested for histological sections 6 days after culture. Proinsulin (Pro)-positive cells (also see inset) were stained with monoclonal anti-proinsulin antibody and peroxidase-conjugated rabbit anti-mouse immunoglobulins (A,B). Insulin (Ins)-positive cells were stained with guinea pig anti-insulin serum and visualised by peroxidase-conjugated rabbit anti-guinea pig immunoglobulins. Ins-positive cells constituted and in some cases appeared to bud off from colonies (C-F). Glucagon (Glu)-positive cells (G) and amylase (Amy)-positive cells (H) were stained with rabbit anti-glucagon and anti-{alpha}-amylase antibodies, respectively, and visualized with peroxidase-conjugated swine anti-rabbit immunoglobulins.

 

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© The Company of Biologists Ltd 2002