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. 2016 Feb;11(2):439-447.
doi: 10.3892/etm.2015.2943. Epub 2015 Dec 16.

Biological Characterization of Metanephric Mesenchymal Stem Cells From the Beijing Duck

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Free PMC article

Biological Characterization of Metanephric Mesenchymal Stem Cells From the Beijing Duck

Jia Chen et al. Exp Ther Med. .
Free PMC article

Abstract

Mesenchymal stem cells (MSCs) possess self-proliferation and multi-directional differentiation abilities. Previous studies on MSCs have mostly focused on the bone marrow, lungs, pancreas and umbilical cord blood, with few studies on metanephric tissues in ducks. For the present study, the Beijing duck was selected as an experimental animal. Duck embryo metanephric mesenchymal stem cells (MMSCs) were studied. MMSC isolation culture, analysis of biological characteristics, induced differentiation and identification were performed in preliminary experiments. In the current study, surface antigens and gene expression patterns were detected using immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. The induced cells, adipocytes, hepatocytes, epithelial cells and islet cells were identified by oil red O staining, periodic acid-Schiff staining, immunofluorescence and dithizone staining, respectively. RT-PCR was performed for detection of specific marker genes. The results suggested that the biological characteristics of MMSCs were similar to those of the MSCs previously analyzed. Primary MMSCs were sub-cultured to passage 21. The induced cells exhibit typical staining and immunofluorescence indicating the expression of specific genes. This demonstrates that MMSCs may be a novel alternative source of MSCs for experimental and clinical applications.

Keywords: Beijing duck; differentiation; metanephric mesenchymal stem cells.

Figures

Figure 1.
Figure 1.
Morphology of primary and subcultured MMSCs and growth curve. (A) Following 16 h of primary culture, MMSCs adhered to plates. Hemocytes mixed with MMSCs were present. (B) Passage 3 MMSCs exhibited polygonal and long shuttle shapes, the majority of which presented protrusions, and gradually reached confluence, with strong cell proliferation ability. (C) Passage 8 MMSCs were fibroblast-like and homogeneous. Scale bars, 100 µm. (D) Growth curve for MMSCs. Cell proliferation processes are similar and typical of the ‘S’ shape. Data are presented as the mean ± standard deviation. n=3/group. MMSCs, metanephric mesenchymal stem cells; OD, optical density.
Figure 2.
Figure 2.
Reverse transcription-polymerase chain reaction analysis demonstrated that metanephric mesenchymal stem cells at passages 5 and 10 expressed fibronectin, Pax2, CD29, CD71 and CD166, but CD34 and CD45 were negative. GAPDH served as the internal control.
Figure 3.
Figure 3.
Immunofluorescence identification of P5 generation cells at the same time points. (A) DAPI/without antibody, and (B) DAPI/with antibody. The results demonstrated that fibronectin, CD29, CD71 and CD73 were positive, whilst CD45 was negative. Scale bar, 50 µm.
Figure 4.
Figure 4.
Flow cytometry analysis. The horizontal line on each histogram indicates the percentage of positive cells for each surface protein. FITC, fluorescein isothiocyanate.
Figure 5.
Figure 5.
Morphology and gene expression of adipocytes following differentiation of MMSCs. (A) Negative control cells cultured in complete medium presented no changes in morphology and were negative for oil red O staining throughout the experimental period. (B) Following induction for 9 days, MMSCs became oblate and formed intracellular lipid droplets. At day 12, the mast cells possessed more apparent shiny fat droplets. (C) Lipid droplets, apparent at days 9 and 12, were stained with oil red O. Scale bars, 100 µm. (D) Expression of adipocyte-specific genes. LPL and PPAR-γ were detected in the induced group following induction for 6, 9 or 12 days, but not expressed in the control group. GAPDH served as the internal control. MMSCs, metanephric mesenchymal stem cells; LPL, lipoprotein lipase; PPAR-γ, peroxisome proliferator-activated receptor-γ.
Figure 6.
Figure 6.
Morphology of hepatocyte differentiation of metanephric mesenchymal stem cells and specific gene expression following induction for 15 days. (A) In the negative control group, cells presented no clear change in form and were negative for periodic acid-Schiff staining. Scale bar, 100 µm. (B and C) In the induced group, the number of round cells significantly increased, presenting a polygonal shape. (B) Scale bar, 100 µm; (C) scale bar, 50 µm. (D) Periodic acid-Schiff staining was successful. Scale bar, 100 µm. (E) Hepatocyte-specific genes ALB and AFP were expressed in the induced group following induction for 8, 12 or 15 days. These genes were not expressed in the control group. GAPDH served as the internal control. With the increase of induction days, gene expression strengthened gradually.
Figure 7.
Figure 7.
Morphology and gene expression of epithelial cells following differentiation of metanephric mesenchymal stem cells. (A) The control group presented no clear change in form. Scale bar, 100 µm. (B) In the induced group, following 10 days, epithelioid cells were plates like pebbles or paving stones, with single-layer adherent growth. Normal clear cell boundary and refraction, stereoscopic, closely linked cells. Scale bar, 100 µm. Immunofluorescence analysis: (C) DAPI/without antibody; (D) CK18+/DAPI; (E) DAPI/without antibody; (F) CK19+/DAPI. Scale bars, 50 µm. (G) Epithelial cell-specific genes CK19 and E-cadherin were expressed in the induced group following 8-, 10- and 12-day induction, but the control group was negative.
Figure 8.
Figure 8.
Morphology and specific gene expression of islet cells following differentiation of metanephric mesenchymal stem cells. (A) The control group presented no significant change in form. (B) Following induction for 10 days, there were several islet-like cell clusters in the induced cell plate. (C) Cell clusters were dyed scarlet by dithizone staining. Scale bars, 100 µm. (D) Islet cell-specific genes PDX-1 and insulin were positive in the induced group, but not in the control. GAPDH served as the internal control.

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