Establishment and characterization of GFP-positive primary cell culture

To determine the fate of the GFP-positive cells, GFP-positive cells were sorted from cell suspensions which were prepared from glandular stomach of transgenic mice in P10.

Approximately, 20.000 GFP-positive cells were isolated by FACS-sorting and plated on mitomycin treated embryonic fibroblast layer in two wells of a 24-well plate. Cells were cultured with ES cell growing medium containing LIF. After 72 hrs of culture, the medium

was changed and the growing medium was changed every 4 days. The cells were passaged every 10 days.

Examination of the cell cultures by fluorescence microscopy revealed that approximately 80% of plated cells were GFP-positive. However, the percentage of GFP-positive cells was dramatically decreased after 24 hrs and no GFP-positive cells could be detected after 72 hrs (Fig. 3.51). In another experiment, we plated the sorted GFP-positive cells on gelatine coated wells of a 24-well plate and cultured them with ES cell growing medium containing LIF. Similar to the results of the first experiment, no GFP-positive cells could be observed after 2 days of culture. This result suggests that the expression of EGFP is downregulated in cells during the culture or that the GFP-positive cells are not able to grow under the mentioned culture condition.

Sorted cells were grown during the first week of culture as layers over the feeder cells.

Cells were dissociated to single cells with trypsin and plated into three gelatine coated wells of a 24-well plate (passage-1). After two days of culture, several cell clones were observed in cell culture of passage-1. These clones were increased in size during the culture.

Treatment of clones with trypsin revealed that the trypsin is not able to dissociate the cell clones into single cells. However, mechanical dissociation of trypsinized cells by vigorous resuspension dissected the clones into several parts. These cells were then plated again on a gelatine coated plate (passage-2). We cultured these cells till passage-4. Due to the failure of dissociation of cell clones, we could not able to determine the proliferation rate of

Figure 3.51 FACS sorted EGFP positive stomach cells during 72 hrs. The FACS sorted EGFP cells were plated on embryonic fibroblast feeder layer with ES cell growing medium. The fluorescence photomicrogpraphs of FACS sorted cell were taken after plating 1 hr, after 24 hrs, and after 72 hrs. The photomicrographs are overlapping with bright field and EGFP filter. Optical magnification: 10X

cultured cells. Collagenase and dispase treatment was not able to dissociate cell clones to single cells.

To determine whether stomach-derived cells in passage-4 represent stem cell characteristics, cells were cultured on slides, fixed with 4% paraformaldehyde for 30 min and stained with rabbit anti-VSIG1 and mouse anti-GFP antibody. Other slides with stomach derived cells were used to determine the expression of alkaline phosphatase, a marker of pluripotent stem cells.

ed

e the specific staining of GFP and VSIG1 antibodies, the cell culture was stained only with the second antibodies as control (Fig. 3.52E-H). The immunostaining without first antibodies showed that some cells in the clones could have artificial signals due to unspecific staining by the secondary antibody (Fig. 3.52E-H)

Figure 3.52 Stomach derived cell clumps were stained with anti-VSIG1 and anti-GFP. Confocal images show that the clumps were stained with anti-VSIG1 and anti-GFP antibodies (A-D). But the immunostaining without the first antibody shows that the stained clumps could have artificial signals due to unspecific staining by secondary antibody. Optical magnification: 10X

As shown in figure 3.52B, cells locating in the centre of the clones were highly stain with anti-GFP antibody as compared to those located in the edge of clones. In contrast to that, VSIG1-stained cells were localized at the edge of clones while cells in the centre of clusters were VSIG1 negative (Fig. 3.52A). Co-localization of GFP and Vsig1 expressing cells revealed the presence of GFP and VSIG1 positive cells (Fig. 5.52D). To prov

To determine the alkaline phosphatase activity in clones of passage-4, cells were subjected to alkaline phosphatase assay (AP-assay). For positive control, we used embryonic stem cells. As shown in figure 3.54A, ES cells have high AP-activity (blue staining). In contrast, clones of stomach-derived cells are not stained with AP (Fig. 3.53B)

Figure 3.53 Alkaline phosphatase detection assay on stomach and ES cells.

Blue color is indication of AP activity and the brown color is the indication of lack of activity of AP. The control ES cell clones show blue color which is the indication of alkaline phosphatase activity, whereas the stomach cell clones show brown color. But in stomach cell culture, some cells which are not aggregated also showed blue color (indicated by black arrow). Optical magnification: 10X

3.7.7 Expression analysis for different marker genes in primary cell culture of stomach

To determine the expression of pluripotency and endodermal stem cell markers in stomach-derived cell culture, RT-PCR analysis was performed. Total RNA was isolated from the cells of passage-4 (P4) and cDNAs were reverse transcribed using the poly-T primers. The ES cell-, stomach-, and brain-cDNAs were used as control. Specific primers for pluripotency genes Oct4, Sox2, Nanog, Rex-2, endodermal stem cell marker genes GATA-4, Lgr5, alpha-fetoprotein (Afp), and early epithelial expressing gene KRT1-18 were used in RT-PCR assay. PCR products were cloned into pGEMT-easy vector and sequenced.

Figure 3.54 mRNA expression analysis of cultured stomach cells (P4) for different markers. RT-PCR was performed on cDNAs from brain, ES cells, passage-4 (P-4), stomach, and blank control. GAPDH primers were used for control amplification.

The RT-PCR analysis showed that pluripotent marker Oct4 and Nanog are not expressed in P-4 and stomach. Other pluripotent markers such as Sox2 and Rex1 are slightly expressed in cells of P-4 and stomach. In case of endodermal markers, only GATA4 expression was found in P4 and stomach, whereas Lgr5, Afp, and KRT expression was not found. In case of control ES cells, high expression of pluripotent markers was found. This result indicates that the stomach derived cultured cells do not express pluripotency markers and early epithelial cell markers.

3.8 Analysis of cell adhesion and cell migration in stable cell lines