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Chapter 6 Comparison Between Human 6One Marrow and Gingivae chapter 6 Comparison Between Human 6one marrow and gingivae mesencfiymaC stem ceCCsfor morpfioCogy and growth characteristics Ik. 6.1 Introduction Results from previous chapter demonstrated gingiva as a potential source of MSCs. The cells derived from gingiva exhibit similar morphology, phenotypic surface marker expression, in vitro differentiation potential and in vivo regeneration ability as expressed by BM-MSCs. Like BM-MSCs, gingival cells show typical fibroblast morphology; follow a sigmoid curve growth pattern. In this chapter the comparison between both cell types for growth characteristic in early and late passages is demonstrated. The stem cells derived from dental tissues exhibit higher rate of proliferation (~ 30-50%) when compared to the growth of cultured BM-MSCs [Gronthos et al., 2000; Shi et al., 2001; Gronthos et al., 2002; Gronthos et al., 2003]. Dental stem cells maintain a higher growth potential beyond 100 PDs (-14-16 passages) in contrast to BM-MSCs that begin to undergo cellular senescence at approximately 50 (~7-8 passages) PDs (about 6-7 PDs per passage) [Izadpanah et al., 2006]. These results suggest that dental stem cells are better than BM- MSCs in terms of proliferation and maintenance of phenotypic characters in long term cultures. All the known MSC sources have their own advantages as well as disadvantages. Dental tissue-derived stem/progenitor cells may be more committed or restricted in their differentiation potential in comparison to BM- MSCs. Dental mesenchyme is termed 'ectomesenchyme' due to its earlier interaction with the neural crest. From this perspective, ectomesenchyme- derived DSCs may possess different characteristics akin to those of neural crest cells. 160 In further studies BM-MSCs and GT-MSCs were compared for growth characteristics such as morphology, population doubling time, cell yield, surface marker expression in early and late passages. Also gene expression studies were done by microarray analysis. 161 6.2 Results 6.2.1 Comparison of growth characteristics of GT-MSCs and BM-MSCs To further investigate whether GT-MSCs are better than BM-MSCs, both MSCs were compared for morphology and growth characteristics. BM-MSCs are initiallv heterogenous population up to 2-3 passages and contain the fraction of hematopoeitic and other type of cells in initial cultures (Figure 6.1 A). On the other hand primary culture of GT-MSCs is a homogenous population and is not contaminated by other cells (Figure 6.1 B). A small biopsy of GT was found to yield a large number of MSCs in few passages, which was much greater than the number of MSCs obtained from BM within same passages. Tale 6.1 indicates the comparative average cell yield from both the cell types at different passages using same cell density. FIGURE 6.1 Growth characteristics of BM-MSCs and GT-MSCs. (A) Pnnnm/ culture of BM- MSCs show presence of different types of cells giving rise to a lieterogowus population {Magnification 10.x). (B) Primary culture of GT-MSCs displaying a Iwinogenous population offthrohlast like cells. 162 Properties BM-MSC GT-MSC BM-MSC GT-MSC BM-MSC GT-MSC P3 P3 P5 P5 P6 P6 Cells seeded 5xl04 5x 104 5xl04 5xl04 10^ 10^ Cells harvested 2.75 X 105 1.2 X106 3xl05 6xl05 3.5 X 105 106 TABLE 6.1 Comparison ofMSCs yield from BM and gingival tissue. BM-MSCs and GT-MSCs of different passages were seeded at equal cell densities and confluency harvested cells were counted. 6.2.2 Growth kinetics of GT-MSCs and BM-MSCs. Stem cells derived from dental tissues have been reported to have a higher rate of proliferation [Gronthos et al., 2000; Shi et al., 2001; Gronthos et al., 2002; Gronthos et al., 2003]. Therefore, the growth kinetics of GT-MSCs and BM- MSCs was compared. The PD time of both BM-MSCs and GT-MSCs was determined. The calculated PD time of GT-MSCs was 39.6 ± 3.2 hours, whereas in BM-MSCs it was 80.4 ±1.2 hours (Figure 6.1 A). Also the proliferation rate of GT-MSCs was significantly higher than BM-MSCs when compared by MTT assay (Figure 6.1 B). Next, the cell yield of both the MSCs was compared. For this passage 3 BM-MSCs or GT-MSCs (5 x 10^) were plated and cell number was counted after 4 days. The cell number in GT-MSCs was 4 fold more than BM-MSCs (Figure 6.1 C). This suggests that cell yield of GT- MSCs is much higher than BM-MSCs, and thus, large number of GT-MSCs can easily be generated in short duration. 163 100, 804 + 12 O 0.05 BM-MSCs (P6) GT-MSCs (P7) (P3-P5) (P3-P5) • BMJIISCS ftstagtJ Pa»age3 FIGURE 6.2 Growth kinetics of BM-MSCs and GT-MSCs (A) BM-MSCs and GT-MSCs wen- seeded at a density of 2-4 x 10^ cells/cm^, han>ested after 3-4 days and counted. The cell yield was used to calcidate the PD time. The average PD time of BM-MSCs was more than that of GT-MSCs. (B) BM-MSCs and GT-MSCs were seeded at a density of 1(P cellsA^'ell in 96 well plate and the proliferation rate was determined after 48 hours by MTT. Tlte rate of proliferation of GT-MSCs was higher as compared to BM-MSCs. (C) 5 x ICP purified BM-MSCs (passage 3) and GT-MSCs (from same passages) were plated and cell number zoas counted after 4 days. The results arc expressed as the mean ± SEM of three independent experiments, *p <0.0T 164 6.2.3 Morphological differences between BM-MSCs and GT-MSCs in long term culture. As a general observation the cells at higher passages tend to show morphological abnormalities, cell enlargement and ultimately proliferation arrest, typical of the Hayflick model of cellular aging and enter a phase of senescence in long term in vitro culture. Both BM-MSCs and GT-MSCs were cultured till 13* passage and observed the cells for morphological changes. Both the cell types displayed typical fibroblast morphology during the primary culture and till initial few passages. It was observed that BM-MSCs reached a phase of senescence, showed flattened and abnormal morphology within 8-10 passage whereas GT-MSCs maintained a stable morphology even during higher passage. Figure 6.3 displays GT-MSCs and BM-MSCs at early and late passage. t. FIGURE 6.3 Morphological 1= b comparison between GT-MSCs and ^ i BM-MSCs during early and late -i-. passages. At early passage both the cell U.r. h/pes display a typical fibroblast like \~ ' h • , .^Mi morphology but during long term culture . I morphology of BM-MSCs get distorted wlwreas GT-MSCs maintain their 1 morphology. 3 I^^HHft GT-.M^C BM-MSC 6.2.4 Comparative analysis of surface marker expression between BM-MSCs and GT-MSCs in long term culture BM-MSCs show a decrease in surface marker expression upon long term culture which may be related to a heterogenous population of cells and 165 senescence. Loss of certain surface markers during culture may influence their homing capability [Karp and Leng Teo, 2009] and thus adversely affect the utility of these cells in therapeutic applications. Unlike BM-MSCs, GT-MSCs were found to show a high expression of all the surface markers even during late passages, thus enhancing their utility in regenerative therapy and other clinical applications. GT-MSCs maintain their differentiation potential in long term culture conditions and thus prove as a potential source of MSCs. Table 6.2 shows the expression profile of MSC markers in GT-MSCs and BM-MSCs during early as well as late passages. BM-MSCs show a decrease in percentage of marker expression with increasing passages whereas GT-MSCs maintain a high percentage of surface marker expression till late passage. TABLE 6.2 Flow cytometry analysis of surface markers expressed by GT-MSCs and BM- MSCs in early and late passages. Analysis of two cell lines each of GT-MSCs and BM-MSCs show that GT-MSCs maintain a high surface marker expression in higher passages but BM-MSCs show a decrease in expression at higher passages. Surface GT-MSCs-1 GT-MSCs-2 BM-MSCs-1 BM-MSCs-2 markers (%) P5 P13 P3 P16 P5 PS P3 PIO CD44 95.25 95.31 95.32 86.79 93.76 92.06 92.60 48.95 CD90 98.32 94.61 96.14 85.81 94.04 79.54 95.57 63.07 CD105 97.16 89.33 95.19 82.39 85.38 9.47 94.07 54.01 CD73 98.03 93.61 96.43 85.80 90.45 36.38 96.35 57.91 CD29 78.74 90.77 92.45 86.51 61.03 29.87 93.27 19.61 CD45 3.21 2.77 2.95 4.52 3.62 2.68 3.14 5.10 CD34 3.37 3.95 4.00 3.80 5.16 1.18 2.69 4.98 166 6.2.5 Comparative analysis of GT-MSCs and BM-MSCs by microarray analysis In order to perform a detailed genetic comparison between GT-MSCs and BM- MSCs, microarray analysis was done. GT-MSCs of passage 5 and 7 and BM- MSCs of passage 7 were used for the purpose. Figure 6.4 displays an overview of all the differentially regulated genes in GT-MSCs as compared to BM-MSCs. Biological analysis was performed using Genotypic's Biointerpreter a web- based Biological interpretation tool. For upregulated genes cut off value of ratio greater than 1.8 was used and ratio less than 0.55 was used for down regulated genes.
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