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 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 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 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. Fold change was calculated as log 2. Fold change used for up regulated genes was > 0.8 and for down regulated genes it was <-0.8. The mapped genes correspond to the probe sets belonging to various categories of molecular and cellular functions. We compared GT-MSCs with BM-MSCs in levels of pleuripotency, expression of surface markers, and expression of transcription factors responsible for multipotency, cytokine production and cytokine receptors. Microarray analysis demonstrated that GT-MSCs show a differential regulation of about 2000-3000 genes when compared to the gene profile of BM-MSCs. BM-MSCs are pluripotent cells capable of differentiating into several cell lineages, oct 3/4, klf4, sox2 and c- are the genes reported for pleuripotency [Takahashi and Yamanaka., 2006; Wemig et al., 2007; Wilson et al., 2009]. The microarray data did not indicate any kind of differential regulation in the expression of pleuripotency genes thus supporting the multilineage differentiation potential of GT-MSCs. Neither the tumor genes nor the tumor suppressor genes showed any kind of differential expression. Further, the genes regulating the telomere length or telomerase activity did not show any change in their expression profile when compared to that of BM-MSCs. However many IL receptors are found to be upregulated in GT- MSCs as compared to BM-MSCs. An upregulation in the expression of genes

167 coding for PDGF , EGF receptor and TGF-p3 receptor was observed in the gene profile of GT-MSCs as compared to that of BM-MSCs. An upregulation of these mitogenic factors may account for an enhanced rate of proliferation of GT-MSCs. In contrast to BM-MSCs, GT-MSCs showed an increased expression of CD14, indicating the periodontal origin of the tissue. CD14 signals expression of MCP-1 in response LPS (component of bacterial cell wall). Microarray data also indicated an upregulation of ephrin type-A receptor 5 (EphA5) and nephroblastoma overexpressed gene (NOV) in GT- MSCs signifying an enhanced cell survival and proliferative capacity of GT- MSCs over BM-MSCs. Our gene profiling results indicated the presence of SCF and upregulation of c- in GT-MSCs which may account for a higher rate of proliferation and higher survival rate in vitro. The presence of c-kit also indicates an ectomesenchymal origin of GT-MSCs. An array of up- and down- regulated genes in C and D is shown in Figures 6.5 and 6.6. Table 6.3 displays the profile of GT-MSCs in comparison to BM-MSCs and the fold change in the gene level of various markers.

FIGURE 6.4 Overview of differentially regulated genes in GT-MSCs as compared to BM-MSCs.

168 FIGURE 6.5 Comparative analysis of up- and down- regulated genes in GT- MSC sample 'C. lUO

1 .U genes were iipregulated

o & and 804 genes icere doumregulated in C as compared to BM-MSCs.

Upregulated genes in C Downregulated genes in C

s.a FIGURE 6.6 •1 Comparative analysis of up- and down- regulated genes in GT- i 1 MSC sample 'D'. 983 1 .a genes were upregulated 0.9

o a and 740 genes were downregulated in C as n n compared to BM-MSCs. E 0.o,2 0.0

Upregulated genes in D Downregulated genes in D

169 TABLE 6.3 The profile ofGT-MSCs in comparison to BM-MSCs and the fold change in the gene level of various markers

Gene Bank Gene name Gene Fold change

Genes for pleuripotency NM_002701 oct3/4 POU5F1

NM_003106 sox2 SOX2 NM_004235 klf4 KLF4 NM_002467 c-myc MYC

Genes for stem cell markers NM_000610 CD44 CD44 1.058665263 NM_006288 CD90 Thyl 1.204452954 NM_000118 CD105 ENG - NM_002526 CD73 NT5E - NM_002211 CD29 ITGBl - NM_002838 CD45 PTPRC - NM^001773 CD34 CD34 - NM_000591 CD14 CD14 0.929027417 S70348 Integrin beta 3 integrin beta 3 -

Genes for multipotency NM_004348 cbfa-1 RUNX2 - NM_000346 sox9 SOX9 1.926128118

NM_138711 PPAR-gamma PPARG 1.644193395 NM 032638 GATA2 GATA2 -

170 Genes for tumor suppression NM_000546 TP53 NM_000321 RBI RBI

Genes for tumorogenesis NM_005343 ras HRAS NM_003946 myc NOL3

Genes for haplotyping

NM_002116 HLAA HLA-A NM_005514 HLAB HLA-B NM_002117 HLAC HLA-C NM_019111 HLADR HLA DRA M38056 HLA HLA

Genes for differentiation markers

Osteogenic NM_199173 Osteocalcin (Ocn) BGLAP NM_152860 Osterix (Osx) SP7 NM_000089 Collagen type I COL1A2 Chondrogenic NM_000089 Collagen type II COL2A1 NM_013227 Aggrecan ACAN -5.62596 NM_175856 Chondroitin synthase CHSY-2 1.268515

171 Adipogenic NM_004104 Fatty acid synthase FASN NM 000237 Lipoprotein lipase LPL

Genes for telomerase NM 007110 Telomerase associated TEPl protein 1

NM 017489 Telomeric repeat binding TERFl factor

NM 198253 Telomerase reverse TERT

Transcriptase

NM 016434 Regulator of telomere RTELl elongation helicase 1

NM 005652 Telomeric repeat binding TERF2 factor

Genes for cytokine receptors

NM_000877 IL1 receptor ILIRI -

NM_000418 IL 4 receptor IL4R -

NM_002183 IL 3 receptor alpha IL3RA -

NM_022970 basic FGF receptor FGFR2 - NM_006206 PDGF receptor PDGFRA 2.6244840 NM_005228 EGF receptor EGFR 2.18608959 NM_004612 TGF beta receptor 1 TGFBRl 1.28854597

NM_003242 TGF beta receptor 2 TGFBR2 - NM_003243 TGF beta receptor 3 TGFBR3 1.3006996

172 Genes for cytokines

NM_000575 IL1 alpha ILIA -

NM_000600 IL6 1L6 -

NM_000880 IL7 1L7 -

NM_000584 IL8 IL8 - NM_000641 ILll ILll 0.956290814

NM_000758 GMCSF CSF2 -

NM_172210 MCSF CSFl - NM_172212 MCSF CSFl 1.06615758

NM_000899 Stem cell factor KITLG - NM_000222 c-kit KIT 2.159894525

173 6.3 Discussion

In contrast to ES cells, human MSCs have already found their way to the clinic, thereby emerging as attractive candidates for various therapeutic applications. Recent advancements in identification and characterization of MSCs from dental tissues have provided a new prospect in terms of stem cell sources. Iliac crest aspiration for harvesting BM is a highly invasive procedure. Harvesting of gingival tissue is simple than harvesting BM and healing of donor site is faster without any morbidity or scar formation.

The low frequency of MSCs in BM necessitates their in vitro expansion in large number for successful clinical applications. However, BM-MSCs enter senescence and lose their stem cell characteristics early in culture. This study demonstrates that human gingiva is a novel and potential source of MSCs for clinical applications. Primary culture of GT-MSCs is a homogenous population, whereas BM-MSCs are initially heterogenous population up to 2-3 passages and contain fraction of hematopoeitic cells in initial cultures. A small biopsy of GT yielded large number of MSCs in primary culture which was much greater than the number of MSCs isolated from BM aspirate. GT-MSCs proliferated faster than BM-MSCs, and thus large number of MSCs can be generated in short period of time.

GT-MSCs were uniformly spindle-shaped fibroblast-like cells and maintained their morphology in long-term culture. However, BM-MSCs showed abnormalities typical of the Hayflick model of cellular aging at 8-10 passages. These findings are in consistence with previous reports that BM-MSCs enter a phase of senescence within 7-12 passages as demonstrated by morphological abnormalities, cell enlargement, and ultimately proliferation arrest [Banfi et al., 2000; Gregory et al, 2005; Bonab et al., 2006; Izadpanah et al, 2006;

174 Izadpanah et al., 2008; Wagner et al., 2008]. GT-MSCs showed the stability of putative MSC markers expression in long-term culture, whereas there was decrease in surface marker expression by BM-MSCs from passage 7 onwards. The loss of surface markers in BM-MSCs may be related to heterogenous population of cells and senescence in long term culture which was not seen in GT-MSCs. Loss of certain surface markers during culture may influence their homing capability [Karp and Leng Teo, 2009]. GT-MSCs are not dependent on growth factors for expansion, where as BM-MSCs need growth factors for continuous proliferation in vitro and to achieve higher population doubling (i.e. > 50 PDs) [Bianchi et al, 2003; Sotiropoulou et al., 2006]. GT-MSCs did not express HLA-DR suggesting their histocompatibility and suitability for allogenous stem cell therapy. GT-MSCs and other DSCs have been shown to possess immunosuppressive properties mediated in part by soluble factors [Wada et al., 2009].

The population doubling time of GT-MSCs remained in the range of 30-50 h from primary to long-term cultures, whereas in BM-MSCs it was in the range of 50-60 h in primary culture and increased upto 160-180 h in long term culture. This supports the previous findings that BM-MSCs has increased population doubling time in long term cultures [Izadpanah et al., 2006]. GT- MSCs showed 80-90% viability, high proliferation rate and differentiation potential into functional osteoblasts, adipocytes and chondrocytes after cryopreservation.

In conclusion, GT-MSCs are easy to isolate, clonogenic, have high proliferation rate, and retain morphology, expression of MSC markers and stem cell characteristics in long term cultures. GT-MSCs have potential for differentiation into cells of mesenchymal lineages, and also for in vivo bone regeneration. Moreover, GT-MSCs are non-tumorigenic; maintain normal

175 karyotype and telomerase activity in long-term cultures. Thus, GT-MSCs have several advantages over BM-MSCs and other dental stem cells, and offer a promising novel source of MSCs for cell therapy in regenerative medicine. This study reveal for the first time that human gingiva is a novel and rich source of MSCs, and large number of clinical grade MSCs can be generated from small tissue of gingiva in a short duration, without any growth factors.

176