5.4 PERIODONTAL LIGAMENT STEM CELL (PDLSC) ISOLATION. Having validated that the isolated PDL stromal cells possessed the major phenotypic

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1 5.4 PERIODONTAL LIGAMENT STEM CELL (PDLSC) ISOLATION Having validated that the isolated PDL stromal cells possessed the major phenotypic characteristics of their tissue of origin, we next focused on identifying and isolating potential adult stem cell niches within the PDL population. For this, a combination of stem cell markers and flow cytometry were employed PDLSC ISOLATED WITH FLOW CYTOMETRY USING STRO-1, CD146 AND ABCG2 GENE MARKERS Immuno-selection with the surface markers was adopted for isolation of PDLSC. (70) Cells were sorted using specific antibodies (STRO-1, CD 146 and ABCG2), which have previously been shown to target the putative PDL stem cell surface markers (Fig 5.4). (50, 85) This project aimed to combine all three antibodies in order to enrich PDLSC isolation as well as to validate the PDLSC presence in the PDL population. Results have shown that with the use of all three antibodies targeting the documented PDL stem cell markers, the isolated population obtained ranged from 1.21% to 4.33%. (Fig 5.5) 81

2 STRO 1 : 6.24 % A CD 146 : 81.6 % B ABCG 2 : % C Fig 5.4 Cell sorting of periodontal ligament cells with respective antibody conjugation in cell sorting. PDL population was sorted with STRO1 antibody using flow cytometry and 6.24% of the population was shown to be positive (A). CD146 marker was expressed by 81.6% of PDL population (B) and ABCG2 marker was expressed by 24.6% of PDL population (C) 82

3 A" B" C" Fig 5.5 The diagram demonstrates the result of the tri-staining putative stem cell markers of PDLSC (STRO-1, CD146 and ABCG2). PDLSC isolated and were stained with all three putative markers. Triple positive staining results ranged from 1.34% (A) to 4.33% (B). C demonstrated the cell sorting scatter plot of tri-staining of PDLSC. 83

4 5.4.2 CONFOCAL MICROSCOPY OF PDLSC WITH STEM CELL MARKERS To complement the flow cytometric analysis and further validate the existence of a specific stem cell population within the periodontal ligaments, PDL derived cells were immunostained with the putative stem cell markers STRO 1, ABCG2 and CD 146. All cells were counter stained with DAPI to visualize nuclei and total cell number and then examined by confocal microscopy. (Fig 5.6) As shown in Figure 5.6, whereas individual antibodies showed selectivity to specific cell sub-populations, only a small subset were triple positive for all stem cells markers in good accord with the flow cytometry data. 84

5 DAPI (A)A ABCG2 (B) B 50μm 50μm CD146 (C) C 50μm OverlayE A+B+C+D Stro1 (D) D 50μm 50μm Fig 5.6 The diagram demonstrates confocal microscopy fluorescence of the stained stem cell markers of PDLSC. DAPI is used as a nuclear stain to detect the nucleus (A). Staining of each stem cell markers with their conjugated Fluorophores have been detected in the population. ABCG2 (B), CD146 (C), STRO1 (D), are the stem cell markers. Overlay (E) demonstrated when all fluorescence are combined including DAPI, ABCG2, CD 146 and STRO1 PDLSC is present with all putative stem cell markers stained. 85

6 5.4.3 MULTI-POTENT DIFFERENTIATION VALIDATION OF PDLSC Multi-potent differentiation is one of the key stem cell criteria and its validation was carried out to further ascertain whether the isolated PDLSCs were indeed stem cell like in nature. Osteogenic, adipogenic and chondrogenic differentiation potential is routinely assessed to validate the multipotent characteristics of MSCs. The key markers for respective lineage s differentiation were detected using quantitative real time polymerase chain reaction (qpcr) upon respective induction in lineage specific supplemented media. In addition, the results from qpcr analysis were further supported by histological evidence validating its committed lineage PDLSC VALIDATION OF OSTEOGENIC LINEAGE DIFFERENTIATION Osteogenic lineage differentiation was validated with qpcr and histological staining once the PDLSC cultures were induced with osteogenic induction medium. The cell cultures were divided into 2 groups with cells seeded at a cell density of 1x10 6 /well. Cells in group 1 were induced for 7 days in osteogenic media whilst group 2 induced for 21 days in osteogenic media and each group includes its respective control wells. Triplicates were carried out to ensure statistical significance. Group 1 was fixed and stained for ALP at the end of the induction period as per manufacturer s protocol. ALP staining intensity was quantified using FIJI image software. Cells in group 2 were fixed after 21 days of culture and subsequently stain for Alizarin red and quantified using colorimetric assay. Quantitative polymerase chain reaction (qpcr) investigating gene expression of osteogenic markers such as alkaline phosphatase (ALP), Osteocalcin (OC), collagen 1 alpha (Coll 1α), 86

7 runt related transcriptional factor 2 (Runx2) were used to validate osteogenic induced cells and comparing it with the control cells. Induction to the osteogenic lineage was carried out according to the protocol described in chapter Osteogenic induction was carried out at 7 and 21 days to assess both early and late osteogenic differentiation. Detection with alkaline phosphatase (ALP) staining at 7 days and alizarin red staining at 21 days induced samples were conducted (Fig 5.7 A, B and C). Osteogenic induction with duration of 7 days demonstrated an increased staining of ALP when compared to control (Figure 5.7A and B). ALP staining was quantified using digital subtraction of the two images by FIJI software. The result demonstrated that osteogenic induced samples at 7 days have statistically greater ALP levels compared with the control (Fig 5.7D). Upon osteogenic induction of the PDLSC at 21 days, a significant contrast of alizarin red staining was evident in the induced group (Fig 5.7 G and H) when compared to the control. This is indicative of PDLSC differentiation along the osteogenic path (Fig 5.7E). In summary the ALP and alizarin staining have evidently supported the PDLSC s multipotency with osteogenic differentiation with induction. In addition to the enzymatic staining findings of ALP and alizarin red, the key osteogenic genes were consistently expressed amongst the induction groups (Fig 5.7 I, J, K and L). 87

8 Results obtained from qpcr demonstrated that OC gene expression were all up regulated at 7 and 21-days induction (Fig 5.7I). ALP gene expression was up regulated at 21-days induction but down regulated at 7 days induction (Fig 5.7J). Collagen 1α gene expression was up regulated for both 7 and 21-day induction (Fig 5.7K). Unexpectedly, however, Runx2 gene expression was markedly down regulated in both 7 and 21-days induction group (Fig 5.7L). All data obtained were made in comparison to their respective controls. 88

9 ALP"stain"7D"Induc5on" A" C" B" Control" 100μm" Induced" ALP"staining"between"induced"and"control" PDLSC" ALP stain intensity with osteogenic induction in hpdl *" Alizarin"red"PDLSC"osteogenic"induc5on" ****" D" E" P"<"0.05*" P"<"0.0001"****" du on tr ce d ol 0 In C Intensity of ALP stain μm" hpdl Osteogenic Induction Alizarin"Red"21D"Induc5on" F" H" G" 100μm" Induced" Control" μm"

10 Fig 5.7 Osteogenic induction of PDLSC with histological staining of ALP and alizarin red and qpcr genes expression. The ALP staining at day 7 for PDLSC in control group (A) and those induced with osteogenic media (C). B shows ALP staining of the control (left) and induced group (right). Alizarin red staining was demonstrated in a 6 wells plate where there was a significant staining among the induced group (G, H) compared to the control (F and G). The ALP staining between the control and induced group of PDLSC were quantified and demonstrated statistical significance with p value <0.05 (D). Alizarin red staining indicative of mineral deposition at latter stage of osteogenic induction of PDLSC were also quantified demonstrated statistical significance of p value < (E). 90

11 OC' Rela%ve'Expression'of'Fold'Changes' 1.80# 1.60# 1.40# 1.20# 1.00# 0.80# 0.60# 0.40# 0.20# *# Rela%ve'Expression'of'Fold'Changes' 1.40# 1.20# A# B# 1.00# P"value"<"0.01"**" P"value"<"0.05"*" 0.80# 0.60# 0.40# 0.20# ALP' *# P"value"<"0.001"**" P"value"<0.01"*" 0.00# Control# Induced# 7D# Induced# 21D# 0.00# Control# Induced# Induced# 7D# 21D# OC# ALP# COLLAGEN'1α' Rela%ve'Expression'of'Fold'Changes' 3.00# 2.50# 2.00# 1.50# 1.00# 0.50# 0.00# Control# Induced# 7D# Induced# 21D# C# 1.00# D# P"value"<"0.001"**" Rela%ve'Expression'of'Fold'Changes' 1.20# 0.80# 0.60# 0.40# 0.20# 0.00# Runx'2' Control# Induced# Induced# 7D# 21D# P"value"<"0.001" **" COLL#1# RUNX2# Fig 5.8 Osteogenic induction of PDLSC with qpcr genes expression. Quantitative polymerase chain reaction demonstrated 3 out of 4 osteogenic genes were up regulated with induced group compared to control. Osteogenic genes OC and Coll 1α were up regulated in both 7 and 21 days while ALP was up regulated in 21 days but down regulated in 7 day induction. Runx2 were down regulated. p value < (A, B, C and D respectively). 91