RESUMO
Limited resources for adult stem cells necessitate their in vitro culture prior to Clinical use. Investigating mitochondrial DNA [mtDNA] and telomere shortening has proved to be important indications of stem cell validity. This study was designed to investigate these indicators in multiple passages of three adult stem cell lines which were produced in our stem cell laboratory. In this study, Dental Pulp Stem Cells [DPSCs], Periapical Follicle Stem Cells [PAFSCs] and Human Foreskin Fibroblast [HFF] cell lines were expanded for 20 passages. After 1, 5, 10, 15 and 20 passages, expanded cells were harvested and DNA was extracted for further studies. Common mtDNA mutation was detected by multiplex PCR and telomere shortening was tested by Southern blot analysis. The common deletion was not detected in any of the stem cells or cell lines after several passages. In addition, Southern blot analysis indicated that the mean difference of telomere length between first and last passage was 0.25 kb in DPSC, 0.1 kb in PAFSC and 0.32 kb in HFF which indicates that the mean telomere length in various passages of the samples showed insignificant changes. Absence of mtDNA mutations in adult stem cell lines indicates good mitochondrial function even after 20 passages. In addition, absence of telomere shortening indicates stem cells validity after multiple passages. It is hoped this information could pave the way for using in vitro expansion of adult stem cells for future Clinical applications
RESUMO
Human dental stem cells have high proliferative potential for self-renewal that is important to the regenerative capacity of the tissue. The aim was to isolate human dental pulp stem cells [DPSC], periodontal ligament stem cells [PDLSC] and periapical follicle stem cells [PAFSC] for their potential role in tissue regeneration. In this experimental study, the postnatal stem cells were isolated from dental pulp, preapical follicle and periodontal ligament .The cells were stained for different stem cell markers by immunocytochemistry. To investigate the mesenchymal nature of cells, differentiation potential along osteoblastic and adipogenic lineages and gene expression profile were performed. For proliferation potential assay, Brdu staining and growth curve tests were performed. Finally, all three cell types were compared together regarding their proliferation, differentiation and displaying phenotype. The isolated cell populations have similar fibroblastic like morphology and expressed all examined cell surface molecule markers. These cells were capable of differentiating into osteocyte with different capability and adipocyte with the same rate. PAFSCs showed more significant proliferation rate than others. Reverse transcriptase PCR [RT-PCR] for nanog, oct4, Alkaline phosphatase [ALP] and glyceraldehydes-3-phosphate dehydrogenease [GADPH] as control gene showed strong positive expression of these genes in all three isolated cell types. PDLSCs, DPSCs and PAFSCs exist in various tissues of the teeth and can use as a source of mesenchymal stem cells for developing bioengineered organs and also in craniomaxillofacial reconstruction with varying efficiency in differentiation and proliferation