The dental pulp stem cell niche based on aldehyde dehydrogenase 1 expression.

AIM: To detect cells expressing the stem cell marker ALDH1 (aldehyde dehydrogenase1) in the pulp of human permanent teeth and to investigate the expression of ALDH1 in isolated dental pulp cells. METHODOLOGY: Pulp tissue was collected and processed for immunohistochemistry to detect ALDH1-, STRO-1- and CD90-positive cells. In addition, cells were isolated and analysed by flow cytometry for ALDH1 activity and for the cell surface markers CD44, CD73, CD90, STRO-1 and CD45. Cells were also examined for multidifferentiation capacity. Within these cells, an ALDH1(+) cell subpopulation was selected and evaluated for multidifferentiation capacity. RESULTS: The immunohistochemistry analyses showed that ALDH1-, CD90- and STRO-1-positive cells were located mainly in the perivascular areas and nerve fibres of dental pulps. Cells on the fifth passage had high expression for CD44, CD73 and CD90, whereas moderate labelling was observed for STRO-1 and ALDH1 in flow cytometry analysis. On the same passages, cells were able to differentiate into osteogenic, adipogenic and chondrogenic lineages. The ALDH1(+) cell subpopulation also demonstrated multilineage differentiation ability. CONCLUSIONS: Dental pulp stem cells reside in the vicinity of blood vessels and nerve fibres, indicating the possible existence of more than one stem cell niche in dental pulps. Furthermore, ALDH1 was expressed by isolated dental pulp cells, which had mesenchymal stem cell characteristics. Thus, it can be suggested that ALDH1 may be used as a DPSC marker.

Expressão da aldeído desidrogenase (ALDH), STRO-1 e CD44 em células-tronco da polpa de dentes decíduos e permanentes / Expression of aldehyde dehydrogenase (ALDH), STRO-1 and CD44 in pulp stem cells from deciduous and permanent teeth

O objetivo deste estudo foi avaliar a expressão da enzima aldeído desidrogenase (ALDH), assim como dos marcadores de membrana celular STRO-1 e CD44 em células-tronco da polpa de dentes decíduos (SHEDs), permanentes (DPSCs) e fibroblastos da polpa (HDPFs), através da citometria de fluxo e do western blot. Para tanto, as células foram cultivadas em meio DMEM/ HEPES, suplementado com soro fetal bovino a 10%, 100U/mL de penicilina, 100µg/mL de estreptomicina e armazenadas em estufa a 37ºC e 5% de CO2. Para a avaliação dos resultados da citometria de fluxo foram utilizados o teste não paramétrico de Kruskal-Wallis (p ≤ 0,05) e o teste de comparação múltipla de Dunn. As três linhagens de células, quando caracterizadas pelo western blot, expressaram fortemente a ALDH, assim como o STRO-1, ao passo que apresentaram uma fraca expressão para o CD44, não apresentando diferença na intensidade das bandas entre as mesmas. Já na análise por citometria de fluxo, todas as células apresentaram valores percentuais altos para o CD44, sem diferença estatística. Para o marcador STRO-1, os valores foram relativamente baixos para os três tipos celulares, havendo diferença apenas entre SHEDs e HDPFs. Os valores percentuais para a atividade da enzima ALDH foram igualmente baixos, havendo diferença estatisticamente significante entre DPSCs e HDPFs. Os resultados deste estudo sugerem que SHEDs, DPSCs e fibroblastos podem compartilhar algumas características, como a expressão de determinados marcadores genéticos. Da mesma forma, indicam que o uso da ALDH pode ser explorado em pesquisas futuras, para caracterização e seleção das MSCs.

The aim of this study was to evaluate the aldehyde dehydrogenase (ALDH) activity, as well as the expression of STRO-1 and CD44 in pulp stem cells from deciduous (SHEDs) and permanent (DPSCs) teeth, as well in pulp fibroblasts, by flow cytometry and western blot. For this purpose, cells were cultured in DMEM/ HEPES, supplemented with fetal bovine serum at 10%, 100U/ml of penicillin, 100µg/mL streptomycin and stored at 37°C and 5% CO2. To verify differences in the flow cytometry analysis between the three cell types, the nonparametric Kruskal-Wallis test was used (p  0,05), along with the Dunn multiple comparison test. In the western blot test, all the cell lineages strongly expressed ALDH, as well as STRO-1, while they presented a weak expression for CD44. There were no differences in the band intensity between the three cell types for the proteins tested. In the flow cytometry analysis, all cells showed high percentages of CD44, with no statistical difference. For the STRO-1 marker, the values were relatively low for all the three cell types, and differences were observed just between SHEDs and HDPFs. The values observed for the ALDH activity were also lower, with statistically significant difference between DPSCs and HDPFs. The results of this study suggest that SHEDs, DPSCs and pulp fibroblasts may share some important characteristics, such as the expression of certain genetic markers. Likewise, indicate that the use of ALDH activity can be exploited in future research for identification and characterization of MSCs.