RESUMO
Dental pulp (DP) can be extracted from child's primary teeth (deciduous), whose loss occurs spontaneously by about 5 to 12 years. Thus, DP presents an easy accessible source of stem cells without ethical concerns. Substantial quantities of stem cells of an excellent quality and at early (2-5) passages are necessary for clinical use, which currently is a problem for use of adult stem cells. Herein, DPs were cultured generating stem cells at least during six months through multiple mechanical transfers into a new culture dish every 3-4 days. We compared stem cells isolated from the same DP before (early population, EP) and six months after several mechanical transfers (late population, LP). No changes, in both EP and LP, were observed in morphology, expression of stem cells markers (nestin, vimentin, fibronectin, SH2, SH3 and Oct3/4), chondrogenic and myogenic differentiation potential, even after cryopreservation. Six hours after DP extraction and in vitro plating, rare 5-bromo-2'-deoxyuridine (BrdU) positive cells were observed in pulp central part. After 72 hours, BrdU positive cells increased in number and were found in DP periphery, thus originating a multicellular population of stem cells of high purity. Multiple stem cell niches were identified in different zones of DP, because abundant expression of nestin, vimentin and Oct3/4 proteins was observed, while STRO-1 protein localization was restricted to perivascular niche. Our finding is of importance for the future of stem cell therapies, providing scaling-up of stem cells at early passages with minimum risk of losing their "stemness".
Assuntos
Separação Celular/métodos , Polpa Dentária/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Biomarcadores/metabolismo , Bromodesoxiuridina/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Criança , Condrogênese/efeitos dos fármacos , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura , Fatores de TempoRESUMO
Dental pulp from deciduous (baby) teeth, which are discarded after exfoliation, represents an advantageous source of young stem cells. Herein, we discuss the methods of deciduous teeth stem cell (DTSC) isolation and cultivation. We show that based on these methods, at least three different stem cell populations can be identified: a population similar to bone marrow-derived mesenchymal stem cells, an epithelial stem-like cells, and/or a mixed population composed of both cell types. We analyzed the embryonic origin and stem cell niche of DTSCs with respect to the advantages they can provide for their future use in cell therapies and regenerative medicine. In vitro and in vivo differentiation of the DTSC populations, their developmental potential, immunological compatibility, tissue engineering, and transplantation use in studies in animal models are also the focus of the current report. We briefly describe the derivation of induced pluripotent stem (iPS) cells from DTSCs, which can be obtained more easily and efficiently in comparison with human fibroblasts. These iPS cells represent an interesting model for the investigation of pediatric diseases and disorders. The importance of DTSC banking is also discussed.
Assuntos
Polpa Dentária/citologia , Células-Tronco/citologia , Dente Decíduo/citologia , Animais , Polpa Dentária/embriologia , Humanos , Imunofenotipagem , Nicho de Células-Tronco , Telômero/metabolismo , Dente Decíduo/embriologiaRESUMO
Mesenchymal stem cells (MSCs) have been described as being able to give rise to several quite different mesenchymal cell phenotypes. However, the ability to differentiate is not the only characteristic that makes these cells attractive for therapeutic purposes. The secretion of a broad range of bioactive molecules by MSCs, such as growth factors, cytokines and chemokines, constitutes their most biologically significant role under injury conditions. Understanding this intricate secretory activity as well as the properties of MSCs in vivo is central to harnessing their clinical potential. Herein, we identify some of the molecules involved in the paracrine effects of MSCs with a perspective that these cells intrinsically belong to a perivascular niche in vivo, and discuss how this knowledge could be advantageously used in clinical applications.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Indutores da Angiogênese/metabolismo , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Cicatriz/prevenção & controle , Humanos , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Comunicação Parácrina/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to develop into different mature mesenchymal cell types. They were originally isolated from bone marrow, but MSC-like cells have also been isolated from other tissues. The common feature of all of these tissues is that they all house blood vessels. It is, thus, possible that MSCs are associated with perivascular locations. The objective of this work was to test the hypothesis that MSCs are associated with blood vessels by verifying if MSC frequency positively correlates with blood vessel density. To this end, samples from highly and poorly vascularized adipose tissue sites of two equine donors were collected and processed for histology and cell isolation. MSC frequency in these samples was estimated by means of CFU-F assays, which were performed under MSC conditions. Culture-adherent cells from equine adipose tissue and bone marrow were culture expanded, tested for differentiation into mesenchymal cell types in vitro, and implanted in vivo in porous ceramic vehicles to assess their osteogenic capacity, using human MSCs and brain pericytes as controls. The differentiation assays showed a difference between adipose tissue-derived cells as compared to equine bone marrow MSCs. While differences in CFU-F frequencies between both donors were evident, the CFU-F numbers correlated directly with blood vessel densities (r(2) = 0.86). We consider these preliminary data as further evidence linking MSCs to blood vessels.
Assuntos
Tecido Adiposo/citologia , Vasos Sanguíneos/citologia , Cavalos/fisiologia , Células-Tronco Mesenquimais/citologia , Adipogenia , Fosfatase Alcalina/metabolismo , Animais , Bioensaio , Células da Medula Óssea/citologia , Cálcio/metabolismo , Contagem de Células , Condrogênese , Ensaio de Unidades Formadoras de Colônias , Fibroblastos/citologia , Células-Tronco Mesenquimais/enzimologia , OsteogêneseRESUMO
In spite of the advances in the knowledge of adult stem cells (ASCs) during the past few years, their natural activities in vivo are still poorly understood. Mesenchymal stem cells (MSCs), one of the most promising types of ASCs for cell-based therapies, are defined mainly by functional assays using cultured cells. Defining MSCs in vitro adds complexity to their study because the artificial conditions may introduce experimental artifacts. Inserting these results in the context of the organism is difficult because the exact location and functions of MSCs in vivo remain elusive; the identification of the MSC niche is necessary to validate results obtained in vitro and to further the knowledge of the physiological functions of this ASC. Here we show an analysis of the evidence suggesting a perivascular location for MSCs, correlating these cells with pericytes, and present a model in which the perivascular zone is the MSC niche in vivo, where local cues coordinate the transition to progenitor and mature cell phenotypes. This model proposes that MSCs stabilize blood vessels and contribute to tissue and immune system homeostasis under physiological conditions and assume a more active role in the repair of focal tissue injury. The establishment of the perivascular compartment as the MSC niche provides a basis for the rational design of additional in vivo therapeutic approaches. This view connects the MSC to the immune and vascular systems, emphasizing its role as a physiological integrator and its importance in tissue repair/regeneration.
Assuntos
Vasos Sanguíneos/citologia , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Pericitos/citologia , Animais , Diferenciação Celular , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Homeostase , Humanos , Sistema Imunitário/fisiologia , Tolerância Imunológica , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/fisiologia , Pericitos/imunologia , Pericitos/fisiologiaRESUMO
We report the isolation of a population of immature dental pulp stem cells (IDPSC), which express embryonic stem cell markers Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 as well as several other mesenchymal stem cell markers during at least 25 passages while maintaining the normal karyotype and the rate of expansion characteristic of stem cells. The expression of these markers was maintained in subclones obtained from these cells. Moreover, in vitrothese cells can be induced to undergo uniform differentiation into smooth and skeletal muscles, neurons, cartilage, and bone under chemically defined culture conditions. After in vivo transplantation of these cells into immunocompromised mice, they showed dense engraftment in various tissues. The relative ease of recovery and the expression profiles of various markers justify further exploration of IDPSC for clinical therapy.