Scaling-Up of Dental Pulp Stem Cells Isolated from Multiple Niches

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”

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). the AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. the expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. in contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo UNIFESP, Dept Farmacol, BR-04044020 São Paulo, BrazilCtr Toxinol Aplicada CEPID, Inst Butantan, São Paulo, BrazilUniv São Paulo, Dept Fisiol & Biofis, São Paulo, BrazilInst Butantan, Genet Lab, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Morfol & Genet, BR-04044020 São Paulo, BrazilAtividades Vet Ltda Genet Aplicada, Celltrovet, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Morfol & Genet, BR-04044020 São Paulo, BrazilWeb of Scienc

Proliferation rate and gene expression of IDPSCs after cultivation in three distinct culture media. A

<p>) Proliferation curve of LP before cryopreservation; <b>B</b>) Proliferation curve of LP after cryopreservation. <b>C</b>) Gene expression of LP after cryopreservation.</p

Characterization of EP and LP of IDPSCs.

<p><b>A1–F1)</b> Flow cytometry showing EP of IDPSCs, which highly expressed such markers as SH2/CD105 (A1); SH3/CD73 (B1); nestin (C1); vimentin (D1); fibronectin (E1). <b>F1)</b> Low expression of Oct3/4 in EP; <b>A2–F2)</b> Flow cytometry showing LP of IDPSCs, which expressed same markers as EP. <b>F2)</b> Higher expression of Oct3/4 in LP, than in F1. <b>A3–F3)</b> Immunofluorescence of LP of IDPSCs using same markers as in (A2–E2). <b>F3)</b> Nuclear localization of Oct3/4 can be observed. A3–F3) Epi-fluorescence, nuclei stained with DAPI (blue). Scale bars: A3, B3, E3, F3 = 5 µm; C3, D3 = 10 µm.</p