Recentemente, células-tronco da polpa dental humana (hNDP-SCs) foram caracterizadas in vitro e foi demonstrado que elas satisfazem critérios que definem células mesenquimais do estroma humana (MSCs), tal como proposto pela Sociedade Internacional para Terapia Celular. No entanto, esses resultados foram alcançados na presença de meio de expansão xenogênico, que tem o potencial de alterar a capacidade funcional das células. Para determinar a validade das características das hNDP-SCs anteriormente relatadas para a terapia celular humana, cultivamos hNDP-SCs em meio de expansão alogênico. Duas linhagens hNDP-SC foram isoladas de dentes natais vitais, doadas por uma recém-nascida saudável, e cultivadas em plasma rico em plaquetas a 2% (PRP). Análises das expressões fenotípicas, taxas de proliferação, viabilidade, comprimento de telomerase e potenciais de diferenciação adipogênica, osteogênica e condrogênica in vitro das duas linhagens hNDP-SC (Zn001 e Zn002) foram realizadas. Ambas as linhagens apresentaram morfologia, taxas de proliferação, potencial de diferenciação adipogênico, condrogênico e osteogênico semelhantes. O encurtamento dos telômeros em 41,0% e 13,49% ocorreu da 3ª até a 14ª passagem para as linhagens Zn001 e Zn002, respectivamente. A viabilidade de ambas as linhagens foi superior a 90%. A citometria de fluxo demonstrou que ambas as linhagens foram positivas para a maioria dos marcadores testados, incluindo marcadores, que foram negativamente expressados quando hNDP-SCs foram previamente cultivadas em meio xenogênico. Usando análise imunocitoquímica, as células mostraram expressar a beta III-tubulina, nestina, neurofilamentos e Nanog. O PRP usado como meio alogênico mostrou-se adequado para o cultivo de hNDP-SCs.
Article • Braz. Dent. J. 29
(5)
• Sep-Oct 2018 • https://doi.org/10.1590/0103-6440201802388 linkcopiar
Characteristics of Human Natal Stem Cells Cultured in Allogeneic Medium
Autoria
person Jakub Suchánek
schoolDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech RepublicUniversity Hospital Hradec KrálovéCzech RepublicCzech RepublicDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic
person Klara Zoe Browne
schoolDepartment of Histology and Embryology, Charles University, Faculty of Medicine in Hradec Králové, Czech RepublicCharles University, Faculty of Medicine in Hradec KrálovéCzech RepublicCzech RepublicDepartment of Histology and Embryology, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic
person Sherine Adel Nasry
person Tereza Suchánková Kleplová
schoolDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech RepublicUniversity Hospital Hradec KrálovéCzech RepublicCzech RepublicDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic
person Nela Pilbauerová
schoolDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech RepublicUniversity Hospital Hradec KrálovéCzech RepublicCzech RepublicDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic
person Jan Schmidt
schoolDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech RepublicUniversity Hospital Hradec KrálovéCzech RepublicCzech RepublicDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic
person Tomáš Soukup
schoolDepartment of Histology and Embryology, Charles University, Faculty of Medicine in Hradec Králové, Czech RepublicCharles University, Faculty of Medicine in Hradec KrálovéCzech RepublicCzech RepublicDepartment of Histology and Embryology, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic
Correspondence: Sherine Adel Nasry, Oral and Dental Research Division, National Research Centre, 3 El Buhouth Str, Dokki, 12622. Tel.: +2 01223134144. e-mail: ghattassherine@gmail.com
SCIMAGO INSTITUTIONS RANKINGS
Department of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech RepublicUniversity Hospital Hradec KrálovéCzech RepublicCzech RepublicDepartment of Dentistry, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic
Department of Histology and Embryology, Charles University, Faculty of Medicine in Hradec Králové, Czech RepublicCharles University, Faculty of Medicine in Hradec KrálovéCzech RepublicCzech RepublicDepartment of Histology and Embryology, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic
Figuras
imageFigure 1 A: lineage Zn002 14 days after isolation: we can observe small colonies containing about 30 cells, B: lineage Zn002 in the second passage 2 days after seeding: cells adhered more to the surface of cultivation dish, became flattened and rounded; C: lineage Zn002 at 2th passage 4 days after seeding: cells reached almost 100 % confluence, maintaining the normal morphology of DPS, being spindle shaped with long processes, D: Zn001 at 14th passage 7 days after seeding: confluence reached was about 60%, cells kept the spindle shape morphology. Phase contrast microscopy magnification 200. open_in_new

imageFigure 2 A: number of population doublings done during each passage, B: Cumulative number population doublings show linear growth during each passage, C: time in hours needed for doubling the population shows decreased proliferation rate after the 7th passage, D-F: comparison of hNDP-SCs’ proliferative capacity in 1st- 7th passage and 8th-14th passage open_in_new

imageFigure 3 Flow cytometry and cell immunofluorescence of cultured hNDP-SCs: A-B: expression of cell surface markers of undifferentiated hNDP-SCs. C: beta3-tubulin and D: nestin were detected within the cytoplasm of all cells, E: neurofilaments were localized surrounding the cell nuclei with fading out concentration towards the cell peripheral sites, F: Sox 2 was mostly detectable just in the area of cell nuclei with very low concentration in the surrounding cytoplasm and G: Nanog was localized just within the area closest to the nuclei and therefore most of it expression is covered by DAPI signal of nuclei. Samples were examined with BX51 microscope (Olympus) equipped with DP71 digital camera. open_in_new

imageFigure 4 Histological slides comparing differentiated hNDP-SCs lineages to negative control: A: osteo-differentiated hNDP-SCs stained by Von Kossa technique - brown color visualized calcified extracellular particles, B: undifferentiated hNDP-SCs stained by Von Kossa technique, C: osteo-differentiated hNDP-SCs stained by Alizarin red technique - red color visualized calcified extracellular particles; D: nondifferentiated hNDP-SCs stained by Alizarin red technique; E: chondro-differentiated hNDP-SCs stained by Alcian blue - blue colour indicate the presence of acid mucopolysaccharides, purple color indicates the cell nuclei; F) undifferentiated hNDP-SCs stained by Alcian blue technique; G: adipo-differentiated hNDP-SCs stained using Oil red staining. Dark red color indicates the presence of lipid vacuoles within the cells; H: undifferentiated hNDP-SCs stained using Oil red staining. All slides were taken using 100x magnification microscope. open_in_new

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