Agradecemos Dra. Leila Shakeri &amp; Dr. Aref Dournaei para sua discussão crítica e Mohammad Reza Sr. Khadem Sharif por seus apoios técnicos.

1. Prepare a solução de enzima e meio de proliferação (PM) <ol><li> Fazer colagenase tipo I Solução: Pesar colagenase tipo I (12 mg / ml) e dissolver em 1 ml de PBS e de filtro usando um filtro de seringa de 0,2 | im. Em seguida, colocá-lo 15 ml de tubo e mantê-lo a -20 ° C até ser necessário. </li><li> Solução fazer dispase: Pesar dispase (16 mg / ml) e dissolver em 1 ml de PBS e de filtro usando um filtro de seringa de 0,2 | im. Em seguida, colocá-lo 15 ml de tubo e manter a 4 ° C até ser necessário. </li><li> Fazer a solução de enzima: Adicione 1 ml de colagenase tipo I de soluções (12 mg / ml) e 1 ml de soluções de dispase (16 mg / ml) na 2 ml de PBS estéril, contendo 100 mg / ml de penicilina, 100 mg / ml de estreptomicina. Concentração total de colagenase e dispase I em volume final deve ser de 4 mg / ml e 3 mg / ml, receptivamente. Em seguida, este volume da alíquota para quatro tubos de 15 ml, contendo cada um deles uma solução de enzima ml. Cada tubo podia ser usado para uma digestão de celulose. </li><li> Fazer a solução de lavagem: Adicionam-se 100 mg / ml de penicilina, 100 mg / mL de estreptomicina em PBS. </li><li> Fazer meio básico: Alpha modificação do meio de Eagle (α-MEM) suplementado com FBS a 10% e 100 unidades / ml de penicilina, estreptomicina 100 mg / ml. </li><li> Fazer a mídia Proliferação (PM): Alpha modificação do meio de Eagle (α-MEM) suplementado com FBS a 10%, 100 uM de ácido L-ascórbico-2-fosfato, 2 mM de L-glutamina, 100 unidades / penicilina ml, 100 mg / ml estreptomicina, 0,25 mg / ml de anfotericina B. </li><li> Fazer media odontogênicos: Alpha modificação do meio de Eagle (α-MEM) suplementado com FBS a 10%, 100 uM de ácido L-ascórbico-2-fosfato, 2 mM de L-glutamina, 100 unidades / penicilina ml, 100 mg / ml de estreptomicina, 0,01 uM dexametasona, 5 mM de fosfato de glicerol β-, 1,8 mM de fosfato monopotássico. </li></ol> 2. Prepare polpa dental humano para Dental Isolatio celular Pulp Stemn <ol><li> Normais terceiros molares humanos foram coletados de adultos (21-29 anos de idade) na Clínica de Odontologia da Imam Ali, sob aprovado Institutional Review Board (IRB). </li><li> Os dentes foram colocados em meio básico (α-MEM suplementado com FBS 10%) e foram transferidos em laboratório, a 4 ° C. </li><li> Sob a condição estéril, trabalhando dentro de um capuz de risco biológico de fluxo laminar, set-up foi feita uma placas de 100 mm de Petri para cada dente a ser processado. Superfícies dos dentes foram limpos por etanol 70%. </li><li> Enquanto trabalha em um dos pratos de Petri-, dente foi mantido por meio de fórceps dentais e uma lâmina de bisturi foi utilizada para limpar os detritos e do ligamento periodontal, nas raízes. </li><li> Cemento-esmalte junção foi lentamente cortar usando esterilizados disco dental para revelar a câmara pulpar. Deve-se considerar que o processo de corte tem de ser realizada lentamente para reduzir o sobreaquecimento do tecido dental. </li><li> Tecido pulpar foi gentilmente separada da coroa e raiz. </li&gt; <li> Polpa foi cortada em pequenos pedaços, com o auxílio de uma lâmina de bisturi. </li></ol> 3. Isolamento Pulp humana Dental <ol><li> Tecidos pulpares foi submetido a dissociação enzimática do tecido pulpar ou conseqüência direta de células a partir de explantes de polpa de tecidos para isolamento de hDPSCs. </li><li> Dissociação enzimática do tecido pulpar: <ol><li> Pequenos pedaços de tecidos de polpa foram transferidos para solução de enzimas, durante 1 hora a 37 ° C. Vortex cada 30 minutos para ajudar a romper o tecido. </li><li> Depois, os agregados celulares grandes foram removidos e as suspensões unicelulares foram obtidos pela passagem de células através de um coador de células de 70 | iM. </li><li> Unicelulares suspensões foram centrifugadas a 1200 rpm durante 5 min à temperatura ambiente. </li><li> Os sobrenadantes foram cuidadosamente pipetado fora e pelete foi re-suspenso em 1 ml de meio de proliferação (PM). (Nota: FBS em meio de proliferação encerrar enzimática disassociação.) </li><li> Unicelulares suspensões de polpa dentária foi semeada em frasco de cultura de 25 cm 2 com AM e, em seguida, incubadas a 37 ° C em 5% de CO 2. </li><li> O meio de cultura foi mudado de três em três dias até a confluência celular foi alcançada. </li></ol></li><li> Conseqüência direta de células a partir de explantes de tecido de celulose: <ol><li> Polpa foi picada em 1-2 mm de fragmentos, e cada peça foi colocada em 25-cm 2 frascos de cultura com PM e, em seguida, incubadas a 37 ° C em 5% de CO 2. Deve-se considerar que o volume total da PM para crescimento de célula deve apoiar a fixação de peças de celulose para crescimento celular ainda (2-3 ml por frasco). </li><li> O meio foi mudado após crescimento foi observado. </li><li> As células crescido em confluência foram sub-cultivadas a uma razão de 1:4 (passagem 1). </li></ol></li></ol> 4. Imunofenotipagem <ol><li> 250.000 células /tubo de ambos os tipos de células na passagem 3 foram incubadas por PE ou FITC-conjugado anti-humano com anticorpos com os seus isotipos durante 30 min a 4 ° C num local escuro. (Nota:. Da concentração de anticorpos foi aplicada de acordo com o protocolo de fabricação) </li><li> Após o tempo de incubação, 100 uL de PBS ou de FACS solução de diluição foi adicionada a cada tubo e centrifugou-se a 1200 rpm durante 5 min, em local escuro. </li><li> Os sobrenadantes foram removidos e pelotas foram re-suspensas em 100 ul de PBS ou de diluição FACS em local escuro. </li><li> Marcadores de superfície alvo (marcadores CD) foram avaliados por BD FACSCalibur. </li></ol> 5. A indução da diferenciação de células em DPSCs mineralizadas e quantificada Ensaio Vermelho de alizarina S <ol><li> DPSCs que foram obtidos por um ou outro dissociação enzimática do tecido pulpar (conhecido como DPSC-ED) ou excrescência celular directa a partir de explantes de tecido de celulose (DPSC-OG) foram sub-cultivadas durante 3 passagens. </li><li> At A passagem 3, as células foram de semente em uma placa de 6 poços a 5000 células / poço. </li><li> Na confluência de 60%, o meio foi substituído por odontogênico PM convencional. Três cavidades foram permaneceu como um controlo negativo pela adição de meio de crescimento. </li><li> Médio mudam a cada 3 dias até o dia 21. </li><li> No dia 21, as células foram lavadas com PBS e foram fixadas em 1 ml / poço de 10% de paraformaldeído durante 15 min à temperatura ambiente. </li><li> Em seguida, foram pipetados paraformaldeído fora, com cuidado e as células foram lavadas 3 vezes (5-10 min para cada vez) com água destilada H 2 O. (Nota: Evite perturbar a monocamada.) </li><li> Após a lavagem, 1 ml / poço de vermelho de alizarina S foram adicionados durante 20 min à temperatura ambiente. </li><li> As células foram lavadas por desionizada H 2 O (quatro vezes 5 min de cada vez, com agitação suave), para remover qualquer vermelho de alizarina adicional. </li><li> 1 ml / poço de H 2 O foram adicionados para evitar que as células de secagem. </li><li> As placas foram assumidos como ins visuaispection e / ou aquisição de imagem. </li><li> Para o ensaio com vermelho de alizarina S quantificação, H 2 O foram substituídos por 1 ml de ácido acético a 10% a cada poço da placa de 6 poços e incubar durante 30 min à temperatura ambiente com agitação. </li><li> Então, monocamada de células / poço foram scrape &amp; tanto o ácido acético e as células foram transferidas para as separar de 1,5 ml de centrífuga de micro-tubos. (Nota:. Cada poço deve ser transferido para um tubo separado ou seja, três poços de teste e três poços de controle para cada grupo de ED &amp; OG) </li><li> Os tubos foram agitados vigorosamente durante 30 sec. </li><li> Aquece-se a 85 ° C durante 10 min. (Nota: para evitar a evaporação, os tubos foram selados com parafilme.) </li><li> Após o aquecimento, os tubos foram transferidos para gelo, durante 5 min. (Nota: Os tubos não deve ser aberta até que se esfriou.) </li><li> As suspensões foram centrifugadas a 20.000 xg durante 15 min. Embora a centrifugação, as normas de vermelho de alizarina foram feitas-se. </li></ol> Padrão vermelho de alizarina S foram preparadas por diluição da primeira tampão de diluição e usado para fazer duas diluições em série com base na tabela. <table border="1"><tbody><tr><td colspan="7"> Gama Alta concentração de corante </td><td colspan="7"> Gama Baixa concentração de corante </td></tr><tr><td> 2 mM </td><td> 1 mM </td><td> 500 uM </td><td> 250 uM </td><td> 125 uM </td><td> 62,5 mM </td><td> 31,3 mM </td><td> 15,6 mM </td><td> 7,8 mM </td><td> 3,9 mM </td><td> 1,9 mM </td><td> 0,9 mM </td><td> 0,4 uM </td><td> Em branco </td></tr></tbody></table><ol start="16"><li> Após a centrifugação, 1 ml de sobrenadante / tubo foram transferidas para novos separados de 1,5 ml de centrífuga de micro-tubos. </li><li> O pH foi neutralizado com 375 ul ~ 10Hidróxido de amónio% por tubo. (Nota: a raiva pH deve ser 4,1-4,5). </li><li> 150 ul de padrão e amostras (inclui testes separados e controlos) foram adicionados a um opaco de parede de fundo transparente, de 96 poços da placa. </li><li> A absorvância foi lida a 405 nm e os valores de OD das amostras foram comparadas com o lote padrão para análise posterior. </li></ol>

<ol>
	<li>Volponi, A. A., Pang, Y., Sharpe, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+biological+tooth+repair+and+regeneration.">Stem cell-based biological tooth repair and regeneration.</a> Trends Cell Biol. 20, 715-722 (2010).</li><li>Nosrat, I. V., Widenfalk, J., Olson, L., Nosrat, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+cells+produce+neurotrophic+factors,+interact+with+trigeminal+neurons+in+vitro,+and+rescue+motoneurons+after+spinal+cord+injury.">Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury.</a> Dev. Biol. 238, 120-132 (2001).</li><li>Gandia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dental+pulp+stem+cells+improve+left+ventricular+function,+induce+angiogenesis,+and+reduce+infarct+size+in+rats+with+acute+myocardial+infarction.">Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction.</a> Stem Cells. 26, 638-645 (2008).</li><li>Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+human+dental+pulp+stem+cells+(DPSCs)+in+vitro+and+in+vivo.">Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.</a> Proc. Natl. Acad. Sci. U. S. A.. 97, 13625-13630 (2000).</li><li>Graziano, A., d'Aquino, R., Laino, G., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells:+a+promising+tool+for+bone+regeneration.">Dental pulp stem cells: a promising tool for bone regeneration.</a> Stem Cell Rev. 4, 21-26 (2008).</li><li>Kerkis, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+transplantation+of+human+immature+dental+pulp+stem+cells+from+baby+teeth+to+golden+retriever+muscular+dystrophy+(GRMD)+dogs:+Local+or+systemic.">Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic.</a> J. Transl. Med. 6, 35 (2008).</li><li>Onyekwelu, O., Seppala, M., Zoupa, M., Cobourne, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+development:+2.+Regenerating+teeth+in+the+laboratory.">Tooth development: 2. Regenerating teeth in the laboratory.</a> Dent. Update. 34, 20-29 (2007).</li><li>Cordeiro, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+tissue+engineering+with+stem+cells+from+exfoliated+deciduous+teeth.">Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth.</a> J. Endod. 34 (08), 962-969 (2008).</li><li>Pierdomenico, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+mesenchymal+stem+cells+with+immunosuppressive+activity+can+be+easily+isolated+from+dental+pulp.">Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp.</a> Transplantation. 80, 836-842 (2005).</li><li>de Mendonca Costa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+large+cranial+defects+in+nonimmunosuppressed+experimental+design+with+human+dental+pulp+stem+cells.">Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells.</a> J. Craniofac. Surg. 19, 204-210 (2008).</li><li>Tirino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+identification,+characterization+and+banking+of+human+DPSCs:+current+strategies+and+perspectives.">Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives.</a> Stem Cell Rev. 7, 608-615 (2011).</li><li>Tirino, V., Paino, F., De Rosa, A., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation,+characterization,+and+banking+of+human+dental+pulp+stem+cells.">Identification, isolation, characterization, and banking of human dental pulp stem cells.</a> Methods Mol. Biol. 879, 443-463 (2012).</li><li>Eslaminejad, M. B., Vahabi, S., Shariati, M., Nazarian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Growth+and+Characterization+of+Stem+Cells+from+Human+Dental+Pulp+of+Deciduous+Versus+Permanent+Teeth.">In vitro Growth and Characterization of Stem Cells from Human Dental Pulp of Deciduous Versus Permanent Teeth.</a> J. Dent. (Tehran). 7, 185-195 (2010).</li><li>Wei, X., Ling, J., Wu, L., Liu, L., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mineralization+markers+in+dental+pulp+cells.">Expression of mineralization markers in dental pulp cells.</a> J. Endod. 33, 703-708 (2007).</li><li>Nombela-Arrieta, C., Ritz, J., Silberstein, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elusive+nature+and+function+of+mesenchymal+stem+cells.">The elusive nature and function of mesenchymal stem cells.</a> Nat. Rev. Mol Cell Biol. 12, 126-131 (2011).</li><li>Huang, G. T., Gronthos, S., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+derived+from+dental+tissues+vs.+those+from+other+sources:+their+biology+and+role+in+regenerative+medicine.">Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine.</a> J. Dent. Res. 88, 792-806 (2009).</li><li>Graziano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold's+surface+geometry+significantly+affects+human+stem+cell+bone+tissue+engineering.">Scaffold's surface geometry significantly affects human stem cell bone tissue engineering.</a> J. Cell Physiol. 214, 166-172 (2008).</li><li>d'Aquino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dental+pulp+stem+cells:+from+biology+to+clinical+applications.">Human dental pulp stem cells: from biology to clinical applications.</a> J. Exp. Zool. B. Mol. Dev. Evol. 312, 408-415 (2009).</li><li>Mitsiadis, T. A., Feki, A., Papaccio, G., Caton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells,+niches,+and+notch+signaling+in+tooth+injury.">Dental pulp stem cells, niches, and notch signaling in tooth injury.</a> Adv. Dent. Res. 23, 275-279 (2011).</li><li>Shi, S., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+niche+of+postnatal+mesenchymal+stem+cells+in+human+bone+marrow+and+dental+pulp.">Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp.</a> J. Bone Miner. Res. 18, 696-704 (2003).</li><li>Tomic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+properties+of+mesenchymal+stem+cells+derived+from+dental+pulp+and+dental+follicle+are+susceptible+to+activation+by+toll-like+receptor+agonists.">Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.</a> Stem Cells Dev. 20, 695-708 (2011).</li><li>Tsukamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mineralized+nodule+formation+by+cultures+of+human+dental+pulp-derived+fibroblasts.">Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.</a> Arch. Oral Biol. 37, 1045-1055 (1992).</li><li>About, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dentin+production+in+vitro.">Human dentin production in vitro.</a> Exp. Cell Res. 258, 33-41 (2000).</li><li>Couble, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odontoblast+differentiation+of+human+dental+pulp+cells+in+explant+cultures.">Odontoblast differentiation of human dental pulp cells in explant cultures.</a> Calcif. Tissue Int. 66, 129-138 (2000).</li><li>Onishi, T., Kinoshita, S., Shintani, S., Sobue, S., Ooshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+proliferation+and+differentiation+of+dog+dental+pulp+cells+in+serum-free+culture+medium+by+insulin-like+growth+factor.">Stimulation of proliferation and differentiation of dog dental pulp cells in serum-free culture medium by insulin-like growth factor.</a> Arch. Oral Biol. 44 (99), 361-371 (1999).</li><li>Huang, G. T., Sonoyama, W., Chen, J., Park, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+human+dental+pulp+cells:+various+isolation+methods+and+culturing+environments.">In vitro characterization of human dental pulp cells: various isolation methods and culturing environments.</a> Cell Tissue Res. 324, 225-236 (2006).</li><li>Bakopoulou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+impact+of+two+different+isolation+methods+on+the+osteo/odontogenic+differentiation+potential+of+human+dental+stem+cells+derived+from+deciduous+teeth.">Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.</a> Calcif. Tissue Int. 88, 130-141 (2011).</li><li>Curtis, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EF1alpha+and+RPL13a+represent+normalization+genes+suitable+for+RT-qPCR+analysis+of+bone+marrow+derived+mesenchymal+stem+cells.">EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.</a> BMC Mol. Biol. 11, 61 (2010).</li><li>Suchanek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells+and+their+characterization.">Dental pulp stem cells and their characterization.</a> Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 153, 31-35 (2009).</li><li>d'Aquino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+postnatal+dental+pulp+cells+co-differentiate+into+osteoblasts+and+endotheliocytes:+a+pivotal+synergy+leading+to+adult+bone+tissue+formation.">Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.</a> Cell Death Differ. 14, 1162-1171 (2007).</li><li>Atari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+pluripotent+stem+cells+from+human+third+molar+dental+pulp.">Isolation of pluripotent stem cells from human third molar dental pulp.</a> Histol. Histopathol. 26, 1057-1070 (2011).</li></ol>

Não há conflitos de interesse declarados.

Este protocolo descreve o processo de isolamento e caracterização de hDPSCs da polpa dentária através de dois métodos, dissociação enzimática e excrescência directa de células estaminais a partir de explantes de tecido de celulose. Além disso, a diferenciação in vitro destas células em odontoblastos, foi avaliada por ensaio quantitativo Vermelho de alizarina S &amp; QPCR. Os protocolos existentes para o isolamento de tecido de polpa de dentes humanos foram uti...

As células-tronco são células que possuem clonogénicos duas características marcantes, conhecidos como multi-potência e auto-renovação 15. Entre todas as células estaminais com potências diferentes replicativas, as células estaminais dentários como as células-tronco pós-natais têm chamado a atenção nos últimos anos por causa da sua acessibilidade, plasticidade e capacidade proliferativa elevada em comparação com as células estaminais adultas outros 16. Caracteristicamente, semelhante para as células estaminais mesenquimais, as células estaminais da polpa dentária são células aderentes clonogénicas que têm a capacidade de diferenciação em múltiplas mesênquima e / ou não-mesênquima linhagens, tanto in vitro como in vivo. 1-8,17,18 DPSCs são identificados pela a sua expressão negativa de antigénios hematopoiéticas (por exemplo, CD45, CD34, CD14), e da expressão de CD90 positivas, CD29, CD73, CD105, CD44 e STRO-1, 19,20. Potencial obtida fácil com o mínimo de dor e morbidade fazer DPSCs humanos como um valiosofonte capaz de MSCs em comparação com as fontes comuns, tais como a da medula óssea as células estaminais mesenquimais 21. Em geral, DPSCs ter sido isolada por qualquer método outgrowth, isto é, a migração de células estaminais a partir de explantes de tecido de celulose (DPSC-OG) 22-24, e / ou por digestão enzimática (DPSC-ED) 4,6,25. Estudos anteriores têm mostrado que o método de isolamento e as condições de cultura podem induzir diferentes populações ou linhagens sob passagens in vitro 26,27. No caso dos dentes permanentes (pDPSCs), Huang et al mostraram que enzimáticas pDPSCs digeridos têm um maior potencial de proliferação em comparação com os DPSCs crescido. 26 Por outro lado, no caso dos dentes de leite (dDPSCs), demonstrou-se que STRO-1 e CD34 marcadores expressa mais em dDPSC-ED em comparação com dDPSC-OG. Além disso, dDPSC-ED apresentado maior taxa de mineralização em meio definido osteo / odonto. 27 Portanto, devido ao excelente potencial de DPSCs em regenerative medicina, mais estudos serão necessários para uma melhor compreensão das possíveis várias populações que são derivadas de diferentes métodos de isolamento. Aqui, ela foi a tentativa de introduzir de maneira fácil a extracção da polpa, utilizando-se um passo de disco de diamante dental para facilitar o processo de extracção da polpa. Além disso, após o isolamento de células-tronco derivadas de celulose, aplicando métodos de ED ou OG, propriedades gerais e capacidade de diferenciação entre os dois grupos também foram investigados.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Catálogo ou Lot. número </td><td> Comentários (opcional) </td></tr><tr><td> α-MEM </td><td> GIBCO </td><td> 11900-073 </td><td></td></tr><tr><td> Colagenase tipo I </td><td> Sigma-Aldrich </td><td> C0130-100mg </td><td></td></tr><tr><td> Dispase </td><td> GIBCO </td><td> 17105-041 </td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> GIBCO </td><td> 15140-122 </td><td></td></tr><tr><td> A anfotericina B </td><td> GIBCO </td><td> 15290-018 </td><td></td></tr><tr><td> Soro de bovino fetal definido (FBS) </td><td> HyClone </td><td> SH30070.03 </td><td></td></tr><tr><td> Ácido L-ascórbico 2-fosfato </td><td> Sigma </td><td> A8960-5G </td><td></td></tr><tr><td> L-glutamina </td><td> GIBCO </td><td> 25030-024 </td><td></td></tr><tr><td> Dexametasona </td><td> Sigma </td><td> D4902 </td><td></td></tr><tr><td> β glicerol-fosfato de hidrato de sal de dissódio, BioUltra </td><td> Sigma </td><td> G9422-100G </td><td></td></tr><tr><td> Fosfato de potássio monobásico </td><td> Sigma-Aldrich </td><td> P5655 </td><td></td></tr><tr><td> Osteogênese Ensaio Kit </td><td> Millipore </td><td> PS01802031 </td><td></td></tr><tr><td> Rato IgG2b-PE isotipo controle </td><td> BD Pharmingen </td><td> 50808088029 </td><td></td></tr><tr><td> FITC rato IgG2b isotipo controle </td><td> BD Pharmingen </td><td> 559532 </td><td></td></tr><tr><td> FITC rato IgG1 κ isotipo </td><td> BD Pharmingen </td><td> 11471471 </td><td></td></tr><tr><td> FITC / PE de murganho anti-humano CD34/CD45 </td><td> BD Pharmingen </td><td>341071 </td><td></td></tr><tr><td> PE CD146 anti-humano </td><td> BD Pharmingen </td><td> 550315 </td><td></td></tr><tr><td> Anticorpo monoclonal de rato anti-humano CD90/FITC </td><td> Daka </td><td> 00034418 </td><td></td></tr><tr><td> PE rato anti-CD73 humano </td><td> BD Pharmingen </td><td> 550257 </td><td></td></tr><tr><td> Anti-h CD105/Endoglin PE </td><td> BD Pharmingen </td><td> FAB10971P </td><td></td></tr><tr><td> PE rato CD11b/Mac1 anti-humano </td><td> BD Pharmingen </td><td> 5553888 </td><td></td></tr><tr><td> CD44 PE rato anti humano </td><td> BD Pharmingen </td><td> 555479 </td><td></td></tr><tr><td> Solução tampão de fosfato (PBS) </td><td> GIBCO </td><td> 003000 </td><td></td></tr><tr><td> 70 mícrons coador celular </td><td> Falcão </td><td> 352360 </td><td></td></tr><tr><td&gt; 0,2 um filtro de seringa </td><td> Millex-GV </td><td> SLGV033RB </td><td></td></tr><tr><td> 25 centímetros frasco de cultura de 2 </td><td> Sigma-Aldrich </td><td> Z707481 </td><td></td></tr><tr><td></td><td></td><td></td><td> EQUIPAMENTOS </td></tr><tr><td> BD FACSCalibur </td><td> BD </td><td> 342975 </td><td></td></tr><tr><td> multiskan microplaca espectrofotômetro </td><td> Thermo Scientific </td><td> 51119200 </td><td></td></tr><tr><td> Microscópio Fleurcense </td><td> Olimpo </td><td></td><td></td></tr><tr><td> Fluindo versão de software 2.3.1 </td><td></td><td></td><td></td></tr></tbody></table>

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

<p class="jove_content"&gt; Dentes do siso em desenvolvimento são de fácil acesso fonte de células-tronco durante a idade adulta, que pode ser obtido por rotina tratamentos ortodônticos. As células-tronco derivadas de celulose (hDPSCs) possuem potencial de proliferação elevado com multi-linhagem capacidade de diferenciação comparar com a fonte normal de células-tronco adultas<sup&gt; 1-8</sup&gt; E, portanto, hDPSCs poderiam ser os bons candidatos para transplante autólogo em engenharia de tecidos e medicina regenerativa. Juntamente com estas vantagens, possuindo as células estaminais mesenquimais (MSC), tais como as características immunolodulatory efeito, fazer hDPSCs mais valioso, mesmo no caso de transplante de aloenxerto<sup&gt; 6,9,10</sup&gt;. Por conseguinte, o primeiro passo para a utilização dessa fonte de células estaminais é seleccionar o melhor protocolo para o isolamento de hDPSCs de tecido de celulose. A fim de atingir esse objetivo, é fundamental para investigar o efeito de diferentes condições de isolamento em diferentes comportamentos celulares, como os seus marcadores de superfície comuns e também a sua capacidade de diferenciação.</p&gt;<p class="jove_content"&gt; Assim, aqui nós separar tecido pulpar humano de terceira impactados dentes molares, e depois usou ambos os protocolos existentes com base na literatura, para hDPSCs isolamento,<sup&gt; 11-13</sup&gt;<em&gt; Ie</em&gt; Dissociação enzimática do tecido pulpar (DPSC-ED) ou conseqüência a partir de explantes de tecido (DPSC-GO). Nesta matéria, tentamos facilitar os métodos de isolamento, utilizando disco de diamante dental. Em seguida, estas células caracterizadas em termos de estroma associadas Marcadores (CD73, CD90, CD105 e CD44), marcadores hematopoiéticas / endotelial (CD34, CD45 e CD11b), marcador perivascular, como CD146 e também STRO-1. Em seguida, estes dois protocolos foram comparadas com base na potência de diferenciação em odontoblastos tanto pela reacção em cadeia da polimerase quantitativa (QPCR) e coloração alizarina vermelha. QPCR foram utilizados para a avaliação da expressão de genes relacionados com a mineralização (fosfatase alcalina; ALP, matriz extracelular phosphoglycoprotein; MEPE &amp; dentina sialophosphoprotein; DSPP).<sup&gt; 14</sup</p

<ol><li> DPSCs que foram obtidos por dissociação enzimática (DPSC-ED) são mostrados aqui no dia 10, 15,18 (Figura 1). As colónias iniciadas de modo a formar em quase 3 a 5 dias após o isolamento. </li><li> DPSCs outgrown (DPSC-OG) são mostrados na Figura 2, no dia 5, 10, 13 &amp; 18. Fibroblast-como as células começaram a migrar a partir do tecido da polpa para o balão através paralelo ordenação por cerca de 5 dias após a semeadura. </li><li> DPSCs a passagem 3, utilizando os dois métodos...

Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

O método descrito o isolamento e caracterização de células pulpares humanas estaminais (hDPSCs) usando<strong&gt; Dissociação enzimática da celulose (DPSC-ED)</strong&gt; Ou<strong&gt; Conseqüência direta de células-tronco a partir de explantes de tecido de celulose (DPSC-GO). Seguido por</strong&gt;<em&gt; In vitro</em&gt; Diferenciação comparativa dos dois tipos de hDPSCs em odontoblastos.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

isolation-characterization-comparative-differentiation-human-dental

Research

JoVE Journal

Medicine

26.2K visualizações.  Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. O método descrito o isolamento e caracterização de células pulpares humanas estaminais (hDPSCs) usando Dissociação enzimática da celulose (DPSC-ED) Ou Conseqüência direta de células-tronco a partir de explantes de tecido de celulose (DPSC-GO). Seguido por In vitro Diferenciação comparativa dos dois tipos de hDPSCs em odontoblastos.

Vídeo: Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicina

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolamento e caracterização funcional de Ventricular Humano cardiomiócitos a partir de amostras cirúrgicas frescos

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Dois métodos para estabelecer células endometriais Humano estromais primários de espécimes de histerectomia

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Isolation and Cellular fenotipagem de células-tronco mesenquimais derivadas do líquido sinovial e Medula Óssea de miniporcos

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Caracterização de células imunes no tecido adiposo humano usando citometria de fluxo

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Formação de biofilme oral em diferentes materiais para implantes dentários

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Um método sem enzimas para isolamento e expansão de células-tronco mesenchymal derivadas do adiposo humano

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Ex Vivo expansão de células-tronco hematopoiéticas do cordão humano hemoderivados CD34+ células usando o ácido valproico

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Caracterização citométrica de fluxo simultâneo de múltiplos tipos de células recuperadas do cérebro do camundongo/medula espinhal através de diferentes métodos de homogeneização

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...