{"id":1010,"date":"2017-02-27T21:26:38","date_gmt":"2017-02-27T21:26:38","guid":{"rendered":"https:\/\/www.progeriaresearch.org\/?page_id=1010"},"modified":"2026-03-17T10:50:58","modified_gmt":"2026-03-17T14:50:58","slug":"induced-pluripotent-stem-cells","status":"publish","type":"page","link":"https:\/\/www.progeriaresearch.org\/pt\/induced-pluripotent-stem-cells\/","title":{"rendered":"C\u00e9lulas-tronco pluripotentes induzidas"},"content":{"rendered":"

[et_pb_section fb_built=\u201d1\u2033 fullwidth=\u201don\u201d disabled_on=\u201doff|off|off\u201d _builder_version=\u201d4.16\u2033 border_width_bottom=\u201d55px\u201d border_color_bottom=\u201d#29327a\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_fullwidth_header _builder_version=\u201d4.16\u2033 title_font=\u201d||||||||\u201d title_font_size=\u201d55\u2033 background_color=\u201d#29327a\u201d background_image=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Jes.jpg\u201d background_position=\u201dcenter_left\u201d custom_padding=\u201d9vw|0px|9vw||true|\u201d custom_padding_tablet=\u201d\u201d custom_padding_phone=\u201d|56px||\u201d custom_padding_last_edited=\u201dno|desktop\u201d title_font_size_tablet=\u201d45px\u201d title_font_size_phone=\u201d40px\u201d title_font_size_last_edited=\u201dno|telefone\u201d z_index_tablet=\u201d500\u2033 custom_css_main_element=\u201dposi\u00e7\u00e3o de fundo: centro 18% !importante;\u201d global_colors_info=\u201d{}\u201d]<\/p>\n

Pluripotente induzido<\/h1>\n

C\u00e9lulas-tronco<\/h1>\n

 <\/p>\n

[\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=”1″ use_custom_gutter=”on” gutter_width=”1″ specialty=”on” padding_left_1=”35px” padding_left_2=”35px” padding_2_tablet=”|||0px” padding_2_phone=”” padding_2_last_edited=”on|desktop” module_class_1=”sidebar-secondary-nav” module_class=”handprint-bg” _builder_version=”4.16″ background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png” parallax=”on” parallax_method=”off” inner_width=”100%” inner_max_width=”100%” custom_padding=”0|0px|54px|0px|false|false” z_index_tablet=”500″ border_width_top=”10px” border_color_top=”#8fd2ed” use_custom_width=”on” width_unit=”off” custom_width_percent=”100%” global_colors_info=”{}”][et_pb_column type=”1_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_sidebar area=”et_pb_widget_area_14″ disabled_on=”on|on|off” module_class=”subpage-sidebars” _builder_version=”4.16″ animation_style=”fade” z_index_tablet=”500″ border_width_right=”5px” locked=”off” global_colors_info=”{}”][\/et_pb_sidebar][\/et_pb_column][et_pb_column type=”3_4″ specialty_columns=”3″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_row_inner custom_padding_last_edited=”on|phone” _builder_version=”4.27.5″ custom_padding=”|35px|0|0px|false|false” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” animation_direction=”top” global_colors_info=”{}”][et_pb_column_inner saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.27.5″ background_size=”initial” background_position=”top_left” background_repeat=”repeat” custom_padding=”||0px||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

Banco de c\u00e9lulas e tecidos da Progeria Research Foundation<\/strong> C\u00e9lulas-tronco pluripotentes induzidas humanas (iPSC)<\/h4>\n
    \n
  1. iPSCs de Prog\u00e9ria <\/strong>Informa\u00e7\u00f5es b\u00e1sicas para o n\u00e3o cientista<\/strong><\/a>\u00a0<\/strong><\/li>\n
  2. Prop\u00f3sito de <\/strong>Gera\u00e7\u00e3o e distribui\u00e7\u00e3o de c\u00e9lulas-tronco pluripotentes induzidas pela The Progeria Research Foundation\u00a0<\/strong>\u00a0<\/a><\/li>\n
  3. Gera\u00e7\u00e3o de c\u00e9lulas-tronco pluripotentes induzidas pela s\u00edndrome de Hutchinson-Gilford Progeria (iPSCs)<\/strong>\u00a0<\/a><\/li>\n
  4. Controle de Qualidade: Valida\u00e7\u00e3o e Caracteriza\u00e7\u00e3o<\/strong>\u00a0<\/a><\/li>\n
  5. Material inicial original do qual os iPSCs foram derivados<\/strong><\/a><\/li>\n
  6. Junte-se \u00e0 nossa lista de e-mail para atualiza\u00e7\u00f5es futuras do iPSC e novas linhas de c\u00e9lulas<\/strong>\u00a0\u00a0<\/strong><\/a><\/li>\n
  7. D\u00favidas? Entre em contato conosco.<\/strong><\/a><\/li>\n
  8. Encomendando linhas iPSC<\/strong><\/a>\u00a0<\/strong><\/li>\n
  9. Prepara\u00e7\u00e3o de meios de cultura HGPS e iPSC de controle<\/strong>\u00a0<\/strong><\/a><\/li>\n
  10. Preparando placas de Matrigel\u00a0<\/strong><\/a><\/li>\n
  11. Descongelamento de c\u00e9lulas hESCs e iPSCs (por crio-frasco)<\/strong><\/a><\/li>\n
  12. Colheita e cuidados com hESC\/iPSC<\/strong>\u00a0<\/strong><\/a><\/li>\n
  13. Passagem <\/strong><\/a>hESC\/iPSC\u00a0<\/a><\/strong><\/li>\n
  14. Congelamento de hESC\/iPSC<\/a><\/strong><\/li>\n<\/ol>\n

    [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner custom_padding_last_edited=”on|phone” _builder_version=”4.27.5″ custom_padding=”42px|35px|0|0px|false|false” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” animation_direction=”top” border_width_top=”10px” border_color_top=”#29327a” global_colors_info=”{}”][et_pb_column_inner saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text admin_label=”iPSC” module_id=”iPSC” _builder_version=”4.27.5″ custom_padding=”||0px||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

    1. Informa\u00e7\u00f5es b\u00e1sicas do iPSC para o n\u00e3o cientista<\/strong><\/h4>\n

    C\u00e9lulas-tronco s\u00e3o c\u00e9lulas \u201cimaturas\u201d que ainda n\u00e3o se comprometeram a se tornar nenhum tipo de c\u00e9lula. Elas s\u00e3o flex\u00edveis porque t\u00eam o potencial de se desenvolver em muitos tipos diferentes de c\u00e9lulas maduras no corpo, como c\u00e9lulas que comp\u00f5em o cora\u00e7\u00e3o ou os vasos sangu\u00edneos, e outros tecidos e \u00f3rg\u00e3os. Em 2007, pesquisadores descobriram uma estrat\u00e9gia para criar c\u00e9lulas-tronco em laborat\u00f3rio reprogramando c\u00e9lulas adultas maduras que normalmente cultivamos para fins de pesquisa.1, 2<\/sup><\/strong>\u00a0. Essas c\u00e9lulas-tronco criadas artificialmente s\u00e3o chamadas de C\u00e9lulas-tronco Pluripotentes Induzidas (\u201ciPSCs\u201d). Para o campo da Progeria, este \u00e9 um grande avan\u00e7o. Pela primeira vez, os cientistas agora podem fazer c\u00e9lulas-tronco de Progeria e fazer perguntas sobre como as c\u00e9lulas-tronco funcionam e se desenvolvem na Progeria. Anteriormente, n\u00e3o havia nenhuma fonte de c\u00e9lulas-tronco humanas de Progeria e, portanto, havia um vazio de informa\u00e7\u00f5es sobre como as c\u00e9lulas-tronco de Progeria funcionam em compara\u00e7\u00e3o com c\u00e9lulas-tronco de pessoas sem Progeria. Al\u00e9m disso, os cientistas podem reprogramar as c\u00e9lulas-tronco de Progeria para criar, pela primeira vez, vasos sangu\u00edneos maduros de Progeria, c\u00e9lulas card\u00edacas e outros tipos de c\u00e9lulas. At\u00e9 agora, n\u00e3o havia nenhuma fonte de c\u00e9lulas card\u00edacas ou de vasos sangu\u00edneos de Progeria humana.\u00a0\u00a0Agora podemos pedir a chave\u00a0<\/strong>perguntas sobre a doen\u00e7a card\u00edaca que leva \u00e0 morte precoce na Prog\u00e9ria por ataques card\u00edacos e derrames. Podemos comparar essas descobertas com a doen\u00e7a card\u00edaca e o envelhecimento na popula\u00e7\u00e3o em geral e descobrir mais sobre o que influencia o envelhecimento em todos n\u00f3s. J\u00e1 houve v\u00e1rios estudos excelentes publicados usando c\u00e9lulas-tronco da Prog\u00e9ria.3-5<\/sup> \u00a0Nosso objetivo na The Progeria Research Foundation \u00e9 facilitar muito mais descobertas usando esta ferramenta inestim\u00e1vel. Para uma cartilha sobre c\u00e9lulas-tronco, consulte este site do governo dos EUA:\u00a0https:\/\/stemcells.nih.gov\/info\/basics.htm<\/a><\/p>\n

     <\/p>\n

    \n
      \n
    1. \n
        \n
      1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Indu\u00e7\u00e3o de c\u00e9lulas-tronco pluripotentes de fibroblastos humanos adultos por fatores definidos.\u00a0C\u00e9lula.\u00a0<\/em>2007;131:861-872.<\/li>\n
      2. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin, II, Thomson JA. Linhas de c\u00e9lulas-tronco pluripotentes induzidas derivadas de c\u00e9lulas som\u00e1ticas humanas.\u00a0Ci\u00eancia.\u00a0<\/em>2007;318:1917-1920.<\/li>\n
      3. Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J, 3rd, Izpisua Belmonte JC. Recapitula\u00e7\u00e3o do envelhecimento prematuro com iPSCs da s\u00edndrome de progeria de Hutchinson-Gilford.\u00a0Natureza.\u00a0<\/em>2011;472:221-225.<\/li>\n
      4. Misteli T. iPSCs derivados de HGPS para todas as eras.\u00a0C\u00e9lula C\u00e9lula-tronco.\u00a0<\/em>2011;8:4-6.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<\/div>\n

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        2. Objetivo da gera\u00e7\u00e3o e distribui\u00e7\u00e3o de c\u00e9lulas-tronco pluripotentes induzidas (iPSC) pela The Progeria Research Foundation<\/strong><\/h4>\n

        A miss\u00e3o da The Progeria Research Foundation \u00e9 descobrir tratamentos e a cura para a S\u00edndrome de Progeria de Hutchinson-Gilford e seus dist\u00farbios relacionados ao envelhecimento. Em 2009, a PRF entrou em colabora\u00e7\u00e3o com uma equipe de cientistas especialistas da Universidade de Toronto, Canad\u00e1, sob a dire\u00e7\u00e3o de William Stanford, PhD, para gerar iPSCs de Progeria de alta qualidade. O Dr. Stanford \u00e9 o Canada Research Chair em Biologia Integrativa de C\u00e9lulas-Tronco. A partir de 2011, a PRF continua a colaborar com o Dr. Stanford na Universidade de Ottawa, Canad\u00e1, onde ele \u00e9 Professor de Medicina Celular e Molecular, Faculdade de Medicina e Cientista S\u00eanior no Sprott Centre for Stem Cell Research do Ottawa Hospital Research Institute.<\/p>\n

        Nosso objetivo \u00e9 fornecer esta ferramenta inestim\u00e1vel para pesquisadores em todo o mundo. Esta nova ferramenta de pesquisa ser\u00e1 usada para gerar pesquisas novas e inovadoras em Progeria, bem como sua rela\u00e7\u00e3o com doen\u00e7as card\u00edacas e envelhecimento.\u00a0\u00a0<\/strong><\/p>\n

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        3. Gera\u00e7\u00e3o de c\u00e9lulas-tronco pluripotentes induzidas pela s\u00edndrome de Hutchinson-Gilford Progeria (iPSCs)<\/strong><\/h4>\n

        C\u00e9lulas-tronco pluripotentes induzidas (iPSCs) foram derivadas usando transdu\u00e7\u00e3o retroviral pseudotipada por VSVG de quatro fatores humanos, Oct4, Sox2, Klf4 e c-Myc em fibroblastos. Col\u00f4nias de iPSC foram derivadas em fibroblastos embrion\u00e1rios de camundongo (MEFs). O procedimento usado foi essencialmente como descrito anteriormente, mas sem o uso do rep\u00f3rter EOS (Nature Protocols 4: 1828-1844, 2009).<\/p>\n

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        4. Controle de Qualidade: Valida\u00e7\u00e3o e Caracteriza\u00e7\u00e3o<\/strong><\/h4>\n

        As linhas atualmente dispon\u00edveis passaram por diversas etapas de valida\u00e7\u00e3o (veja os PDFs para download abaixo):<\/p>\n

        \n
          \n
        1. \n
            \n
          1. Teste de Mycoplasma para cada linha: O laborat\u00f3rio do Dr. Stanford realizou an\u00e1lise de micoplasma por PCR para cada linha celular. Al\u00e9m disso, ap\u00f3s a expans\u00e3o e antes do envio das c\u00e9lulas, as linhas ser\u00e3o testadas novamente para micoplasma.<\/li>\n
          2. Imunocolora\u00e7\u00e3o para marcadores de pluripot\u00eancia Tra-1-60, Tra-1-81 e SSEA4.<\/li>\n
          3. Colora\u00e7\u00e3o de fosfatase alcalina como indicador de pluripot\u00eancia<\/li>\n
          4. Forma\u00e7\u00e3o do corpo embrion\u00e1rio e subsequente imunocolora\u00e7\u00e3o para marcadores das tr\u00eas camadas germinativas. Os marcadores testados foram \u03b2III-Tubulina (Ectoderma), Actina de M\u00fasculo Liso (Mesoderma) e Gata4 ou AFP (Endoderma)<\/li>\n
          5. An\u00e1lise de cari\u00f3tipo.<\/li>\n
          6. Reexpress\u00e3o da l\u00e2mina A em c\u00e9lulas diferenciadas<\/li>\n
          7. Ensaios de teratoma<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n

            Valida\u00e7\u00e3o adicional em andamento:<\/strong>
            Algumas linhas conclu\u00edram ensaios de teratoma, conforme mostrado nos dados de suporte. Para todas as outras linhas, os ensaios de teratoma est\u00e3o em andamento e o status ser\u00e1 atualizado conforme esses ensaios forem conclu\u00eddos.<\/p>\n<\/div>\n

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            5. Material inicial original do qual essas c\u00e9lulas iPS foram derivadas<\/strong><\/h4>\n

            As iPSCs foram derivadas de linhas celulares de fibroblastos n\u00e3o transformadas do PRF Cell & Tissue Bank.<\/p>\n

            O m\u00e9todo de transdu\u00e7\u00e3o usado para todas as linhas iPS foi o Retrovirus MKOS.<\/p>\n

            <\/p>\n

            [\/et_pb_text][et_pb_text admin_label=”Table” module_id=”original” _builder_version=”4.27.5″ custom_padding=”||0px|||” hover_enabled=”0″ z_index_tablet=”500″ global_colors_info=”{}” sticky_enabled=”0″]<\/p>\n

            \n\n\n\n\t\n\n\t\n\t\n\t\n\t\n\t\n\t\n\t\n\t\n\t
            ID da linha iPSC<\/b><\/th>Muta\u00e7\u00e3o<\/b><\/th>G\u00eanero e Doa\u00e7\u00e3oIdade<\/b><\/th>Tipo de c\u00e9lula de origem Clique aqui<\/a>.<\/b><\/th>Dados de suporte<\/b><\/th>\n<\/tr>\n<\/thead>\n
            HGADFN003 iPS 1B<\/td> LMNAExon 11,
            \n1824 C>T<\/td>            		Masculino 2 anos 0 meses<\/td> Fibroblastos D\u00e9rmicos
            \nHGADFN003<\/td>            		003 iPS1B<\/a><\/td>\n<\/tr>\n
            HGADFN003 iPS 1C<\/td> Ex\u00e3o 11 do LMNA,
            \n1824 C>T<\/td>            		Masculino 2 anos 0 meses<\/td> Fibroblastos D\u00e9rmicos
            \nHGADFN003<\/td>            		003 iPS1C<\/a><\/td>\n<\/tr>\n
            HGDFN003
            \niPS 1D<\/td>            		Ex\u00e3o 11 do LMNA,
            \n1824 C>T<\/td>            		Masculino 2 anos 0 meses<\/td> Fibroblastos D\u00e9rmicos
            \nHGADFN003<\/td>            		003 iPS1D<\/a><\/td>\n<\/tr>\n
            HGADFN167 iPS 1J<\/td> LMNA Exon 11, 1824 C>T<\/td>Masculino 8 anos e 5 meses<\/td>Fibroblastos D\u00e9rmicos HGADFN167<\/td>167 PS 1J<\/a><\/td>\n<\/tr>\n
            HGADFN167 iPS 1Q<\/td> LMNA Exon 11, 1824 C>T<\/td>Masculino 8 anos e 5 meses<\/td>Fibroblastos D\u00e9rmicos HGADFN167<\/td>167 iPS1Q<\/a><\/td>\n<\/tr>\n
            HGMDFN090 iPS 1B<\/td> M\u00e3e de HGADFN167 (n\u00e3o afetado)<\/td>Mulher 37 anos 10 meses<\/td>Fibroblastos D\u00e9rmicos
            \nHGMDFN090<\/td>            		090 iPS1B<\/a><\/td>\n<\/tr>\n
            HGMDFN090 iPS 1C<\/td> M\u00e3e de HGADFN167 (n\u00e3o afetado)<\/td>Mulher 37 anos 10 meses<\/td>Fibroblastos D\u00e9rmicos
            \nHGMDFN090<\/td>            		090 iPS1C<\/a><\/td>\n<\/tr>\n
            HGFDFN168 iPS1 D2<\/td>Pai de HGADFN167 (n\u00e3o afetado)<\/td>Masculino 40 anos
            \n5 meses<\/td>            		Fibroblastos D\u00e9rmicos HGFDFN168<\/td>168 iPS1 D2<\/a><\/td>\n<\/tr>\n
            HGFDFN168 iPS1P<\/td>Pai de HGADFN167 (n\u00e3o afetado)<\/td>Masculino 40 anos
            \n5 meses<\/td>            		Fibroblastos D\u00e9rmicos
            \nHGFDFN168<\/td>            		168 iPS1P<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/h4>\n

            [\/et_pb_text][et_pb_button button_url=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2023\/05\/PRF-AVAILABLE-CELL-LINES-5-24-23.pdf” button_text=”PRF Available Cell Lines” button_alignment=”left” admin_label=”Cell lines pdf” _builder_version=”4.27.5″ background_layout=”dark” custom_margin=”||25px||false|false” hover_enabled=”0″ z_index_tablet=”500″ global_colors_info=”{}” sticky_enabled=”0″][\/et_pb_button][et_pb_text admin_label=”join” module_id=”join” _builder_version=”4.27.5″ custom_padding=”||||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

            6. Junte-se \u00e0 nossa lista de e-mail para futuras atualiza\u00e7\u00f5es do iPSC e novas linhas de c\u00e9lulas<\/strong><\/h4>\n

            Continuamos a gerar linhas de iPSC. Se voc\u00ea quiser atualiza\u00e7\u00f5es peri\u00f3dicas sobre iPSCs mantidas no PRF Cell & Tissue Bank, inscreva-se em nossa lista de e-mail clicando em\u00a0aqui<\/a><\/strong><\/p>\n

            [\/et_pb_text][et_pb_text admin_label=”questions” module_id=”questions” _builder_version=”4.27.5″ custom_padding=”||||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

            7. D\u00favidas?<\/strong><\/h4>\n

            <\/a>Entre em contato com Leslie Gordon, MD, PhD, Diretora M\u00e9dica, para quaisquer d\u00favidas ou necessidades, em lgordon@progeriaresearch.org<\/a>\u00a0ou 978-535-2594<\/p>\n

            [\/et_pb_text][et_pb_text admin_label=”ordering” module_id=”ordering” _builder_version=”4.27.5″ custom_padding=”||0px||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

            8. Solicita\u00e7\u00e3o de linhas de c\u00e9lulas iPS<\/strong><\/h4>\n

            Em 2014, a PRF instituiu uma pol\u00edtica de n\u00e3o fazer altera\u00e7\u00f5es em nosso MTA. Este \u00e9 o resultado de 12 anos de acordos contratuais com 70 equipes de pesquisa trabalhando em institui\u00e7\u00f5es em 14 pa\u00edses. A PRF e seu advogado levaram em considera\u00e7\u00e3o as quest\u00f5es que surgiram naquele per\u00edodo e editaram o acordo de acordo, resultando no que consideramos termos justos e razo\u00e1veis.<\/p>\n

            Para perguntas ou informa\u00e7\u00f5es sobre institui\u00e7\u00f5es do governo federal dos EUA, entre em contato com Wendy Norris em: wnorris@brownhealth.org<\/a><\/a>\u00a0ou\u00a0401-274-1122 x 48063<\/a>.<\/p>\n

            Etapa 1: Preencha um requerimento e um acordo de transfer\u00eancia de material
            <\/strong>
            <\/a>            Aplica\u00e7\u00e3o e Acordo para Institui\u00e7\u00f5es N\u00e3o Governamentais<\/a><\/p>\n

            Acordo de Transfer\u00eancia de Material para Institui\u00e7\u00f5es N\u00e3o Governamentais<\/a><\/p>\n

            Passo 2: Devolva o requerimento preenchido e o acordo de transfer\u00eancia de material para Wendy Norris em wnorris@brownhealth.org<\/a>. Ap\u00f3s a aprova\u00e7\u00e3o, voc\u00ea receber\u00e1 um e-mail confirmando seu pedido e a data prevista de envio.<\/b>\u00a0<\/strong><\/a><\/p>\n

            Etapa 3: <\/strong>O laborat\u00f3rio do Dr. Stanford est\u00e1 atualmente distribuindo linhas em criotubos congelados. Seu laborat\u00f3rio enviar\u00e1 um e-mail quando a cultura for enviada, com informa\u00e7\u00f5es de envio e rastreamento. Pesquisadores inexperientes s\u00e3o direcionados a obter treinamento em cursos especializados essenciais para o trabalho com c\u00e9lulas-tronco embrion\u00e1rias humanas\/iPSCs.<\/p>\n

            O Human Pluripotent Stem Cell Facility (dirigido pelo Dr. Stanford) no Ottawa Hospital Research Institute oferece treinamento virtual individual sobre os fundamentos das t\u00e9cnicas de cultura de iPSC espec\u00edficas para as linhas de c\u00e9lulas iPSC Progeria. As op\u00e7\u00f5es de treinamento e o formato s\u00e3o flex\u00edveis, dependendo do n\u00edvel de experi\u00eancia dos cientistas. \u00a0Para mais informa\u00e7\u00f5es, envie um e-mail para hpscf@ohri.ca<\/a>.<\/u><\/strong><\/p>\n

            Etapa 4: A Universidade de Ottawa cobrar\u00e1 diretamente de voc\u00ea cada linha iPSC, al\u00e9m dos custos de entrega, se houver.<\/strong><\/p>\n

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            9. Prepara\u00e7\u00e3o do meio de cultura de c\u00e9lulas HGPS e iPS de controle<\/strong><\/h4>\n

            iPSC e ESCs precisam ser alimentados com mTeSR Plus da Stem Cell Technologies (cat# 5825). Siga as recomenda\u00e7\u00f5es do fornecedor para armazenamento.<\/p>\n

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            10. Preparando placas de Matrigel<\/strong><\/h4>\n

            Observa\u00e7\u00e3o<\/strong>:Todas as etapas que envolvem o matrigel devem ser realizadas o mais r\u00e1pido poss\u00edvel e mantidas o mais frias poss\u00edvel.<\/p>\n

              \n
            1. Descongele a garrafa de matrigel \u00e0s 4\u00b0<\/strong>C. Verifique o certificado de an\u00e1lise desse lote para encontrar sua concentra\u00e7\u00e3o de prote\u00edna.<\/li>\n
            2. Adicione meio DMEM\/F12 frio suficiente ao matrigel descongelado para atingir uma concentra\u00e7\u00e3o final de 5 mg\/mL.<\/li>\n
            3. Fa\u00e7a al\u00edquotas de 1 mL do matrigel preparado na etapa 2 em tubos Falcon de 15 mL pr\u00e9-refrigerados.<\/li>\n
            4. Congele e armazene todas as al\u00edquotas a -20\u00b0<\/strong>C.<\/li>\n
            5. Para fazer placas de matrigel, remova uma al\u00edquota de matrigel (1 mL) de -20\u00b0<\/strong>C e adicione 10mL de DMEM\/F12 frio. Misture bem at\u00e9 que o pellet descongele (sem criar bolhas, e sempre mantenha a solu\u00e7\u00e3o fria).<\/li>\n
            6. Transfira para um tubo de 50 mL e adicione 20 mL de DMEM\/F12 frio (1 mL de al\u00edquota de matrigel \u00e9 dilu\u00edda em 30 mL de DMEM\/F12) e misture bem.<\/li>\n
            7. Placa (1 mL\/po\u00e7o para placa de 6 po\u00e7os, 0,5 mL\/po\u00e7o para placa de 12 po\u00e7os, 0,25 mL\/po\u00e7o para placa de 24 po\u00e7os). Certifique-se de que a solu\u00e7\u00e3o esteja cobrindo toda a \u00e1rea da superf\u00edcie agitando a placa suavemente. N\u00e3o recongele nenhum matrigel restante.<\/li>\n
            8. Se usar o prato imediatamente, deixe-o em temperatura ambiente por 1 hora (ou 30 minutos a 37\u00b0<\/strong>C), observe matrigel sob o microsc\u00f3pio. Matrigel deve estar bem disperso e n\u00e3o \u201cgrumoso\u201d.<\/li>\n
            9. Se usar os pratos em outro momento, envolva a borda do prato com parafilme e guarde a 4\u00b0<\/strong>C por at\u00e9 2 semanas.<\/li>\n<\/ol>\n

              Matrigel \u2013 BD\/Fisher, cat#CB-40230<\/p>\n

              DMEM\/F12 \u2013 Tecnologias da Vida, cat#11330-057<\/p>\n

              Dr. William Stanford-2022<\/p>\n

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              11. <\/strong>Descongelamento de c\u00e9lulas ES ou iPS (por crio-frasco)<\/strong><\/h4>\n
                \n
              1. Retire uma placa de matrigel de 4\u00b0C e aque\u00e7a em temperatura ambiente por uma hora, ou fa\u00e7a uma nova placa de matrigel (veja o protocolo de prepara\u00e7\u00e3o da placa de matrigel).<\/li>\n
              2. Aque\u00e7a 4 mL de mTeSR Plus em um tubo Falcon de 15 mL.<\/li>\n
              3. Remova as c\u00e9lulas do tanque de nitrog\u00eanio l\u00edquido e gire em um banho de 37\u00b0C at\u00e9 que reste apenas um pequeno peda\u00e7o de gelo. O frasco deve descongelar em 1-2 minutos. Esta etapa deve ser feita rapidamente.<\/li>\n
              4. Tubo de c\u00e9lula de etanol e tubo de meio Falcon e coloque na capela.<\/li>\n
              5. Use um 1mL ponta da boca larga para lentamente <\/u>adicione c\u00e9lulas a 4 mL de meio pr\u00e9-aquecido (evite misturar a suspens\u00e3o de c\u00e9lulas).<\/li>\n
              6. Centrifugue a 130 rcf por 5 minutos.<\/li>\n
              7. Remova o sobrenadante.<\/li>\n
              8. Adicione 2 mL de meio PSC e com uma ponta de boca larga quebre os aglomerados suavemente<\/u>. Transfira a m\u00eddia para um po\u00e7o de uma placa de 6 po\u00e7os e adicione 2uL de inibidor ROCK (Y27632, concentra\u00e7\u00e3o final de 10uM). Coloque na placa em um po\u00e7o revestido de matrigel (de uma placa de 6 po\u00e7os).<\/li>\n
              9. Balance as c\u00e9lulas suavemente para distribuir uniformemente as c\u00e9lulas e coloque-as em uma incubadora hip\u00f3xica (5%O2<\/sub>, 10% CO2<\/sub>). Evite perturbar a placa por 24 horas ap\u00f3s a semeadura.OBSERVA\u00c7\u00c3O: <\/strong>\u00c9 muito importante evitar a quebra excessiva de aglomerados ou pipetagem agressiva. Isso pode reduzir significativamente a taxa de sobreviv\u00eancia. As c\u00e9lulas devem permanecer em peda\u00e7os de 100-300 c\u00e9lulas grandes no momento da semeadura. Embora seja gentil, tente trabalhar rapidamente assim que as c\u00e9lulas forem descongeladas para minimizar o tempo em que elas ficam em contato com o crioprotetor.<\/li>\n
              10. Remova a m\u00eddia ap\u00f3s 24 horas e adicione 2 mL de m\u00eddia PSC (para uma placa de 6 po\u00e7os, 1 mL para 12 po\u00e7os e 0,5 mL para uma placa de 24 po\u00e7os). Veja o protocolo Harvesting and Caring for hESC\/iPSC.<\/li>\n<\/ol>\n

                M\u00eddia PSC<\/strong><\/p>\n

                mTeSR Plus da Stem Cell Technologies (cat# 5825). Siga as recomenda\u00e7\u00f5es do fornecedor para armazenamento.<\/p>\n

                O que \u00e9 o inibidor ROCK Y27632?<\/strong><\/p>\n

                O inibidor ROCK Y27632 \u00e9 um inibidor seletivo da cinase p160 ROCK associada a Rho. O tratamento com o inibidor ROCK Y27632 previne a apoptose induzida por dissocia\u00e7\u00e3o de c\u00e9lulas-tronco embrion\u00e1rias humanas (hESC) e c\u00e9lulas-tronco pluripotentes induzidas por humanos (iPSC), aumentando a taxa de sobreviv\u00eancia e mantendo a pluripot\u00eancia durante o subcultivo e descongelamento de hESCs e hiPSCs. O inibidor ROCK Y27632 tamb\u00e9m demonstrou aumentar a taxa de sobreviv\u00eancia de c\u00e9lulas-tronco durante a criopreserva\u00e7\u00e3o. Observe que as al\u00edquotas do inibidor Rock s\u00e3o sens\u00edveis \u00e0 luz e aos ciclos repetitivos de congelamento e descongelamento. Certifique-se de usar essas al\u00edquotas dentro do prazo de validade recomendado pelo fornecedor.<\/p>\n

                Dr. William Stanford-2022<\/p>\n

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                12. <\/strong>Colheita e cuidados com hESC\/iPSC<\/strong><\/h4>\n
                  \n
                1. No dia seguinte ao descongelamento das c\u00e9lulas, observe-as no microsc\u00f3pio para determinar a taxa de sobreviv\u00eancia. NOTA: \u00e9 normal observar um alto n\u00famero de c\u00e9lulas soltas. Enquanto algumas c\u00e9lulas estiverem presas, col\u00f4nias podem surgir delas dentro de 3 a 7 dias.<\/li>\n
                2. Remova a m\u00eddia dos po\u00e7os e pipete 2 mL (para uma placa de 6 po\u00e7os, 1 mL para 12 po\u00e7os e 0,5 mL para uma placa de 24 po\u00e7os) de meio PSC fresco e morno por po\u00e7o. Retorne a placa para a incubadora.<\/li>\n
                3. As c\u00e9lulas s\u00e3o alimentadas at\u00e9 a conflu\u00eancia de 60-70% (siga as recomenda\u00e7\u00f5es do fornecedor).<\/li>\n
                4. A partir do segundo dia, as c\u00e9lulas devem ser observadas e limpas de quaisquer c\u00e9lulas diferenciadas que possam estar crescendo.<\/li>\n
                5. Para limpar as c\u00e9lulas, use a tampa de coleta e raspe as c\u00e9lulas diferenciadas com a ponta de uma pipeta.<\/li>\n
                6. Depois que as c\u00e9lulas estiverem limpas, troque o meio conforme feito nas etapas acima.<\/li>\n<\/ol>\n

                  (Veja Congelando hESC\/iPSC ou Passando hESC\/iPSC)<\/p>\n

                  OBSERVA\u00c7\u00c3O:<\/strong><\/p>\n

                  A m\u00eddia PSC foi determinada como mais eficiente em um ambiente hip\u00f3xico. Tamb\u00e9m observamos que ocorre menos diferencia\u00e7\u00e3o quando as c\u00e9lulas s\u00e3o cultivadas em incubadoras hip\u00f3xicas versus norm\u00f3xicas. Por fim, as c\u00e9lulas semeadas t\u00eam uma melhor taxa de sobreviv\u00eancia ao usar uma incubadora hip\u00f3xica.<\/p>\n

                  Norm\u00f3xico: 37\u00b0<\/strong>C, 21%O2<\/sub>, 5% CO2<\/sub><\/p>\n

                  Hip\u00f3xico: 37\u00b0<\/strong>C, 5%O2<\/sub>, 10% CO2<\/sub><\/p>\n

                  Dr. William Stanford-2022<\/p>\n

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                  13. <\/strong>Passando hESC\/iPSC<\/strong><\/h4>\n
                    \n
                  1. Adicione 2 mL de meio PSC a uma placa revestida com matrigel de 6 po\u00e7os e reserve.<\/li>\n
                  2. Pegue a placa a ser passada e remova o meio do po\u00e7o e lave uma vez com 1 mL de PBS(-\/-).<\/li>\n
                  3. Adicione 1 mL da solu\u00e7\u00e3o de EDTA ao po\u00e7o e deixe por 3-4 minutos em temperatura ambiente. N\u00e3o mova a placa, pois as c\u00e9lulas podem come\u00e7ar a se desprender.<\/li>\n
                  4. Remova a solu\u00e7\u00e3o de EDTA e adicione 1 mL de meio PSC. N\u00e3o deixe o EDTA nas c\u00e9lulas por mais de 4 minutos, pois isso far\u00e1 com que as c\u00e9lulas se soltem.<\/li>\n
                  5. Raspe as c\u00e9lulas usando um raspador de c\u00e9lulas e divida as c\u00e9lulas entre os 6 po\u00e7os da sua placa contendo meio PSC. Evite a quebra excessiva dos peda\u00e7os da col\u00f4nia e tente ser gentil com a raspagem. Tente manter as c\u00e9lulas em peda\u00e7os grandes. Use uma ponta de pipeta de boca larga para quebrar os aglomerados, se necess\u00e1rio. A quebra excessiva de c\u00e9lulas pode causar morte celular ou diferencia\u00e7\u00e3o espont\u00e2nea excessiva ap\u00f3s a passagem.<\/li>\n
                  6. Incubar a 37\u00b0<\/strong>C ap\u00f3s distribuir uniformemente as c\u00e9lulas em cada po\u00e7o (agita\u00e7\u00e3o em forma de 8 ou L). Evite perturbar a placa por 24 horas ap\u00f3s a passagem.<\/li>\n<\/ol>\n

                    OBSERVA\u00c7\u00c3O<\/strong>: Depois que as c\u00e9lulas forem raspadas, voc\u00ea deve transferi-las para a nova placa o mais r\u00e1pido poss\u00edvel, pois elas se reconectar\u00e3o rapidamente (em 5 minutos).<\/p>\n

                    Se o EDTA for deixado nas c\u00e9lulas por mais de 4 minutos, elas podem come\u00e7ar a se desprender. Se isso acontecer, simplesmente colete as c\u00e9lulas em um Falcon de 15 mL com 4 mL de meio PSC. Centrifugue as c\u00e9lulas a 130 rcf por 5 minutos. Ressuspenda o pellet com 1 mL de meio e divida uniformemente entre uma placa revestida de matrigel de 6 po\u00e7os (160 uL por po\u00e7o).<\/p>\n

                    Solu\u00e7\u00e3o de EDTA: Adicione 500 uL de EDTA 0,5 M (pH 8,0) em 500 mL de DPBS (-\/-). Adicione 0,9 g de NaCl. Filtre a solu\u00e7\u00e3o para esterilizar e armazene a 4 \u00b0C por at\u00e9 6 meses. <\/strong><\/p>\n

                    Do artigo:<\/p>\n

                    Passagem e expans\u00e3o de col\u00f4nias de c\u00e9lulas-tronco pluripotentes humanas por dissocia\u00e7\u00e3o livre de enzimas em condi\u00e7\u00f5es de cultura quimicamente definidas<\/strong><\/p>\n

                    Cervejas Jeanette<\/a>,1<\/sup> Daniel R. Gulbranson<\/a>,2,3<\/sup> Nicole George<\/a>,4<\/sup>Lauren I. Siniscalchi<\/a>,1<\/sup> Jeffrey Jones<\/a>,4,5<\/sup> James A. Thomson<\/a>,2,3,6<\/sup> e Guokai Chen<\/a>1,2<\/sup><\/p>\n

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                    14. <\/strong>Congelamento de hESC\/iPSC <\/strong><\/h4>\n
                      \n
                    1. Ligue o Bio-Cool (congelador de taxa controlada) e ajuste a temperatura para -7\u00b0C.<\/li>\n
                    2. Remova as c\u00e9lulas da incubadora e observe a conflu\u00eancia e a morfologia no microsc\u00f3pio.<\/li>\n
                    3. Se os po\u00e7os forem confluentes com 70%, remova o meio antigo e lave uma vez com PBS(-\/-) e adicione 1 mL de solu\u00e7\u00e3o de EDTA (veja passagem com solu\u00e7\u00e3o de EDTA) por po\u00e7o.<\/li>\n
                    4. Incube em temperatura ambiente por 3-4 minutos.<\/li>\n
                    5. Aspire a solu\u00e7\u00e3o de EDTA e adicione 1 mL de meio mFreSR frio (cat#05855, Stem Cell Technologies).<\/li>\n
                    6. Use um raspador de c\u00e9lulas para levantar as c\u00e9lulas gentilmente. Mantenha as c\u00e9lulas em peda\u00e7os grandes o m\u00e1ximo poss\u00edvel e evite pipetar para cima e para baixo.<\/li>\n
                    7. Transfira c\u00e9lulas\/mFreSR para um criotubo usando uma ponta de boca larga. Mantenha os frascos no gelo at\u00e9 que estejam prontos para a etapa 8.<\/li>\n
                    8. Coloque os tubos no Bio-Cool e incubar por 10 minutos.<\/li>\n
                    9. Obtenha nitrog\u00eanio l\u00edquido.<\/li>\n
                    10. Ap\u00f3s 10 minutos, semeie as c\u00e9lulas mergulhando uma esp\u00e1tula em nitrog\u00eanio l\u00edquido e tocando na lateral do frasco criog\u00eanico por aproximadamente 10 a 30 segundos ou at\u00e9 ver um cristal se formar na lateral do frasco criog\u00eanico.<\/li>\n
                    11. Inicie o programa 1 pressionando o bot\u00e3o \u201cPROG\u201d e prossiga com o programa pressionando o bot\u00e3o novamente e voc\u00ea dever\u00e1 ver a taxa de 0,5\u00b0C\/min., ent\u00e3o pressione \u201cRUN\u201d.<\/li>\n
                    12. Quando a temperatura atinge -65\u00b0C, os criotubos podem ser transferidos e armazenados em nitrog\u00eanio l\u00edquido.<\/li>\n<\/ol>\n

                      Alternativas<\/p>\n

                      Outra op\u00e7\u00e3o \u00e9 colocar os criotubos em um recipiente de congelamento (Biocision-CoolCell) e armazenar a -80\u00b0C durante a noite. Mova os criotubos para nitrog\u00eanio l\u00edquido (fase l\u00edquida ou vapor) no dia seguinte.<\/p>\n

                      Dr. William Stanford-2022<\/p>\n

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                      Inscrever-se<\/h2>\n

                      para o nosso<\/h2>\n

                      Atualiza\u00e7\u00f5es!<\/h2>\n

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                      Juntos, n\u00f3s<\/h2>\n

                      VAI<\/em><\/h2>\n

                      encontre a cura!<\/h2>\n

                      [\/et_pb_cta][\/et_pb_column][et_pb_column type=”1_2″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_image src=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2026\/03\/2026-footer-image-copy.png” title_text=”2026 footer image copy” _builder_version=”4.27.5″ _module_preset=”default” custom_margin=”35px||||false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>","protected":false},"excerpt":{"rendered":"

                      [et_pb_section fb_built=\u201d1\u2033 fullwidth=\u201don\u201d disabled_on=\u201doff|off|off\u201d _builder_version=\u201d4.16\u2033 border_width_bottom=\u201d55px\u201d border_color_bottom=\u201d#29327a\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_fullwidth_header _builder_version=\u201d4.16\u2033 title_font=\u201d||||||||\u201d title_font_size=\u201d55\u2033 background_color=\u201d#29327a\u201d background_image=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Jes.jpg\u201d background_position=\u201dcenter_left\u201d custom_padding=\u201d9vw|0px|9vw||true|\u201d custom_padding_tablet=\u201d\u201d custom_padding_phone=\u201d|56px||\u201d custom_padding_last_edited=\u201dno|desktop\u201d title_font_size_tablet=\u201d45px\u201d title_font_size_phone=\u201d40px\u201d title_font_size_last_edited=\u201dno|telefone\u201d z_index_tablet=\u201d500\u2033 custom_css_main_element=\u201dposi\u00e7\u00e3o de fundo: centro 18% !importante;\u201d global_colors_info=\u201d{}\u201d] C\u00e9lulas-tronco pluripotentes induzidas [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 use_custom_gutter=\u201don\u201d gutter_width=\u201d1\u2033 specialty=\u201don\u201d padding_left_1=\u201d35px\u201d padding_left_2=\u201d35px\u201d padding_2_tablet=\u201d|||0px\u201d padding_2_phone=\u201d\u201d padding_2_last_edited=\u201don|desktop\u201d module_class_1=\u201dsidebar-secondary-nav\u201d module_class=\u201dhandprint-bg\u201d _builder_version=\u201d4.16\u2033 background_image=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png\u201d parallax=\u201don\u201d parallax_method=\u201doff\u201d largura_interna=\u201d100%\u201d largura_m\u00e1xima_interna=\u201d100%\u201d preenchimento_personalizado=\u201d0|0px|54px|0px|falso|falso\u201d z_index_tablet=\u201d500\u2033 largura_da_borda_superior=\u201d10px\u201d cor_da_borda_superior=\u201d#8fd2ed\u201d [\u2026]<\/p>","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"\t\t\t\t[vc_row][vc_column][vc_custom_heading text=\"Induced Pluripotent Stem Cells\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\n\r\n\r\n\r\n
                      \"\"<\/td>\r\n<\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\nThe Progeria Research Foundation Cell & Tissue Bank\r\n<\/strong>Human Induced Pluripotent Stem Cells (iPSC)\r\n
                        \r\n \t
                      1. Progeria iPSCs <\/strong>Background Information for the Non-scientist<\/strong><\/a>\u00a0<\/strong><\/li>\r\n \t
                      2. Purpose of <\/strong>Induced Pluripotent Stem Cell Generation and Distribution by The Progeria Research Foundation\u00a0<\/strong>\u00a0<\/a><\/li>\r\n \t
                      3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs)<\/strong>\u00a0<\/a><\/li>\r\n \t
                      4. Quality Control: Validation and Characterization<\/strong>\u00a0<\/a><\/li>\r\n \t
                      5. Original Starting Material from which iPSCs Were Derived<\/strong><\/a><\/li>\r\n \t
                      6. Join our Email List for Future iPSC Updates and New Cell Lines<\/strong>\u00a0\u00a0<\/strong><\/a><\/li>\r\n \t
                      7. Questions?\u00a0 Contact us.<\/strong><\/a><\/li>\r\n \t
                      8. Ordering iPSC Lines<\/strong><\/a>\u00a0<\/strong><\/li>\r\n \t
                      9. HGPS and Control iPSC Culture Media Preparation<\/strong>\u00a0<\/strong><\/a><\/li>\r\n \t
                      10. Preparing Madrigal Plates\u00a0<\/strong><\/a><\/li>\r\n \t
                      11. Thawing hESCs and iPSCs<\/strong><\/a><\/li>\r\n \t
                      12. Harvesting and Caring for hESCs and iPSCs<\/strong>\u00a0<\/strong><\/a><\/li>\r\n \t
                      13. Passaging with EDTA solution<\/strong><\/a><\/li>\r\n \t
                      14. Freezing hESCs and iPSCs<\/a><\/strong><\/li>\r\n<\/ol>\r\n\r\n
                        \r\n\r\n                        <\/a>1. iPSC Background information for the non-scientist<\/strong>\r\nStem cells are \u201cimmature\u201d cells that have not yet committed to becoming any one cell type.\u00a0 They are pliable because they have the potential to develop into many different types of mature cells in the body, such as cells that make up the heart or blood vessels, and other tissues and organs.\u00a0 In 2007, researchers discovered a strategy for creating stem cells in the laboratory by reprogramming mature adult cells that we commonly grow for research purposes.1, 2<\/sup><\/strong> . These artificially created stem cells are called Induced Pluripotent Stem Cells (\u201ciPSCs\u201d). For the field of Progeria, this is a huge breakthrough.\u00a0 For the first time, scientists can now make Progeria stem cells and ask questions about how stem cells function and develop in Progeria.\u00a0 Previously there was no source of human Progeria stem cells, and there was therefore a void of information about how Progeria stem cells function compared with stem cells from people without Progeria.\u00a0 In addition, scientists can re-program the Progeria stem cells to create, for the first time, mature Progeria blood vessels, heart cells, and other cell types.\u00a0 Until now, there was no source of human Progeria heart or blood vessel cells.\u00a0 We can now ask key <\/strong>questions about the heart disease that leads to early death in Progeria from heart attacks and strokes. We can compare these discoveries with the heart disease and aging in the general population and discover more about what influences aging in all of us.\u00a0 Already there have been several excellent studies published using Progeria stem cells.3-5<\/sup> \u00a0Our goal at The Progeria Research Foundation is to facilitate many more discoveries using this invaluable tool.\u00a0 For a primer on stem cells, please see this US government website:\u00a0https:\/\/stemcells.nih.gov\/info\/basics.htm<\/a>\r\n
                        \r\n
                          \r\n \t
                        1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. <\/em>2007;131:861-872.<\/li>\r\n \t
                        2. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin, II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. <\/em>2007;318:1917-1920.<\/li>\r\n \t
                        3. Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J, 3rd, Izpisua Belmonte JC. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature. <\/em>2011;472:221-225.<\/li>\r\n \t
                        4. Misteli T. HGPS-derived iPSCs for the ages. Cell Stem Cell. <\/em>2011;8:4-6.<\/li>\r\n<\/ol>\r\n<\/div>\r\n<\/a>2.\u00a0Purpose of induced pluripotent stem cell (iPSC) generation and distribution by The Progeria Research Foundation<\/strong>\r\nThe mission of The Progeria Research Foundation is to discover treatments and the cure for Hutchinson-Gilford Progeria Syndrome and its aging-related disorders. In 2009, PRF entered into a collaboration with an expert team of scientists at the University of Toronto, Canada, under the direction of William Stanford, PhD, to generate high quality Progeria iPSCs. Dr. Stanford is the Canada Research Chair in Integrative Stem Cell Biology. As of 2011, PRF continues to collaborate with Dr. Stanford at the University of Ottawa, Canada where he is Professor of Cellular and Molecular Medicine, Faculty of Medicine, and Senior Scientist at Ottawa Hospital Research Institute's Sprott Centre for Stem Cell Research.<\/a>\r\n\r\nOur goal is to provide this invaluable tool to researchers throughout the world.\u00a0 This new research tool will be used to generate new and innovative research in Progeria, as well as its relationship to heart disease and aging. \u00a0<\/strong>\r\n\r\n3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs)<\/strong><\/a>\r\nInduced-Pluripotent Stem Cells (iPSCs) were derived using VSVG-pseudotyped retroviral transduction of four human factors, Oct4, Sox2, Klf4, and c-Myc into fibroblasts. \u00a0iPSC colonies were derived on mouse-embryonic fibroblasts (MEFs). The procedure used was essentially as previously described but without the use of the EOS reporter (Nature Protocols 4: 1828-1844, 2009). \u00a0<\/strong>\r\n\r\n4. Quality Control: Validation and Characterization<\/strong>\r\nThe lines that are currently available have undergone several validation steps (see downloadable PDFs below):\r\n
                          \r\n
                            \r\n \t
                          1. Mycoplasma Testing for each line: Dr. Stanford\u2019s lab has performed mycoplasmaanalysis by PCR for each cell line.\u00a0 In addition, after expansion and prior to shipping cells, the lines will be retested for mycoplasma.<\/li>\r\n \t
                          2. Immunostaining for pluripotency markers Tra-1-60, Tra-1-81, and SSEA4.<\/li>\r\n \t
                          3. Alkaline Phosphatase Staining as an indicator of pluripotency<\/li>\r\n \t
                          4. Embryoid body formation and subsequent immunostaining for markers of the three germ-layers. Markers tested were \u03b2III-Tubulin (Ectoderm), Smooth-Muscle Actin (Mesoderm), and Gata4 or AFP (Endoderm)<\/li>\r\n \t
                          5. Karyotype analysis.<\/li>\r\n \t
                          6. Re-expression of lamin A in differentiated cells<\/li>\r\n \t
                          7. Teratoma assays<\/li>\r\n<\/ol>\r\n<\/div>\r\n<\/a>\r\nAdditional validation in process:<\/strong>\r\nSome lines have completed teratoma assays as shown in supporting data. For all other lines, teratoma assays are in process and status will be updated as these assays are completed.\r\n\r\n5.\u00a0\u00a0 Original starting material from which these iPS cells were derived<\/strong>\r\niPSCs were derived from PRF Cell & Tissue Bank non-transformed fibroblast cell lines.\r\n\r\nThe transduction method used for all iPS lines was Retrovirus MKOS.\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n
                            \r\n
                            iPSC Line<\/strong> ID<\/strong>\r\n<\/strong><\/div><\/td>\r\n
                            		\r\n
                            Mutation<\/strong>\r\n<\/strong><\/div><\/td>\r\n
                            		\r\n
                            Gender<\/strong> and Donation<\/strong> Age\r\n<\/strong><\/div><\/td>\r\n
                            		\r\n\r\n
                            Supporting Data\u00a0\"\"<\/strong><\/div><\/td>\r\n<\/tr>\r\n
                            \r\n
                            HGADFN003 iPS 1B<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA<\/em>Exon 11,\r\n1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 2yr 0mo<\/div><\/td>\r\n
                            		\r\n
                            \u00a0<\/strong>Dermal Fibroblasts\r\nHGADFN003<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGADFN003 iPS 1C<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA<\/em> Exon 11,\r\n1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 2yr 0mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGADFN003<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGDFN003\r\niPS 1D<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA <\/em>Exon 11,\r\n1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 2yr 0mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGADFN003<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGADFN167 iPS 1J<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA <\/em>Exon 11, 1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 8yr 5mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts HGADFN167<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGADFN167 iPS 1Q<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA<\/em> Exon 11, 1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 8yr 5mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts HGADFN167<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGMDFN090 iPS 1B<\/div><\/td>\r\n
                            		\r\n
                            \u00a0<\/em>Mother of HGADFN167 (unaffected)<\/div><\/td>\r\n
                            		\r\n
                            Female 37yr 10mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGMDFN090<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGMDFN090 iPS 1C<\/div><\/td>\r\n
                            		\r\n
                            \u00a0<\/em>Mother of HGADFN167 (unaffected)<\/div><\/td>\r\n
                            		\r\n
                            Female 37yr 10mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGMDFN090<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGFDFN168 iPS1 D2<\/div><\/td>\r\n
                            		\r\n
                            \u00a0Father of HGADFN167 (unaffected)<\/div><\/td>\r\n
                            		\r\n
                            Male 40yr\r\n5mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts HGFDFN168<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGFDFN168 iPS1P<\/div><\/td>\r\n
                            		\r\n
                            Father of HGADFN167 (unaffected)<\/div><\/td>\r\n
                            		\r\n
                            Male 40yr<\/div>\r\n
                            5mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGFDFN168<\/div><\/td>\r\n
                            		<\/a>\r\n
                            168 iPS1P<\/a><\/div><\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n
                            <\/div>\r\n\"\"<\/a>\u00a0PRF AVAILABLE CELL LINES<\/a><\/strong>\u00a0<\/strong>\r\n\r\n6.\u00a0Join our email list for future iPSC updates and new cell lines<\/strong>\r\nWe are continuing to generate iPSC lines.\u00a0 If you would like periodic updates on iPSCs held in the PRF Cell & Tissue Bank,\u00a0please join our emailing list by clicking here<\/a><\/strong>\r\n\r\n7.\u00a0Questions?<\/strong><\/a>\r\nPlease contact Leslie Gordon, MD, PhD, Medical Director, with any questions or needs, at lgordon@progeriaresearch.org<\/a> or 978-535-2594\r\n<\/strong>\r\n\r\n8.\u00a0 Ordering iPS cell lines<\/strong>\r\n\r\nIn 2014, PRF instituted a policy of no changes to our MTA. This is the result of 12 years of contractual arrangements with 70 research teams working at institutions in 14 countries. PRF and its counsel have taken into consideration the issues that have arisen in that time period and edited the agreement accordingly, resulting in what we feel are fair and reasonable terms.\r\n\r\nFor U.S. Federal Government Institutions or questions, please contact<\/span>\u00a0<\/span>Wendy Norris at:\u00a0<\/span>wnorris@lifespan.org<\/a><\/span>\u00a0or\u00a0401-274-1122 x 48063<\/a>.\r\n\r\nStep 1: Complete an application and material transfer agreement\r\n<\/strong>\r\n<\/a>\u00a0 Application and Agreement for Non-government Institutions<\/a>\r\n\r\n\u00a0Material Transfer Agreement for Non-government Institutions*<\/a>\r\n\r\nStep 2: Return the completed application and material transfer agreement to PRF at info@progeriaresearch.org<\/a>.\u00a0 Once approved, you will receive an email confirming your order and anticipated shipping date.\u00a0<\/strong><\/a>\r\n\r\nStep 3: <\/strong>Dr. Stanford\u2019s laboratory is currently distributing lines\u00a0in frozen cryovials.\u00a0 His laboratory will email you when the culture has been shipped, with shipping and tracking information. Inexperienced researchers are directed to obtain training at specialized courses essential to human embryonic stem cell\/iPSCs work.<\/a>\r\n\r\nStep 4: The University of Ottawa will charge $84.00 per iPSC line plus courier costs, if any, and will send you a bill directly.<\/strong>\r\n\r\n9.\u00a0 HGPS and Control iPS Cell Culture Media Preparation<\/strong>\r\niPSC and ESCs need to be fed with either TeSR-E8 from Stem Cell\u00a0Technologies (cat# 05990) or Essential 8 medium from ThermoFisher Scientific (cat#A1517001). Please follow the supplier's recommendations for storage.\r\n\r\n10.Preparing Madrigal Plates<\/strong>\r\n\r\nNote<\/strong>: All steps involving matrigel should be done as quickly as possible and stay as cold as possible.\r\n
                              \r\n \t
                            1. Thaw matrigel bottle (10ml) at 4C.<\/li>\r\n \t
                            2. Take matrigel bottle and add 10ml of cold DMEM\/F12 media.<\/li>\r\n \t
                            3. Aliquot 1ml of matrigel in pre-chilled 15ml falcon tubes.<\/li>\r\n \t
                            4. Freeze all aliquots at -20C<\/li>\r\n \t
                            5. To make matrigel plate, remove one aliquot of matrigel (1ml) from -20C and add 18ml of cold DMEM\/F12. Let pellet thaw and once thawed mix well (without creating bubbles) and plate (1ml\/well for 6 well plate, 0.5ml\/well for 12 well plate, 0.25ml\/well for 24 well plate). These plates are labelled as MG1.<\/li>\r\n \t
                            6. If using plate immediately, let plate sit at room temperature for 1 hour, observe matrigel under the microscope. Matrigel should be well dispersed and not \u201cclumpy\u201d.<\/li>\r\n \t
                            7. Once 1 hour is passed transfer matrigel to a second plate (this plate is labelled MG2). If using right away, incubate at room temperature for 1 hour.<\/li>\r\n \t
                            8. If using plates at another time, wrap edge of plate with paraffin and store at 4 degrees.<\/li>\r\n<\/ol>\r\n<\/a>\r\nMatrigel \u2013 BD\/Fisher, cat#CB-40230\r\nDMEM\/F12 \u2013 Life Technologies, cat#11330-057\r\n\r\nDr. William Stanford-2018\r\n\r\n11. Thawing hESCs and iPSCs<\/strong>\r\n\r\nThawing ES or iPS cells (per cryo-vial)<\/u><\/strong>\r\n
                                \r\n \t
                              1. Take matrigel plate out of 4C and warm at room temperature for one hour or make fresh matrigel plate (see preparing matrigel plate protocol<\/strong>).<\/li>\r\n \t
                              2. Warm 4ml of PSC media (pluripotent stem cell media) in a 15ml falcon tube.<\/li>\r\n \t
                              3. Remove cells from freezer and swirl in 37 degree water bath until only a small ice chunk is left. This step must be done quickly.<\/li>\r\n \t
                              4. Ethanol cell tube and falcon media tube and place in the hood.<\/li>\r\n \t
                              5. Use a 1ml wide mouth tip to slowly add cells to 4ml of prewarmed media<\/li>\r\n \t
                              6. Spin at 850rpm for 5min<\/li>\r\n \t
                              7. Remove supernatant<\/li>\r\n \t
                              8. Add 2ml of PSC media and with a\u00a0wide mouth tip break up clumps. Transfer media to one well of a 6 well plate add\u00a02ul of\u00a0ROCK inhibitor (Y27632, final concentration of 10uM)\u200b.<\/li>\r\n \t
                              9. Remove media after 24 hours and add 2ml of PSC media (for a 6 well, 1ml for 12 well and 0.5ml for 24 well plate). See Harvesting and Caring for hESC\/iPSC <\/strong>protocol<\/li>\r\n<\/ol>\r\nPSC media\r\n<\/u><\/strong>TeSR-E8 from Stem Cell Technologies, catalogue # 05990 or Essential 8 Medium from ThermoFisher Scientific, catalogue # A1517001\r\n\r\nWhat is ROCK Inhibitor Y27632?<\/a>\r\n<\/u><\/strong>ROCK Inhibitor Y27632 is a selective inhibitor of the Rhoassociated kinase p160ROCK. Treatment with ROCK Inhibitor Y27632 prevents dissociationinduced apoptosis of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), increasing the survival rate and maintaining pluripotency during subcultivation and thawing of hESCs and hiPSCs. ROCK Inhibitor Y27632 also has been shown to enhance the survival rate of stem cells during cryopreservation.\r\n\r\nDr. William Stanford-2018\r\n\r\n12.Harvesting and Caring for hESCs and iPSCs<\/strong>\r\n
                                  \r\n \t
                                1. Remove media from 4C and place in 37C water bath.<\/li>\r\n \t
                                2. The day after cells were thawed take a look at them under the microscope to determine survival rate. (If cells were frozen only with mFreSR the survival rate is appr. 20%, if cells were frozen using the Bio-Cool survival rate is appr. 60%).<\/li>\r\n \t
                                3. Remove media from the wells and pipette 2ml (for a 6 well, 1ml for 12 well and 0.5ml for 24 wells plate) of fresh PSC media per well.<\/li>\r\n \t
                                4. Place plate in 37C incubator (hypoxic condition: 10% CO2 and 5% O2).<\/li>\r\n \t
                                5. Cells are fed every day for the next 3-5 days until 80% confluent.<\/li>\r\n \t
                                6. Cells should be observed and cleaned of any differentiated cells that might be growing.<\/li>\r\n \t
                                7. To clean cells use the picking hood and \u201cscrap off\u201d differentiated cells with a pipette tip.<\/li>\r\n \t
                                8. Once cells are cleaned changed media as done in above steps.<\/li>\r\n<\/ol>\r\n(See Freezing hESC\/iPSC or Passing hESC\/iPSC<\/strong>)\r\n\r\nNOTE:\r\n<\/u><\/strong>PSC media was determine to be more efficient\u00a0 in a hypoxic environment. We have also observed that less differentiation occurs when cells are grown in hypoxic incubators versus normoxic. Also when seeding cells there\u2019s a better survival rate when using a hypoxic incubator.<\/a>\r\n\r\nNormoxic: 5% CO2\r\nHypoxic: 5%O2, 10% CO2\r\n\r\nDr. William Stanford-2018\r\n\r\n13.Passaging with EDTA solution<\/strong>\r\n
                                    \r\n \t
                                  1. Add 2ml of PSC media to a 6 well matrigel coated plate and set aside.<\/li>\r\n \t
                                  2. Take the plate to be passaged and remove the media from the well and wash once with 1ml of PBS(-\/-).<\/li>\r\n \t
                                  3. Add 1ml of\u00a0 the EDTA solution to the well and leave for 3-4min at room temperature. \u00a0Don\u2019t move the plate around as cells can start detaching.<\/li>\r\n \t
                                  4. Once 4 min. is up remove EDTA solution and add 1ml of PSC media. Do not leave EDTA on the cells for more than 4 min as this will cause the cells to lift off.<\/li>\r\n \t
                                  5. Scrape cells (with cell scrapper) and divide cells amongst the 6 wells of your plate containing PSC media (I\u2019ve been taking 160ul into each well). Avoid breaking up the pieces in very small pieces. Try to keep large chunks. Preferably use a wide mouth pipette tip.<\/li>\r\n<\/ol>\r\nNOTE<\/u><\/strong>: Once the cells have been scraped you want to transfer them to the new plate as soon as possible because the cells will reattach quickly (within 5min).\r\n\r\nIf EDTA is left on the cells for more than 4mins. the cells can start to detach. If this happen simply scrap cells and collect in a 15ml falcon with 4ml of PSC media. Spin cells at 850rpm for 5 min. Resuspend the pellet with 1ml of media and divide evenly among a 6 well matrigel coated plate (160ul per well).\r\n\r\nEDTA solution<\/u><\/strong>:\u00a0 Add 500ul of 0.5M EDTA (pH 8.0) into 500ml of DPBS (-\/-). Add 0.9g of NaCl and adjust the osmolarity to 340 mOsm. Filter the solution to sterilize and store it at 4C for up to 6 months. We want the least amount of disturbance for the cells during dissociation therefore the EDTA solution is at the same osmolarity as the PSC media. <\/u><\/strong>\r\n\r\nFrom the paper:<\/a>\r\nPassaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions\r\n<\/strong>Jeanette Beers<\/a>,1<\/sup>\u00a0Daniel R. Gulbranson<\/a>,2,3<\/sup>\u00a0Nicole George<\/a>,4<\/sup>Lauren I. Siniscalchi<\/a>,1<\/sup>\u00a0Jeffrey Jones<\/a>,4,5<\/sup>\u00a0James A. Thomson<\/a>,2,3,6<\/sup>\u00a0and\u00a0Guokai Chen<\/a>1,2<\/sup>\r\n\r\n14.Freezing hESCs and iPSCs<\/strong>\r\n
                                      \r\n \t
                                    1. Turn on Bio-Cool (Controlled Rate Freezer) and adjust SP temperature to -7C.<\/li>\r\n \t
                                    2. Remove cells from incubator and observe confluency and morphology under the microscope.\r\nNOTE:<\/strong> Looking for ~80% confluency<\/li>\r\n \t
                                    3. If confluent, remove old media and wash once with PBS(-\/-) then add 1 ml of EDTA solution (see passaging with EDTA solution) per well.<\/li>\r\n \t
                                    4. Incubate at room temperature for 3-4 minutes.<\/li>\r\n \t
                                    5. Aspirate the EDTA solution and add 1 ml of mFreSR media (cat#05855, Stem Cell Technologies)<\/li>\r\n \t
                                    6. Use cell scraper to detach the cells from the well (gently).<\/li>\r\n \t
                                    7. Transfer cells\/mFreSR to a cryotube using a wide mouth tip.<\/li>\r\n \t
                                    8. Place tubes in Bio-Cool and incubate for 10min.<\/li>\r\n \t
                                    9. Get liquid nitrogen.<\/li>\r\n \t
                                    10. After 10min. seed the cells by dipping a spatula into liquid nitrogen and touching the side of the cryo-vial for approximately 10 seconds or until you see a crystal form on the side of the cryo-vial.<\/li>\r\n \t
                                    11. Start program 1 by pushing \u201cPROG\u201d button and going through the program by pushing the button again and you should see the rate of 0.5C\/min. , then press \u201cRUN\u201d.<\/li>\r\n \t
                                    12. Once temperature reaches -65C the cryo-tubes can be transferred and stored in liquid nitrogen.<\/li>\r\n<\/ol>\r\nAlternatives\r\n<\/u><\/strong>1-Another option is to place the cryo-tubes in a freezing container (Biocision-CoolCell) and store at -80C overnight. The next day store cryo-tubes in liquid nitrogen.\r\n\r\nDr. William Stanford-2018[\/vc_column_text][\/vc_column][\/vc_row]\t\t","_et_gb_content_width":"","footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-1010","page","type-page","status-publish","hentry"],"yoast_head":"\nInduced | The Progeria Research Foundation<\/title>\n<meta name=\"description\" content=\"To learn more about Induced Pluripotent Stem Cells, please contact Leslie Gordon, MD, PhD, Medical Director at lgordon@progeriaresearch.org.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.progeriaresearch.org\/pt\/induced-pluripotent-stem-cells\/\" \/>\n<meta property=\"og:locale\" content=\"pt_PT\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Induced | The Progeria Research Foundation\" \/>\n<meta property=\"og:description\" content=\"To 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