ES2582342T3 - Reparación y regeneración de tejido blando usando células derivadas del posparto - Google Patents
Reparación y regeneración de tejido blando usando células derivadas del pospartoInfo
- Publication number
- ES2582342T3 ES2582342T3 ES04777286.8T ES04777286T ES2582342T3 ES 2582342 T3 ES2582342 T3 ES 2582342T3 ES 04777286 T ES04777286 T ES 04777286T ES 2582342 T3 ES2582342 T3 ES 2582342T3
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- Rheumatology (AREA)
Abstract
Células que se pueden obtener obtener del tejido del cordón umbilical humano sustancialmente libre de sangre para su uso en la inducción de angiogénesis, en donde dichas células son capaces de autorrenovación y diferenciación, y tienen las siguientes características: a. potencial de experimentar al menos 40 duplicaciones en el cultivo; b. unión y expansión en un recipiente de cultivo de tejido recubierto o sin recubrir, en donde el recipiente de cultivo de tejido recubierto comprende un recubrimiento de gelatina, laminina, colágeno, poliornitina, vitronectina o fibronectina; c. producción de vimentina y actina del músculo liso alfa; d. producción de cada uno de CD10, CD13, CD44, CD73, HLA-A,B,C, CD90, HLA-A,B,C, PD-L2 y, PDGFralfa, como se detectan por citometría de flujo; e) expresión aumentada de genes endógenos que codifican interleucina 8; reticulon 1; ligando 1 de quimiocinas (motivo C-X-C) (actividad estimulante del crecimiento de melanoma, alfa); ligando 6 de quimiocinas (motivo C-X-C) (proteína 2 quimiotáctica de granulocitos); ligando 3 de quimiocinas (motivo C-XC); y proteína 3 inducida por el factor de necrosis tumoral alfa en relación a la expresión endógena en una célula humana que es un fibroblasto, una célula madre citoblasto mesenquimatoso, o una célula de la médula ósea de las crestas ilíacas, como se caracterizan en una matriz génica; f) falta de producción de CD31, CD34, CD45, CD80, CD86, CD 117, CD141, CD178, HLA-G, B7-H2 y HLADR, DP,DQ como se detectan por citometría de flujo; g) crecimiento de aproximadamente el 5% a aproximadamente el 20% de oxígeno h). requieren L-valina para el crecimiento; i) expresión disminuida de los genes caja homeótica 2 de la baja estatura; proteína 2 de choque térmico de 27 kDa; ligando 12 de quimiocina (motivo C-X-C) (factor 1 derivado de células del estroma); elastina; ADNc DKFZp586M2022 (del clon DKFZp586M2022); caja homeótica 2 de mesénquima; homólogo 1 de caja homeótica de "sine oculis"; cristalina, alfa B; activador de morfogénesis 2 asociado a "dishevelled"; proteína DKFZP586B2420; similar a neuralin 1; tetranectina; dominio 3 de homología con src (SH3) y rico en cisteínas; gen 1 de translocación de linfocitos B, antiproliferativa; colesterol 25-hidroxilasa; factor de transcripción 3 relacionado con el enanismo; proteína hipotética FLJ23191; receptor de interleucina 11, alfa; potenciador de procolágeno C-endopeptidasa; homólogo 7 de "frizzled"; gen hipotético BC008967; colágeno, tipo VIII, alfa 1; tenascina C; proteína de caja homeótica iroquois 5; hefaestina; integrina, beta 8; glicoproteína 2 de vesícula sináptica; ADNc FLJ12280 fis, clon MAMMA1001744; factor 1 similar al receptor de citocinas; canal de potasio activado por calcio de conductancia intermedia/baja, subfamilia N, miembro 4; integrina, alfa 7; proteína DKFZP586L151; coactivador de la transcripción con motivo de unión a PDZ (TAZ); homólogo 2 de caja homeótica de "sine oculis"; proteína KIAA1034; respuesta de crecimiento precoz 3; caja homeótica de "distal less" 5; proteína hipotética FLJ20373; familia 1 de aldo-ceto reductasas, miembro C3 (3-alfa hidroxiesteroide deshidrogenasa, tipo II); biglicano; fibronectina 1; proencefalina; integrina, tipo beta 1 (con dominios de repetición similares a EGF); clon de ADNc EUROIMAGE 1968422; EphA3; proteína KIAA0367; receptor de péptido natriurético C/guanilato ciclasa C (receptor del péptido atrial natriurético C); proteína hipotética FLJ14054; ADNc DKFZp564B222 (del clon DKFZp564B222); proteína de membrana asociada a vesícula 5; proteína 1 de la matriz extracelular tipo fibulina que contiene EGF; similar a proteína 3 de interacción con BCL2/adenovirus E1B de 19 kDa; proteína 1 de unión a AE; polipéptido 1 de la subunidad VIIa de la citocromo c oxidasa (músculo); neuroblastoma, supresión de tumorigenicidad 1; y proteína 2 de unión al factor de crecimiento insulinoide, 36 kDa en relación a una célula humana que es un fibroblasto, una citoblasto mesenquimatoso, o una célula de la médula ósea de las crestas ilíacas, como se caracterizan en una matriz génica; j) expresan GCP-2 y NOGO-A como se ensayan por inmunocitoquímica; k) secretan MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO y TIMP1, como se determinan por ELISA; l. no secretan SDF-1 alfa, TGF-beta2, ANG2, PDGFbb y VEGF, como se determinan por ELISA, en donde dicha inducción de angiogénesis comprende exponer una población de células de tejido blando a dichas células que se pueden obtener a partir de tejido del cordón umbilical.
Description
reversible por cualquier agente biológico, químico o viral.
Como se usa en el presente documento, el término medio de crecimiento se refiere a un medio de cultivo suficiente para la expansión de células derivadas del posparto. El medio de cultivo del medio de crecimiento contiene 5 preferentemente medio esencial modificado por Dulbecco (DMEM). Más preferentemente, el medio de crecimiento contiene glucosa. El medio de crecimiento contiene preferentemente DMEM-baja glucosa (DMEM-LG) (Invitrogen, Carlsbad, CA). El medio de crecimiento contiene preferentemente aproximadamente 15 % (v/v) de suero (por ejemplo, suero bovino fetal, suero bovino definido). El medio de crecimiento contiene preferentemente al menos un agente antibiótico y/o agente antimicótico (por ejemplo, penicilina, estreptomicina, anfotericina B, gentamicina,
10 nistatina; preferentemente 50 unidades/mililitro de penicilina G de sodio y 50 microgramos/mililitro de sulfato de estreptomicina). El medio de crecimiento contiene preferentemente 2-mercaptoetanol (Sigma, St. Louis MO). Lo más preferentemente, el medio de crecimiento contiene DMEM-baja glucosa, suero, 2-mercaptoetanol y un agente antibiótico.
15 Como se usa en el presente documento, condiciones de crecimiento estándar se refieren a condiciones atmosféricas estándar que comprenden aproximadamente 5 % de CO2, una temperatura de aproximadamente 35-39 ºC, más preferentemente 37 ºC, y una humedad relativa de aproximadamente el 100 %.
El término aislado se refiere a una célula, componente celular o una molécula que se ha sacado de su 20 entorno nativo.
El término aproximadamente se refiere a una aproximación de un valor establecido dentro de un intervalo de ±10 %.
25 Tejido blando, como se usa en el presente documento, se refiere generalmente a estructuras extraesqueléticas encontradas en todo el cuerpo e incluye, pero no se limita a, tejido de cartílago, tejido de menisco, tejido de ligamento, tejido de tendón, tejido de disco intervertebral, tejido periodontal, tejido de piel, tejido vascular, tejido de músculo, tejido de fascia, tejido del periostio, tejido ocular, tejido pericárdico, tejido de pulmón, tejido sinovial, tejido nervioso, tejido de riñón, médula ósea, tejido urogenital, tejido intestinal, tejido de hígado, tejido de
30 páncreas, tejido del bazo, tejido adiposo, y combinaciones de los mismos.
Afección de tejido blando (o lesión o enfermedad) es un término incluyente que engloba afecciones, trastornos o enfermedades agudos y crónicos de tejido blando. Por ejemplo, el término engloba afecciones producidas por enfermedad o traumatismo o fallo del tejido para desarrollarse normalmente. Ejemplos de afecciones
35 de tejido blando incluyen, pero no se limitan a, hernias, daño al suelo pélvico, desgarro o rotura de un tendón o ligamento, heridas de la piel (por ejemplo, cicatrices, heridas traumáticas, quemaduras graves, úlceras de la piel (por ejemplo, úlceras de decúbito (presión), úlceras venosas y úlceras diabéticas) y heridas quirúrgicas tales como aquellas asociadas a la extirpación de cánceres de piel); afección vascular (por ejemplo, enfermedad vascular tal como enfermedad arterial periférica, aneurisma aórtico abdominal, enfermedad de la carótida y enfermedad venosa;
40 lesión vascular, desarrollo vascular inapropiado); y enfermedades musculares (por ejemplo, miopatías congénitas; miastenia grave; enfermedades musculares inflamatorias, neurogénicas y miogénicas; y distrofias musculares tales como distrofia muscular de Duchenne, distrofia muscular de Becker, distrofia miotónica, distrofia muscular de cinturas, distrofia muscular facioescapulohumeral, distrofias musculares congénitas, distrofia muscular oculofaríngea, distrofia muscular distal y distrofia muscular de Emery-Dreifuss).
45 El término tratar (o tratamiento de) una afección de tejido blando se refiere a mejorar los efectos de, o retrasar, detener o invertir el progreso de, o retrasar o prevenir la aparición de, una afección de tejido blando como se define en el presente documento e incluye reparación de tejido blando, reconstrucción, abultamiento, tratamiento cosmético, tratamiento terapéutico, aumento de tejido y sellado de tejido.
50 El término cantidad eficaz se refiere a una concentración de un reactivo o composición farmacéutica, tal como un factor de crecimiento, agente de diferenciación, factor trófico, población de células u otro agente, que es eficaz para producir un resultado previsto, que incluye crecimiento y/o diferenciación celular in vitro o in vivo, o tratamiento de una afección de tejido blando como se describe en el presente documento. Con respecto a factores
55 de crecimiento, una cantidad eficaz puede oscilar de aproximadamente 1 nanogramo/mililitro a aproximadamente 1 microgramo/mililitro. Con respecto a PPDC como se administran a un paciente in vivo, una cantidad eficaz puede oscilar de tan solo varios cientos o menos a nada menos que varios millones o más. En realizaciones específicas, una cantidad eficaz puede oscilar de 103-1011. Se apreciará que el número de células que va a administrarse variará dependiendo de los detalles del trastorno que va a tratarse, que incluyen, pero no se limitan a, tamaño o volumen
60 total/área superficial que va a tratarse, además de la proximidad del sitio de administración a la localización de la región que va a tratarse, entre otro factores conocidos para el biólogo medicinal.
Los términos periodo eficaz (o tiempo) y condiciones eficaces se refieren a un periodo de tiempo u otras condiciones controlables (por ejemplo, temperatura, humedad para métodos in vitro), necesarias o preferidas para 65 un agente o composición farmacéutica para lograr su resultado previsto.
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8; reticulon 1; ligando 1 de quimiocinas (motivo C-X-C) (actividad estimulante del crecimiento de melanoma, alfa); ligando 6 de quimiocinas (motivo C-X-C) (proteína 2 quimiotáctica de granulocitos); ligando 3 de quimiocinas (motivo C-X-C); y proteína 3 inducida por el factor de necrosis tumoral alfa.
5 Las PPDC de la divulgación pueden tener expresión, que con respecto a una célula humana que es un fibroblasto, un citoblasto mesenquimatoso, o una célula de la médula ósea de las crestas ilíacas, es reducida para al menos uno de: homeocaja 2 de baja estatura; proteína 2 de 27 kDa de choque térmico; ligando 12 de quimiocinas (motivo C-X-C) (factor 1 derivado de células del estroma); elastina; ADNc DKFZp586M2022 (del clon DKFZp586M2022); homeocaja 2 del mesénquima; homólogo 1 de la homeocaja del seno ocular; cristalina, alfa B;
10 activador asociado a dishevelled de la morfogénesis 2; proteína DKFZP586B2420; similar a neuralina 1; tetranectina; dominio de homología tres con src (SH3) y rico en cisteína; gen 1 de translocalización de linfocitos B, antiproliferativo; colesterol 25-hidroxilasa; factor de transcripción 3 relacionado con runt; proteína hipotética FLJ23191; receptor de interleucina 11, alfa; potenciador de la procolágeno C-endopeptidasa; homólogo 7 de frizzled; gen hipotético BC008967; colágeno, tipo VIII, alfa 1; tenascina C; proteína 5 de la homeocaja de iroquois; hefaestina;
15 integrina, beta 8; glucoproteína 2 de las vesículas sinápticas; ADNc FLJ12280 fis, clon MAMMA1001744; factor 1 de tipo receptor de citocinas; canal de potasio activado por calcio de conductancia intermedia/pequeña, subfamilia N, miembro 4; integrina, alfa 7; proteína DKFZP586L151; co-activador transcripcional con motivo de unión a PDZ (TAZ); homólogo 2 de la homeocaja del seno ocular; proteína KIAA1034; respuesta 3 de crecimiento precoz; homeocaja 5 de distal-less; proteína hipotética FLJ20373; familia 1 de la aldo-ceto reductasa, miembro C3 (3-alfa-hidroxiesteroide
20 deshidrogenasa, tipo II); biglicano; fibronectina 1; proencefalina; integrina, 1 de tipo beta (con dominios de repetición de tipo EGF); clon de ADNc EUROIMAGE 1968422; EphA3; proteína KIAA0367; receptor C del péptido natriurético/guanilato ciclasa C (receptor C del péptido atrionatriurético); proteína hipotética FLJ14054; ADNc DKFZp564B222 (del clon DKFZp564B222); proteína 5 de membrana asociada a la vesícula; proteína 1 de la matriz extracelular tipo fibulina que contiene EGF; tipo 3 de proteína de 19 kDa de interacción BCL2/adenovirus E1B;
25 proteína 1 de unión a AE; polipéptido 1 de la subunidad VIIa de la citocromo c oxidasa (músculo); neuroblastoma, supresión de tumorigenicidad 1; y proteína 2 de unión al factor de crecimiento similar a la insulina, 36 kDa; el experto apreciará que la expresión de una amplia variedad de genes se caracteriza convenientemente sobre una matriz de genes, por ejemplo, sobre una GENECHIP de Affymetrix.
30 Las PPDC pueden secretar una variedad de factores bioquímicamente activos, tales como factores de crecimiento, quimiocinas, citocinas y similares. Células preferidas de la divulgación secretan al menos uno de MCP1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES y TIMP1. Las PPDC de la divulgación pueden caracterizarse por su falta de secreción de al menos uno de TGF-beta2, ANG2, PDGFbb, MIP1b, I309, MDC y VEGF, como se detecta por ELISA. Estas y otras características están disponibles para identificar y caracterizar las
35 células, y distinguir las células de la invención de otras conocidas en la técnica.
En realizaciones preferidas de la divulgación, la célula comprende dos o más de las características anteriores. Son más preferidas aquellas células que comprenden, tres, cuatro o cinco o más de las características. Todavía más preferidas son aquellas células derivadas del posparto que comprenden seis, siete u ocho o más de las
40 características. Todavía más preferidas actualmente son aquellas células que comprenden las nueve características reivindicadas.
También se prefieren actualmente por la divulgación células que producen al menos dos de GCP-2, NOGO-A, factor de tejido, vimentina y alfa-actina de músculo liso. Son más preferidas aquellas células que producen tres, 45 cuatro o cinco de estas proteínas.
El experto apreciará que los marcadores celulares están sujetos a variar algo bajo condiciones de crecimiento ampliamente diferentes, y que las generalmente descritas en el presente documento son caracterizaciones en medio de crecimiento, o variaciones de las mismas. Las células derivadas del posparto que se
50 producen de al menos una, dos, tres o cuatro de CD10, CD13, CD44, CD73, CD90, PDGFr-alfa, PD-L2 y HLA-A, B, C son células preferidas de la divulgación. Son más preferidas aquellas células que producen cinco, seis o siete de estos marcadores de la superficie celular. Las células derivadas del posparto de la inevnción pueden producir las ocho de las anteriores proteínas de marcador de la superficie celular.
55 Las PPDC que carecen de producción de al menos una, dos, tres, o cuatro de las proteínas CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G y HLA-DR,DP,DQ, como se detecta por citometría de flujo, son células preferidas de la divulgación. Se prefieren las PPDC que carecen de producción de al menos cinco, seis, siete u ocho o más de estos marcadores. Son más preferidas las células que carecen de producción de al menos nueve o diez de los marcadores de la superficie celular. Las células de la invención carecen de producción de
60 las once de las anteriores proteínas de identificación.
Las células de la invención producen cada uno de CD10, CD13, CD44, CD73, CD90, PDGFr-alfa y HLA-A, B, C, y no producen ninguno de CD31, CD34, CD45, CD117, CD141 o HLA-DR,DP,DQ, como se detecta por citometría de flujo.
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reducir el riesgo de rechazo del tejido implantado.
Una vez las células de la invención se han manipulado genéticamente, pueden implantarse directamente en el paciente para permitir el tratamiento de una afección de tejido blando o para producir un producto génico antiinflamatorio tal como, por ejemplo, péptidos o polipéptidos correspondientes al idiotipo de anticuerpos neutralizantes para GM-CSF, TNF, IL-1, IL-2, u otras citocinas inflamatorias.
Alternativamente, las células genéticamente manipuladas pueden usarse para producir nuevo tejido in vitro, que entonces se implanta en el sujeto, como se describe en el presente documento.
La secreción de factores de crecimiento por las PPDC puede proporcionar soporte trófico para un segundo tipo de célula in vitro o in vivo. Las PPDC de la divulgación pueden secretar, por ejemplo, al menos uno de proteína 1 quimiotáctica de monocitos (MCP-1), interleucina-6 (IL6), interleucina 8 (IL-8), GCP-2, factor de crecimiento de hepatocitos (HGF), factor de crecimiento de queratinocitos (KGF), factor de crecimiento de fibroblastos (FGF), factor de crecimiento epidérmico de unión a la heparina (HB-EGF), factor neurotrófico derivado del cerebro (BDNF), trombopoyetina (TPO), proteína 1 alfa inflamatoria de macrófagos (MIP1a), RANTES e inhibidor de tejido de metaloproteinasa 1 de matriz (TIMP1), que pueden aumentarse mediante una variedad de técnicas, que incluyen cultivo ex vivo de las células en medio químicamente definido.
Como se demuestra por el Ejemplo 14 en el presente documento, las PPDC tienen la capacidad de soportar la supervivencia, crecimiento y diferenciación de otros tipos de células en co-cultivo. Por consiguiente, en otra realización, las PPDC se co-cultivan in vitro para proporcionar soporte trófico al otro tipo de célula deseado, que incluye, pero no se limita a, células epiteliales (por ejemplo, células de la mucosa bucal, tubo gastrointestinal, epitelio nasal, epitelio de las vías respiratorias, epitelio vaginal, epitelio corneal), células de la médula ósea, adipocitos, queratinocitos, células endoteliales vasculares (por ejemplo, células endoteliales aórticas, células endoteliales de la arteria coronaria, células endoteliales de la arteria pulmonar, células endoteliales de la arteria ilíaca, células endoteliales microvasculares, células endoteliales de la arteria umbilical, células endoteliales de la vena umbilical y progenitores endoteliales (por ejemplo, células CD34+, CD34+/CD117+)), mioblastos, miocitos, células del estroma y otras células de tejido blando, y mezclas de las mismas. Para el co-cultivo, puede ser deseable que las PPDC y las otras células deseadas se co-cultiven en condiciones en las que los dos tipos de células estén en contacto. Esto puede lograrse, por ejemplo, sembrando las células como una población de células heterogéneas en medio de cultivo o sobre un sustrato de cultivo adecuado. Alternativamente, las PPDC pueden cultivarse primero hasta confluencia y emplearse como sustrato para el segundo tipo de células deseadas en cultivo. En esta última realización, las células pueden separarse adicionalmente físicamente, por ejemplo, por una membrana o dispositivo similar, de forma que el otro tipo de célula pueda eliminarse y usarse por separado tras el periodo de co-cultivo. En otras realizaciones de la divulgación, las otras células deseadas se cultivan en contacto con el medio acondicionado, matriz extracelular y/o lisado celular de las PPDC. El uso de las PPDC en co-cultivo para promover la expansión y diferenciación de otros tipos de células puede encontrar aplicabilidad en investigación y en áreas clínicas/terapéuticas. Por ejemplo, la divulgación contempla que el co-cultivo de PPDC pueda utilizarse para facilitar el crecimiento y la diferenciación de células de un fenotipo dado en cultivo, por ejemplo, células de un fenotipo de tejido blando, para, por ejemplo, fines de investigación básica o para su uso en ensayos de cribado de fármacos. También puede utilizarse co-cultivo de PPDC para la expansión ex vivo de células de un fenotipo de tejido blando para la posterior administración para fines terapéuticos. Por ejemplo, pueden recogerse células de un individuo, expandirse ex vivo en co-cultivo con las PPDC, luego devolverse a ese individuo (transferencia autóloga) u otro individuo (transferencia singénica o alógena). En estas realizaciones, se apreciará que, tras la expansión ex vivo, la población de células que comprende las PPDC podría administrarse a un paciente en necesidad de tratamiento, por ejemplo, de una afección de tejido blando como se describe en el presente documento. Alternativamente, en situaciones en las que la transferencia autóloga es apropiada o deseable, las poblaciones de células co-cultivadas pueden separarse físicamente en cultivo, permitiendo la eliminación de las células autólogas para administración al paciente.
En algunas realizaciones, las PPDC inducen la angiogénesis en co-cultivo con células tales como, pero no se limitan a, células epiteliales (por ejemplo, células de la mucosa bucal, tubo gastrointestinal, epitelio nasal, epitelio de las vías respiratorias, epitelio vaginal, epitelio corneal), células de la médula ósea, adipocitos, queratinocitos, células endoteliales vasculares (por ejemplo, células endoteliales aórticas, células endoteliales de la arteria coronaria, células endoteliales de la arteria pulmonar, células endoteliales de la arteria ilíaca, células endoteliales microvasculares, células endoteliales de la arteria umbilical, células endoteliales de la vena umbilical y progenitores endoteliales (por ejemplo, células CD34+, CD34+/CD117+)), mioblastos, miocitos, células del estroma y otras células de tejido blando. Por ejemplo, factores angiogénicos, que incluyen pero no se limitan a, EPO, TIMP1, ANG2, PDGF-bb, TPO, KGF, HGF, FGF, VEGF y HBEGF, son liberados por las PPDC. Los métodos de inducción de la angiogénesis exponiendo una célula de tejido blando a una PPDC o producto de PPDC pueden realizarse in vitro o in vivo. Ejemplos de células de tejido blando que forman redes endoteliales según los métodos de la invención incluyen células endoteliales aórticas, células endoteliales de la arteria coronaria, células endoteliales de la arteria
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Pueden añadirse uno o varios de otros componentes a las células trasplantadas, que incluyen componentes seleccionados de la matriz extracelular, tales como uno o más tipos de colágeno conocidos en la técnica, y/o factores de crecimiento, plasma rico en plaquetas y fármacos. Alternativamente, las células de la invención pueden manipularse genéticamente para expresar y producir factores de crecimiento. Factores bioactivos que pueden incorporarse útilmente en la formulación de células incluyen agentes antiapoptósicos (por ejemplo, EPO, mimeticuerpo de EPO, TPO, IGF-I y IGF-II, HGF, inhibidores de la caspasa); agentes antiinflamatorios (por ejemplo, inhibidores de p38 MAPK, inhibidores de TGF-beta, estatinas, inhibidores de IL-6 e IL-1, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS y AINE (fármacos antiinflamatorios no esteroideos; por ejemplo, TEPOXALIN, TOLMETIN, SUPROFEN); agentes inmunosupresores/inmunomoduladores (por ejemplo, inhibidores de la calcineurina, tales como ciclosporina, tacrolimus; inhibidores de mTOR (por ejemplo, SIROLIMUS, EVEROLIMUS); antiproliferativos (por ejemplo, azatioprina, micofenolato mofetilo); corticosteroides (por ejemplo, prednisolona, hidrocortisona); anticuerpos tales como anticuerpos monoclonales anti-receptor IL-2Ralfa (por ejemplo, basiliximab, daclizumab), anticuerpos policlonales anti-linfocitos T (por ejemplo, globulina anti-timocitos (ATG); globulina anti-linfocitos (ALG); anticuerpo monoclonal anti-linfocitos T OKT3)); agentes antitrombogénicos (por ejemplo, heparina, derivados de heparina, urocinasa, PPack (dextrofenilalanina prolina arginina clorometilcetona), compuestos antitrombina, antagonistas de los receptores de plaquetas, anticuerpos anti-trombina, anticuerpos antireceptores de plaquetas, aspirina, dipiridamol, protamina, hirudina, inhibidores de la prostaglandina e inhibidores de plaquetas); y antioxidantes (por ejemplo, probucol, vitamina A, ácido ascórbico, tocoferol, coenzima Q-10, glutatión, L-cisteína, N-acetilcisteína), además de anestésicos locales. Como otro ejemplo, las células pueden coadministrarse con factor inhibidor de cicatrices como se describe en la patente de EE.UU. nº 5.827.735.
En una realización no limitante, una formulación que comprende las células de la invención se prepara para administración directamente al sitio en el que se desea la producción de nuevo tejido blando. Por ejemplo, y no a modo de limitación, las células de la invención pueden suspenderse en una disolución de hidrogel para inyección. Ejemplos de hidrogeles adecuados para su uso en la invención incluyen péptidos de auto-ensamblaje, tales como RAD16. Alternativamente, puede dejarse que la disolución de hidrogel que contiene las células endurezca, por ejemplo en un molde, para formar una matriz que tiene células dispersadas en ella antes de la implantación. O, una vez la matriz se ha endurecido, las formaciones de células pueden cultivarse de manera que las células se expandan mitóticamente antes de la implantación. El hidrogel es un polímero orgánico (natural o sintético) que se reticula mediante enlaces covalentes, iónicos o de hidrógeno para crear una estructura tridimensional de red cristalina abierta que atrapa moléculas de agua para formar un gel. Ejemplos de materiales que pueden usarse para formar un hidrogel incluyen polisacáridos tales como alginato y sales del mismo, péptidos, polifosfazinas y poliacrilatos, que se reticulan iónicamente, o polímeros de bloque tales como copolímeros de bloque de poli(óxido de etileno)polipropilenglicol que se reticulan por temperatura o pH, respectivamente. En algunas realizaciones, el soporte para las PPDC de la invención es biodegradable.
En algunas realizaciones de la invención, la formulación comprende un gel polimerizable in situ, como se describe, por ejemplo, en la publicación de solicitud de patente de EE.UU. 2002/0022676; Anseth y col., J. Control Release, 78(1-3):199-209 (2002); Wang y col., Biomaterials, 24(22):3969-80 (2003).
En algunas realizaciones, los polímeros son al menos parcialmente solubles en disoluciones acuosas, tales como agua, soluciones salinas tamponadas o disoluciones alcohólicas acuosas, que tienen grupos laterales cargados, o una sal iónica monovalente de los mismos. Ejemplos de polímeros con grupos laterales ácidos que pueden hacerse reaccionar con cationes son poli(fosfacenos), poli(ácidos acrílicos), poli(ácidos metacrílicos), copolímeros de ácido acrílico y ácido metacrílico, poli(acetato de vinilo) y polímeros sulfonados, tales como poliestireno sulfonado. También pueden usarse copolímeros que tienen grupos laterales ácidos formados haciendo reaccionar ácido acrílico o metacrílico y monómeros de éter vinílico o polímeros. Ejemplos de grupos ácidos son grupos ácido carboxílico, grupos ácido sulfónico, grupos alcohol halogenado (preferentemente fluorado), grupos OH fenólico y grupos OH ácido.
Ejemplos de polímeros con grupos laterales básicos que pueden hacerse reaccionar con aniones son poli(vinilaminas), poli(vinilpiridina), poli(vinilimidazol), y algunos polifosfacenos sustituidos con imino. La sal de amonio o cuaternaria de los polímeros también puede formarse a partir de los nitrógenos del esqueleto o grupos imino laterales. Ejemplos de grupos laterales básicos son grupos amino e imino.
El alginato puede reticularse iónicamente con cationes divalentes, en agua, a temperatura ambiente, para formar una matriz de hidrogel. Debido a estas condiciones suaves, el alginato ha sido el polímero más comúnmente usado para la encapsulación de células de hibridoma, como se describe, por ejemplo, en la patente de EE.UU. nº
4.352.883 a Lim. En el proceso de Lim, una disolución acuosa que contiene los materiales biológicos que van a encapsularse se suspende en una disolución de un polímero soluble en agua, la suspensión se forma en gotitas que se configuran en microcápsulas discretas por contacto con cationes multivalentes, entonces la superficie de las microcápsulas se reticula con poliaminoácidos para formar una membrana semipermeable alrededor de los
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secretan elastina.
La trama tridimensional sembrada o inoculada de la invención puede usarse en una variedad de aplicaciones. Éstas incluyen, pero no se limitan a, trasplante o implantación de tanto las células cultivadas obtenidas 5 de la matriz como la propia matriz cultivada in vivo. Los andamiajes tridimensionales pueden, según la invención, usarse para sustituir o aumentar el tejido existente, para introducir tejido nuevo o alterado, para modificar próstesis artificiales, o para unir juntos tejidos biológicos o estructuras. Por ejemplo, y no a modo de limitación, realizaciones específicas de la invención incluyen, pero no se limitan a, estructuras planas e implantes tridimensionales de tejido tubular para reparación o regeneración, por ejemplo, del tubo gastrointestinal, vías genitourinarias, vasos
10 sanguíneos, músculos, ligamentos, tendones, piel, suelo pélvico, fascia y hernias.
Las PPDC pueden inocularse sobre un andamiaje plano. El andamiaje se incuba preferentemente en medio 15 de cultivo antes de la implantación. Dos o más tramas planas pueden ponerse la una encima de la otra y suturarse juntas para generar una trama multicapa.
20 Por ejemplo y no a modo de limitación, la trama tridimensional puede usarse para construir tejidos tubulares de una sola capa y multi-capa in vitro que pueden servir de sustitución para tejido tubular dañado o enfermo in vivo.
25 Las siguientes subsecciones describen el uso de una trama sembrada para preparar tubos que pueden implantarse en el cuerpo.
30 Puede cortarse un andamiaje en una tira (por ejemplo, forma rectangular) de la que la anchura es aproximadamente igual a la circunferencia interna del órgano tubular en el que se insertará por último lugar. Las células pueden inocularse sobre el andamiaje e incubarse flotando o suspendiendo en medios líquidos. En la etapa apropiada de confluencia, el andamiaje puede enrollarse en un tubo uniendo juntos los bordes largos. La costura puede cerrarse suturando juntos los dos bordes usando fibras de un material adecuado de un diámetro apropiado.
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Según la invención, puede formarse un andamiaje como tubo, inocularse con las PPDC y suspenderse en medios en una cámara de incubación. Con el fin de prevenir que las células obstruyan la luz, uno de los extremos 40 abiertos de la trama tubular puede fijarse a una boquilla. Los medios líquidos pueden ser forzados a pasar a través de esta boquilla desde una cámara de fuente conectada a la cámara de incubación para crear una corriente a través del interior de la trama tubular. El otro extremo abierto puede fijarse a una apertura de flujo de salida que conduce a una cámara de recogida de la que los medios pueden recircularse a través de la cámara de fuente. El tubo puede separarse de la boquilla y la abertura de flujo de salida cuando se completa la incubación. Este método se describe
45 por Ballermann, B. J., y col., solicitud internacional nº WO 94/25584 y en la solicitud de EE.UU. nº de serie 08/430.768.
50 En general, pueden combinarse dos tramas tridimensionales en un tubo según la invención usando cualquiera de los siguientes métodos.
55 Dos o más tramas planas pueden ponerse la una sobre la otra y suturarse juntas. Esta hoja de dos capas puede entonces enrollarse, y, como se ha descrito anteriormente, unirse juntas y fijarse.
60 Un andamiaje tubular que sirve como capa interna capa puede inocularse con las PPDC e incubarse. Puede cultivarse un segundo andamiaje como tira plana con anchura ligeramente mayor que la circunferencia externa de la trama tubular. Después de obtenerse el crecimiento apropiado, la trama plana puede enrollarse alrededor del exterior del andamiaje tubular, seguido del cierre de la costura de los dos bordes de la trama plana y, preferentemente, fijando la trama placa al tubo interno.
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Se cultivaron PPDC de tejido posparto de un neonato masculino en medio de crecimiento (DMEM-baja glucosa (Gibco Carlsbad, CA), 15 % (v/v) de suero bovino fetal (FBS) (Hyclone, Logan, UT), 0,001 % (v/v) de beta5 mercaptoetanol (Sigma, St. Louis, MO) y 50 unidades/mililitro de penicilina, 50 microgramos/mililitro de estreptomicina (Gibco, Carlsbad, CA)). Se seleccionó tejido posparto de un neonato masculino (X,Y) para permitir la distinción entre células derivadas de neonato y células derivadas de la madre (X,X). Las células se sembraron a
5.000 células por centímetro cuadrado en medio de crecimiento en un matraz T25 (Corning, Corning, NY) y se expandieron a aproximadamente el 80 % de confluencia. Se llenó un matraz T25 que contenía células hasta el cuello 10 con medio de crecimiento. Las muestras se entregaron a un laboratorio de citogenética por mensajería (el tiempo de transporte estimado de laboratorio a laboratorio es una hora). El análisis de cromosomas se realizó por el Center for Human & Molecular Genetics at the New Jersey Medical School, Newark, NJ. Las células se analizaron durante la metafase cuando los cromosomas se visualizan mejor. De veinte células contadas en la metafase, cinco se analizaron para el número de cariotipos homogéneos normales (dos). Una muestra de células se caracterizó como
15 homogénea si se observaron dos cariotipos. Una muestra de células se caracterizó como heterogénea si se observaron más de dos cariotipos. Se contaron células en metafase adicionales y se analizaron cuando e identificó un número de cariotipos heterogéneos (cuatro).
20 Todas las muestras de células enviadas para el análisis de cromosomas se interpretaron por el personal del laboratorio de citogenética como que presentaban un aspecto normal. Tres de las dieciséis líneas celulares analizadas presentaron un fenotipo heterogéneo (XX y XY), que indica la presencia de células derivadas de tanto orígenes neonatales como maternos (Tabla 6-1). Las células derivadas del tejido Placenta-N se aislaron del aspecto
25 neonatal de la placenta. En el pase cero, esta línea celular apareció XY homogénea. Sin embargo, en el pase nueve, la línea celular fue heterogénea (XX/XY), que indica una presencia previamente sin detectar de células de origen materno.
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Tabla 6-1. Resultados del cariotipo de las PPDC.
- Tejido
- Pasaje Conteo de Células Metafase Análisis de Células Metafase No. de cariotipos ISCN cariotipo
- Placenta
- 22 20 5 2 46,XX
- Umbilical
- 23 20 5 2 46,XX
- Umbilical
- 6 20 5 2 46,XY
- Placenta
- 2 20 5 2 46,XX
- Umbilical
- 3 20 5 2 46,XX
- Placenta-N
- 0 20 5 2 46,XY
- Placenta-V
- 0 20 5 2 46,XY
- Placenta-M
- 0 21 5 4 46,XY[18]/46,XX[3]
- Placenta-M
- 4 20 5 2 46,XX
- Placenta-N
- 9 25 5 4 46,XY[5]/46,XX[20]
- Placenta-N C1
- 1 20 5 2 46,XY
- Placenta-N C3
- 1 20 6 4 46,XY[2]/46,XX[18]
- Placenta-N C4
- 1 20 5 2 46,XY
- Placenta-N C15
- 1 20 5 2 46,XY
- Placenta-N C20
- 1 20 5 2 46,XY
- Placenta-N C22
- 1 20 5 2 46,XY
- Clave: N-cara neonatal; V-región vellosidades: M-cara materna; C-Clon
Resumen. El análisis de cromosomas identifica PPDC derivadas de la placenta y del cordón umbilical cuyos cariotipos parecen normales como se interpreta por un laboratorio citogenético clínico. El análisis del cariotipo 60 también identifica líneas celulares libres de células maternas, como se ha determinado por cariotipo homogéneo.
EJEMPLO 7 Evaluación de marcadores de la superficie celular derivados del posparto humanos por citometría de flujo
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Tabla 8-5. Genes que se mostró que tenían reducida expresión en células derivadas del cordón umbilical y de la placenta en comparación con las otras líneas celulares ensayadas.
- Genes disminuidos en las células derivadas de cordón umbilical y de la placenta
- ID del conjunto de sondas
- Nombre del gen Número de registro del NCBI
- 210135_s_at
- caja homeótica 2 de la baja estatura AF022654.1
- 205824_at
- proteína 2 de choque térmico de 27kDa NM_001541.1
- 209687_at
- ligando 12 de quimiocina (motivo C-X-C) (factor 1 derivado de células del estroma) U19495.1
- 203666_at
- ligando 12 de quimiocina (motivo C-X-C) (factor 1 derivado de células del estroma) NM_000609.1
- 212670_at
- elastina (estenosis aórtica supravalvular, síndrome de Williams-Beuren) AA479278
- 213381_at
- ARNm de Homo sapiens; ADNc DKFZp586M2022 (del clon DKFZp586M2022) N91149
- 206201_s_at
- caja homeótica 2 de mesénquima (caja homeótica específica de la detención del crecimiento) NM_005924.1
- 205817_at
- homólogo 1 de caja homeótica de “sine oculis” (Drosophila) NM_005982.1
- 209283_at
- cristalina, alfa B AF007162.1
- 212793_at
- activador de morfogénesis 2 asociado a “dishevelled” BF513244
- 213488_at
- proteína DKFZP586B2420 AL050143.1
- 209763_at
- similar a neuralin 1 AL049176
- 205200_at
- tetranectina (proteína de unión a plasminógeno) NM_003278.1
- 205743_at
- dominio tres de homología con src (SH3) y rico en cisteínas NM_003149.1
- 200921_s_at
- gen 1 de translocación de linfocitos B, antiproliferativa NM_001731.1
- 206932_at
- colesterol 25-hidroxilasa NM_003956.1
- 204198_s_at
- factor de transcripción 3 relacionado con el enanismo AA541630
- 219747_at
- proteína hipotética FLJ23191 NM_024574.1
- 204773_at
- receptor de interleucina 11, alfa NM_004512.1
- 202465_at
- potenciador de procolágeno C-endopeptidasa NM_002593.2
- 203706_s_at
- homólogo 7 de “frizzled” (Drosophila) NM_003507.1
- 212736_at
- gen hipotético BC008967 BE299456
- 214587_at
- colágeno, tipo VIII, alfa 1 BE877796
- 201645_at
- tenascina C (hexabraquion) NM_002160.1
- 210239_at
- proteína de caja homeótica iroquois 5 U90304.1
- 203903_s_at
- hefaestina NM_014799.1
- 205816_at
- integrina, beta 8 NM_002214.1
- 203069_at
- glicoproteína 2 de vesícula sináptica NM_014849.1
- 213909_at
- ADNc de Homo sapiens FLJ12280 fis, clon MAMMA1001744 AU147799
- 206315_at
- factor 1 similar al receptor de citocinas NM_004750.1
- 204401_at
- canal de potasio activado por calcio de conductancia intermedia/baja, subfamilia N, miembro 4 NM_002250.1
- 216331_at
- integrina, alfa 7 AK022548.1
- 209663_s_at
- integrina, alfa 7 AF072132.1
- 213125_at
- proteína DKFZP586L151 AW007573
- 202133_at
- coactivador de la transcripción con motivo de unión a PDZ (TAZ) AA081084
- 206511_s_at
- homólogo 2 de caja homeótica de “sine oculis” (Drosophila) NM_016932.1
- 213435_at
- proteína KIAA1034 AB028957.1
- 206115_at
- respuesta de crecimiento precoz 3 NM_004430.1
- 213707_s_at
- caja homeótica de “distal less” 5 NM_005221.3
- 218181_s_at
- proteína hipotética FLJ20373 NM_017792.1
- 209160_at
- familia 1 de aldo-ceto reductasas, miembro C3 (3-alfa hidroxiesteroide deshidrogenasa, tipo II) AB018580.1
- 213905_x_at
- biglicano AA845258
- 201261_x_at
- biglicano BC002416.1
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- 202132_at
- coactivador de la transcripción con motivo de unión a PDZ (TAZ) AA081084
- 214701_s_at
- fibronectina 1 AJ276395.1
- 213791_at
- proencefalina NM_006211.1
- 205422_s_at
- integrina, tipo beta 1 (con dominios de repetición similares a EGF) NM_004791.1
- 214927_at
- clon de ADNc de inserto de longitud completa de ARNm de Homo sapiens EUROIMAGE 1968422 AL359052.1
- 206070_s_at
- EphA3 AF213459.1
- 212805_at
- proteína KIAA0367 AB002365.1
- 219789_at
- receptor de péptido natriurético C/guanilato ciclasa C (receptor del péptido atrial natriurético C) A1628360
- 219054_at
- proteína hipotética FLJ14054 NM_024563.1
- 213429_at
- ARNm de Homo sapiens; ADNc DKFZp564B222 (del clon DKFZp564B222) AW025579
- 204929_s_at
- proteína de membrana asociada a vesícula 5 (miobrevina) NM_006634.1
- 201843_s_at
- proteína 1 de la matriz extracelular tipo fibulina que contiene EGF NM_004105.2
- 221478_at
- similar a proteína 3 de interacción con BCL2/adenovirus E1B de 19 kDa AL132665.1
- 201792_at
- proteína 1 de unión a AE NM_001129.2
- 204570_at
- polipéptido 1 de la subunidad VIIa de la citocromo c oxidasa (músculo) NM_001864.1
- 201621_at
- neuroblastoma, supresión de tumorigenicidad 1 NM_005380.1
- 202718_at
- proteína 2 de unión al factor de crecimiento insulinoide, 36 kDa NM_000597
Tabla 8-6. Genes que se mostró que tenían elevada expresión en fibroblastos en comparación con las otras líneas celulares ensayadas.
Las Tablas 8-6, 8-7,y 8-8 muestran la elevada expresión de genes en fibroblastos humanos (Tabla 8-6), células 35 ICBM (Tabla 8-7) y MSC (Tabla 8-8).
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- Genes fibrobastos elevados
- fosfatasa de especificidad dual 2
- KIAA0527 proteina
- Homo sapiens cADN: FLJ23224 fis, clon ADSU02206
- dineína, citoplasmática, polipéptido intermedio 1
- ankyrin 3, el nodo de Ranvier (ankyrin G)
- inhibina, beta A (activina A, activina AB polipéptido alfa)
- pirofosfatasa ectonucleotide / fosfodiesterasa 4 (función putativa)
- KIAA1053 proteina
- proteína asociada a microtúbulos 1A
- proteína con dedos de zinc 41
- HSPC019 proteina
- Homo sapiens cADN: FLJ23564 fis, cloe LNG10773
- ARNm sapiens Homo; ADNc DKFZp564A072 (del clon DKFZp564A072)
- Proteína LIM (similar al enigma de proteína quinasa C de rata vinculante)
- inhibidor de la luz kappa polipéptido potenciador del gen en las células B, la proteína-quinasa asociada complejo
- proteína hipotética FLJ22004
- (clon CTG-A4) secuencia de ARNm Humana
- EST, Moderadamente similar al factor de citoquinas similar al receptor de 2; precursor CRL2 receptor de citoquinas [Homo sapiens]
- factor de crecimiento transformante, beta 2
- MGC29643 hipotética proteína
- antígeno identificado por el anticuerpo monoclonal MRC OX-2
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Reacción de linfocitos mixtos. Viales criopreservados de PPDC derivadas del cordón umbilical del pase 10 etiquetadas como la línea celular A y PPDC derivadas de la placenta del pase 11 etiquetadas como la línea celular B se enviaron sobre nieve carbónica a CTBR (Senneville, Quebec) para realizar una reacción de linfocitos mixtos usando CTBR SOP no. CAC-031. Se recogieron células mononucleares de sangre periférica (PBMC) de
5 múltiples donantes voluntarios masculinos y femeninos. Se trataron PBMC alógenas estimulantes (donantes), PBMC autólogas y líneas celulares derivadas del posparto con mitomicina C. Las células estimulantes autólogas y tratadas con mitomicina C se añadieron a PBMC respondedoras (receptoras) y se cultivaron durante 4 días. Después de la incubación, se añadió [3H]timidina a cada muestra y se cultivaron durante 18 horas. Tras la recogida de las células, se extrajo ADN radiomarcado, y la incorporación de [3H]-timidina se midió usando un contador de centelleo.
10 El índice de estimulación para el donante alógeno (SIAD) se calculó como la proliferación media del receptor más el donante alógeno tratado con mitomicina C dividido entre la proliferación de referencia del receptor. El índice de estimulación de las células derivadas del posparto se calculó como la proliferación media del receptor más la línea celular derivada del posparto tratada con mitomicina C dividido entre la proliferación de referencia del
15 receptor.
Reacción de linfocitos mixtos -Placenta. Se cribaron siete donantes de sangre voluntarios humanos
20 para identificar un único donante alógeno que presentaría una respuesta a la proliferación robusta en una reacción de linfocitos mixtos con los otro seis donantes de sangre. Este donante se seleccionó como el donante de control positivo alógeno. Los seis donantes de sangre restantes se seleccionaron como receptores. El donante de control positivo alógeno y las líneas celulares derivadas de la placenta se trataron con mitomicina C y se cultivaron en una reacción de linfocitos mixtos con los seis receptores alógenos individuales. Las reacciones se realizaron por
25 triplicado usando dos placas de cultivo celular con tres receptores por placa (Tabla 11-2). El índice de estimulación promedio osciló de 1,3 (placa 2) a 3 (placa 1) y los controles positivos de donante alógenos oscilaron de 46,25 (placa 2) a 279 (placa 1) (Tabla 11-3).
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Tabla 11-2. Datos de la reacción linfocitaria mixta -Línea celular B (placenta) 30
- DMP para el ensayo de proliferación
- ID de placa: Placa 1
- Número del análisis
- Sistema de cultivo Replicaciones
- 1
- 2 3 Media DE CV
- IM03-7769
- Medida inicial de la proliferación del receptor 79 119 138 112,0 30,12 26,9
- Control de autoestimulación (células autólogas tratadas con mitomicina C)
- 241 272 175 229,3 49,54 21,6
- Donante alogénico de MLR IM03-7768 (tratadas con mitomicina C)
- 23971 22352 20921 22414,7 1525,97 6,8
- MLR con línea celular (célula tipo B tratada con mitomicina C)
- 664 559 1090 771,0 281,21 36,5
- SI (donante)
- 200
- SI (línea celular)
- 7
- IM03-7770
- Medida inicial de la proliferación del receptor 206 134 262 200,7 64,17 32,0
- Control de autoestimulación (células autólogas tratadas con mitomicina C)
- 1091 602 524 739,0 307,33 41,6
- Donante alogénico de MLR IM03-7768 (tratadas con mitomicina C)
- 45005 43729 44071 44268,3 660,49 1,5
- MLR con línea celular (célula tipo B tratada con mitomicina C)
- 533 2582 2376 1830,3 1128,24 61,6
- SI (donante)
- 221
60
5
10
15
20
25
30
35
40
45
50
55
60
65
- SI (línea celular)
- 9
- IM03-7771
- Medida inicial de la proliferación del receptor 157 87 128 124,0 35,17 28,4
- Control de autoestimulación (células autólogas tratadas con mitomicina C)
- 293 138 508 313,0 185,81 59,4
- Donante alogénico de MLR IM03-7768 (tratadas con mitomicina C)
- 24497 34348 31388 30077,7 5054,53 16,8
- MLR con línea celular (célula tipo B tratada con mitomicina C)
- 601 643 a 622,0 29,70 4,8
- SI (donante)
- 243
- SI (línea celular)
- 5
- IM03-7772
- Medida inicial de la proliferación del receptor 56 98 51 68,3 25,81 37,8
- Control de autoestimulación (células autólogas tratadas con mitomicina C)
- 133 120 213 155,3 50,36 32,4
- Donante alogénico de MLR IM03-7768 (tratadas con mitomicina C)
- 14222 20076 22168 18822,0 4118,75 21,9
- MLR con línea celular (célula tipo B tratada con mitomicina C)
- a a a a a a
- SI (donante)
- 275
- SI (línea celular)
- a
- IM03-7768 (donante alogénico)
- Medida inicial de la proliferación del receptor 84 242 208 178,0 83,16 46,7
- Control de autoestimulación (células autólogas tratadas con mitomicina)
- 361 617 304 427,3 166,71 39,0
- Línea celular tipo B
- Medida inicial de la proliferación del receptor 126 124 143 131,0 10,44 8,0
- Control de autoestimulación (células autólogas tratadas con mitomicina)
- 822 1075 487 794,7 294,95 37,1
- ID de placa: Placa 2
- Número del análisis
- Sistema de cultivo Replicaciones
- 1
- 2 3 Media DE CV
- IM03-7773
- Medida inicial de la proliferación del receptor 908 181 330 473,0 384,02 81,2
- Control de autoestimulación (células autólogas tratadas con mitomicina C)
- 269 405 572 415,3 151,76 36,5
- Donante alogénico de MLR IM03-7768 (tratadas con mitomicina C)
- 29151 28691 28315 28719,0 418,70 1,5
- MLR con línea celular (célula tipo B tratada con mitomicina C)
- 567 732 905 734,7 169,02 23,0
- SI (donante)
- 61
- SI (línea celular)
- 2
61
- IM03-7774
- Medida inicial de la proliferación del receptor 893 1376 185 818,0 599,03 73,2
- Control de autoestimulación (células autólogas tratadas con mitomicina C)
- 261 381 568 403,3 154,71 38,4
- Donante alogénico de MLR IM03-7768 (tratadas con mitomicina C)
- 53101 42839 4828 3 48074,3 5134,18 10,7
- MLR con línea celular (célula tipo B tratada con mitomicina C)
- 515 789 294 532,7 247,97 46,6
- SI (donante)
- 59
- SI (línea celular)
- 1
- IM03-7775
- Medida inicial de la proliferación del receptor 1272 300 544 705,3 505,69 71,7
- Control de autoestimulación (células autólogas tratadas con mitomicina C)
- 232 199 484 305,0 155,89 51,1
- Donante alogénico de MLR IM03-7768 (tratadas con mitomicina C)
- 23554 10523 2896 5 21014,0 9479,74 45,1
- MLR con línea celular (célula tipo B tratada con mitomicina C)
- 768 924 563 751,7 181,05 24,1
- SI (donante)
- 30
- SI (línea celular)
- 1
- IM03-7776
- Medida inicial de la proliferación del receptor 1530 137 1046 904,3 707,22 78,2
- Control de autoestimulación (células autólogas tratadas con mitomicina C)
- 420 218 394 344,0 109,89 31,9
Tabla 11-3. Índice medio de estimulación de células de la placenta y un donante alogénico en una reacción linfocitaria mixta con seis receptores alogénicos individuales.
- Índice medio de estimulación
- Receptor
- Placenta
- Placa 1 (receptores 1-3)
- 279 3
- Placa 2 (receptores 4-6)
- 46,25 1,3
Reacción de linfocitos mixtos -Cordón umbilical. Se cribaron seis donantes de sangre voluntarios humanos para identificar un único donante alógeno que presentaría una respuesta a la proliferación robusta en una reacción de linfocitos mixtos con los otro cinco donantes de sangre. Este donante se seleccionó como el donante de control positivo alógeno. Los cinco donantes de sangre restantes se seleccionaron como receptores. El donante de control positivo alógeno y las líneas celulares derivadas del cordón umbilical se trataron con mitomicina C y se cultivaron en una reacción de linfocitos mixtos con los cinco receptores alógenos individuales. Las reacciones se realizaron por triplicado usando dos placas de cultivo celular con tres receptores por placa (Tabla 11-4). El índice de estimulación promedio osciló de 6,5 (placa 1) a 9 (placa 2) y los controles positivos de donante alógenos oscilaron de 42,75 (placa 1) a 70 (placa 2) (Tabla 11-5).
62 63 64
Tabla 11-4. Datos de la reacción linfocitaria mixta -Línea Celular A (cordón umbilical)
- DMP para el ensayo de proliferación ID de placa: Placa 1
- Número del análisis
- Sistema de cultivo Replicaciones 1 2 3 Media DE CV
- Medida inicial de la
- proliferación del receptor Control de autoestimulación
- 1074 406 391 623,7 390,07 62,5
- (células autólogas tratadas
- 672 510 1402 861,3 475,19 55,2
- IM04-2478
- con mitomicina C) Donante alogénico de
- MLR IM04-2477 (tratadas con mitomicina C) MLR con línea celular
- 43777 48391 38231 43466,3 5087,12 11,7
- (célula tipo A tratada con mitomicina C)
- 2914 5622 6109 4881,7 1721,36 35,3
- SI (donante) SI (línea celular)
- 70 8
- Medida inicial de la
- proliferación del receptor Control de autoestimulación
- 530 508 527 521,7 11,93 2,3
- (células autólogas tratadas
- 701 567 1111 793,0 283,43 35,7
- IM04-2479
- con mitomicina C) Donante alogénico de
- MLR IM04-2477 (tratadas con mitomicina C) MLR con línea celular
- 25593 24732 22707 24344,0 1481,61 6,1
- (célula tipo A tratada con mitomicina C)
- 5086 3932 1497 3505,0 1832,21 52,3
- SI (donante) SI (línea celular)
- 47 7
- Medida inicial de la
- proliferación del receptor Control de autoestimulación
- 1192 854 1330 1125,3 244,90 21,8
- (células autólogas tratadas
- 2963 993 2197 2051,0 993,08 48,4
- IM04-2480
- con mitomicina C) Donante alogénico de
- MLR IM04-2477 (tratadas con mitomicina C) MLR con línea celular
- 25416 29721 23757 26298,0 3078,27 11,7
- (célula tipo A tratada con mitomicina C)
- 2596 5076 3426 3699,3 1262,39 34,1
- SI (donante) SI (línea celular)
- 23 3
- Medida inicial de la
- proliferación del receptor Control de autoestimulación
- 695 451 555 567,0 122,44 21,6
- (células autólogas tratadas
- 738 1252 464 818,0 400,04 48,9
- IM04-2481
- con mitomicina C) Donante alogénico de
- MLR IM04-2477 (tratadas con mitomicina C) MLR con línea celular
- 13177 24885 15444 17835,3 6209,52 34,8
- (célula tipo A tratada con mitomicina C)
- 4495 3671 4674 4280,0 534,95 12,5
- SI (donante) SI (línea celular)
- 31 8
- ID de placa: Placa 2
- Número del análisis
- Sistema de cultivo Replicaciones 1 2 3 Media DE CV
- Medida inicial de la
- proliferación del receptor Control de autoestimulación
- 432 533 274 413,0 130,54 31,6
- (células autólogas tratadas
- 1459 633 598 896,7 487,31 54,3
- IM04-2482
- con mitomicina C) Donante alogénico de
- MLR IM04-2477 (tratadas con mitomicina C) MLR con línea celular
- 24286 30823 31346 28818,3 3933,82 13,7
- (célula tipo A tratada con mitomicina C)
- 2762 1502 6723 3662,3 2724,46 74,4
- SI (donante) SI (línea celular)
- 70 9
- IM04-2477 (donante alogénico)
- Medida inicial de la proliferación del receptor Control de autoestimulación (células autólogas tratadas con mitomicina) 312 419 349 567 604 374 360,0 515,0 54,34 123,50 15,1 24,0
- Medida inicial de la
- Línea celular tipo A
- proliferación del receptor Control de autoestimulación (células autólogas tratadas con mitomicina) 5101 3735 2973 1924 4570 2153 3936,3 2882,3 1078,19 1466,04 27,4 50,9
Tabla 11-5. Índice medio de estimulación de células derivadas de cordón umbilical y un donante alogénico en una reacción linfocitaria mixta con cinco receptores alogénicos individuales.
- Índice medio de estimulación
- Receptor
- Cordón umbilical
- Placa 1 (receptores 1-4)
- 42,75 6,5
- Placa 2 (receptor 5)
- 70 9
Tabla 12-3. Resultados del ensayo ELISA multiplexado SearchLight
- MIP1a
- MIP1b MCP1 RANTES I309 TARC Eotaxina MDC IL8
- hFB
- ND ND 39,6 ND ND 0,1 ND ND 204,9
- P1
- 79,5 ND 228,4 4,1 ND 3,8 12,2 ND 413,5
- U1
- ND 8,0 1694,2 ND 22,4 37,6 ND 18,9 51930,1
- P3
- ND ND 102,7 ND ND 0,4 ND ND 63,8
- U3
- ND 5,2 2018,7 41,5 11,6 21,4 ND 4,8 10515,9
- Leyenda: hFB (fibroblastos humanos), P1 (PPDC derivada de la placenta (042303)), U1 (PPDC derivada de cordón umbilical (022803)), P3 (PPDC derivada de la placenta (071003)), U3 (PPDC derivada de cordón umbilical (071003)). ND: No detectado.
Resumen. Células del cordón umbilical secretaron cantidad significativamente mayor de factores tróficos que las células derivadas de la placenta y fibroblastos. Algunos de estos factores tróficos, tales como HGF, bFGF, MCP-1 y IL-8, desempeñan funciones importantes en la angiogénesis. Otros factores tróficos, tales como BDNF y IL6, tienen funciones importantes en la regeneración neural. Bajo estas condiciones, la expresión de algunos factores se confinó a células derivadas del cordón umbilical, tales como MIP1b, Rantes, I309 y FGF.
Le Belle JE, Svendsen CN.(2002) Stem cells for neurodegenerative disorders: where can we go from here? BioDrugs. 16;389-401 Rosen EM, Lamszus K, Laterra J, Polverini PJ, Rubin JS, Goldberg ID. (1997) HGF/SF in angiogenesis. Ciba Found Symp. 212;215-26. Salcedo R, Ponce ML, Young HA, Wasserman K, Ward JM, Kleinman HK, Oppenheim JJ, Murphy WJ. (2000) Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression. Blood. 96;34-40. Li A, Dubey S, Varney ML, Dave BJ, Singh RK (2003) IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol. 170;3369-76 Hughes GC, Biswas SS, Yin B, Coleman RE, DeGrado TR, Landolfo CK, Lowe JE, Annex BH, Landolfo KP. (2004) Therapeutic angiogenesis in chronically ischemic porcine myocardium: comparative effects of bFGF and VEGF. Ann Thorac Surg. 77;812-8. Cheng A, Wang S, Cai J, Rao MS, Mattson MP (2003) Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain. Dev Biol.258;319-33. Sebire G, Emilie D, Wallon C, Hery C, Devergne O, Delfraissy JF, Galanaud P, Tardieu M. (1993) In vitro production of IL-6, IL-1 beta, and tumor necrosis factor-alpha by human embryonic microglial and neural cells. J Immunol. 150;1517-23.
EJEMPLO 13:
Ensayo de coagulación del plasma
La terapia de células puede inyectarse sistémicamente para ciertas aplicaciones en las que las células pueden dirigirse al sitio de acción. Es importante que las células inyectadas no produzcan trombosis, que puede ser letal. El factor de tejido, una glucoproteína procoagulante unida a la membrana, es el iniciador de la cascada de coagulación extrínseca, que es la vía de coagulación predominante in vivo. El factor de tejido también desempeña una función importante en la formación de vasos embrionarios, por ejemplo, en la formación de la pared vascular primitiva (Brodsky y col. (2002) Exp. Nephrol. 10:299-306). Para determinar las posibilidades de las PPDC para iniciar la coagulación, PPDC derivadas del cordón umbilical y de la placenta se evaluaron para la expresión del factor de tejido y su capacidad para iniciar la coagulación del plasma.
67
Tabla 13-1. Se evaluó el efecto del factor tisular humano (SIMPLASTIN), las células derivadas de la placenta (Pla) y las células derivadas de cordón umbilical (Umb) sobre la coagulación de plasma. Como unidad de medida se utilizó el tiempo hasta la mitad de absorbancia máxima (T ½ a máx) en la meseta en segundos.
- Dilución de Simplastin®
- T ½ a máx (segundos)
- 1:2
- 61
- 1:4
- 107
- 1:8
- 147
- 1:16
- 174
- 1:32
- 266
- 1:64
- 317
- 1:128
- 378
- 0 (control negativo)
- 1188
- Células J-82
- 100.000
- 122
- 50.000
- 172
- 25.000
- 275
- Pla P5
- 50.000
- 757
- Umb P5
- 50.000
- 833
- Umb P18
- 50.000
- 443
Resumen. PPDC derivadas de la placenta y del cordón umbilical expresan factor de tejido, que puede inducir la coagulación. La adición de un anticuerpo a factor de tejido puede inhibir el factor de tejido. El factor de tejido normalmente se encuentra en células en una conformación que es inactiva, pero se activa por tensión mecánica o química (por ejemplo, LPS) (Sakariassen y col. (2001) Tromb. Res. 104:149-74; Engstad y col. (2002) Int. Immunopharmacol. 2:1585-97). Así, la minimización de la tensión durante el proceso de preparación de las PPDC puede prevenir la activación del factor de tejido. Además de la actividad trombogénica, el factor de tejido se ha asociado a actividad angiogénica. Así, la actividad del factor de tejido puede ser beneficiosa cuando las PPDC derivadas del cordón umbilical o de la placenta se trasplantan en tejido, pero deben inhibirse cuando las PPDC se inyectan intravenosamente.
Doshi and Marmur, Critical Care Med., 30:S241-S250 (2002)
Moll and Ortel, Ann. Intern. Med., 127:177-185 (1997)
EJEMPLO 14
Ensayo de formación de redes endoteliales
La angiogénesis, o formación de nueva vasculatura, es necesaria para el crecimiento de tejido nuevo. La inducción de la angiogénesis es un objetivo terapéutico importante en muchas afecciones patológicas. El presente estudio pretendió identificar la posible actividad angiogénica de las células derivadas del posparto en ensayos in vitro. El estudio siguió un método bien establecido de sembrar células endoteliales sobre una placa de cultivo recubierta con MATRIGEL (BD Discovery Labware, Bedford, MA), un extracto de la membrana basal (Nicosia y Ottinetti (1990) In Vitro Cell Dev. Biol. 26(2): 119-28). El tratar células endoteliales sobre MATRIGEL (BD Discovery Labware, Bedford, MA) con factores angiogénicos estimulará las células para formar una red que es similar a capilares. Esto es un ensayo in vitro común para probar estimulantes e inhibidores de la formación de vasos sanguíneos (Ito y col. (1996) Int. J. Cancer 67(1):148-52). Los presentes estudios hicieron uso de un sistema de cocultivo con las células derivadas del posparto sembradas sobre insertos de pocillos de cultivo. Estos insertos permeables permiten el intercambio pasivo de componentes del medio entre los medios de células de cultivo endoteliales y derivadas del posparto.
Material y métodos
Cultivo celular.
69
congeló inmediatamente y se guardó a -80 ºC, y se analizó por el ensayo de ELISA de múltiplex SearchLight (Pierce Chemical Company, Rockford, IL). Los resultados mostrados son los promedios de mediciones por duplicado. Los resultados muestran que las células derivadas del posparto no liberan niveles detectables de factor de crecimiento derivado de plaquetas-bb (PDGF-bb) o factor de crecimiento epidérmico de unión a la heparina (HBEGF). Las células liberan cantidades medibles de inhibidor de tejido de metaloproteasa-1 (TIMP-1), angiopoyetina 2 (ANG2), trombopoyetina (TPO), factor de crecimiento de queratinocitos (KGF), factor de crecimiento de hepatocitos (HGF), factor de crecimiento de fibroblastos (FGF) y factor de crecimiento endotelial vascular (VEGF).
Tabla 14-1: Resultados de la diferenciación osteogénica mediante tinción de von Kossa para el Protocolo 2. Se cultivaron células derivadas de cordón umbilical (Umb), células derivadas de la placenta (Pla), citoblastos mesenquimatosos (MSC), fibroblastos (Fib) y células de médula ósea de cresta ilíaca (ICBM) en medio osteogénico (OM) solo o complementado con BMP2 o BMP2 y BMP4.
- Número
- Línea celular Condiciones von Kossa Cometarios
- 1
- Umb 071003 O1P3 Medio osteogénico (OM) Neg
- 2
- Umb 071003 O1P3 OM, BMP2 Neg
- 3
- Umb 071003 O1P3 OM, BMP4 Neg
- 4
- ICBM 070203 O1P3 Medio osteogénico (OM) Neg Normal 02
- 5
- ICBM 070203 O1P3 OM, BMP2 Pos Normal 03
- 6
- ICBM 070203 O1P3 OM, BMP4 Pos Normal 04
- 7
- MSC 092903 Medio osteogénico (OM) Neg mucha grasa
- 8
- MSC 092903 OM, BMP2 Neg mucha grasa
- 9
- MSC 092903 OM, BMP4 Pos mucha grasa
- 10
- Pla 101603 O1P4 Medio osteogénico (OM) Neg
- 11
- Pla 101603 O1P4 OM, BMP2 Pos
- 12
- Pla 101603 O1P4 OM, BMP4 Pos
- 13
- MSC 012104 O1P4 Medio osteogénico (OM) Neg Grasa
- 14
- MSC 012104 O1P4 OM, BMP2 Neg Grasa
- 15
- MSC 012104 O1P4 OM, BMP2, BMP4 Neg Grasa
- 16
- Umb 022803 O1P4 Medio osteogénico (OM) Neg
- 17
- Umb 022803 O1P4 OM, BMP2 Neg
- 18
- Umb 022803 O1P4 OM, BMP2, BMP4 Neg
- 19
- Pla 100703 O1P4 Medio osteogénico (OM) Neg
- 20
- Pla 100703 O1P4 OM, BMP2 Neg
- 21
- Pla 100703 O1P4 OM, BMP2, BMP4 Neg
- 22
- Fib 1F1853 O1P11 Medio osteogénico (OM) Neg
- 23
- Fib 1F1853 O1P11 OM, BMP2 Neg
- 24
- Fib 1F1853 O1P11 OM, BMP2, BMP4 Neg
La Tabla 14-2 muestra niveles de factores angiogénicos conocidos liberados por las células derivadas del posparto. Las células derivadas del posparto se sembraron sobre insertos como se ha descrito anteriormente. Las células se cultivaron en medio de crecimiento a 5 % de oxígeno durante 48 horas sobre los insertos y a continuación se cambiaron a 2 % de medio FBS y volvieron a incubación con 5 % de O2 durante 24 horas. Se eliminó el medio, se congeló inmediatamente y se guardó a -80 ºC, y se analizó por el ensayo de ELISA múltiplex SearchLight (Pierce Chemical Company, Rockford, IL). Los resultados mostrados son los promedios de mediciones por duplicado. Los resultados muestran que las células derivadas del posparto no liberan niveles detectables de factor de crecimiento derivado de plaquetas-bb (PDGF-bb) o factor de crecimiento epidérmico de unión a la heparina (HBEGF). Las células liberan cantidades medibles de inhibidor de tejido de metaloproteasa-1 (TIMP-1), angiopoyetina 2 (ANG2), trombopoyetina (TPO), factor de crecimiento de queratinocitos (KGF), factor de crecimiento de hepatocitos (HGF), factor de crecimiento de fibroblastos (FGF) y factor de crecimiento endotelial vascular (VEGF).
Tabla 14-2. Posibles factores angiogénicos liberados de células derivadas del posparto. Células derivadas del posparto se cultivaron 24 horas en medio con 2 % de FBS en 5 % de oxígeno. Se eliminó el medio y se ensayó por el ensayo de ELISA múltiplex SearchLight (Pierce). Los resultados son las medias de un análisis por duplicado. Los valores son concentraciones en el medio informadas en picogramos por mililitro de medio de cultivo.
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ES04756395.2T Active ES2564044T3 (es) | 2003-06-27 | 2004-06-25 | Células posparto derivadas de tejido del cordón umbilical y métodos de preparación y uso de las mismas |
ES10184593.1T Active ES2542070T3 (es) | 2003-06-27 | 2004-06-25 | Células posparto derivadas de tejido del cordón umbilical y métodos de preparación y uso de las mismas para la reparación y regeneración de tejido blando |
ES10183911.6T Active ES2552226T3 (es) | 2003-06-27 | 2004-06-25 | Reparación y regeneración de cartílago y hueso utilizando células derivadas posparto |
ES10183053.7T Active ES2550267T3 (es) | 2003-06-27 | 2004-06-25 | Células derivadas de cordón umbilical post-parto para su uso en el tratamiento de enfermedad del corazón y el sistema circulatorio |
ES10184137.7T Active ES2542069T3 (es) | 2003-06-27 | 2004-06-25 | Regeneración y reparación de tejido neural usando células del postparto derivadas del cordón umbilical |
ES10184221.9T Active ES2541604T3 (es) | 2003-06-27 | 2004-06-25 | Reparación y regeneración de tejido ocular usando células derivadas del cordón umbilical post parto |
ES04777231.4T Active ES2597837T3 (es) | 2003-06-27 | 2004-06-25 | Células posparto derivadas de tejido de la placenta, y métodos de fabricación y utilización de los mismos |
ES04777287.6T Active ES2564045T3 (es) | 2003-06-27 | 2004-06-25 | Células derivadas del post-parto para su uso en el tratamiento de enfermedad del corazón y el sistema circulatorio |
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ES04809466.8T Active ES2565582T3 (es) | 2003-06-27 | 2004-06-25 | Reparación y regeneración de cartílago y hueso utilizando células derivadas posparto |
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- 2016-11-28 US US15/362,453 patent/US10039793B2/en active Active
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2017
- 2017-02-27 US US15/443,718 patent/US10195233B2/en active Active
- 2017-02-27 US US15/443,602 patent/US10220059B2/en active Active
- 2017-02-27 US US15/443,733 patent/US10383898B2/en not_active Expired - Fee Related
- 2017-07-27 US US15/661,878 patent/US10500234B2/en not_active Expired - Fee Related
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2018
- 2018-08-06 US US16/056,146 patent/US10758576B2/en active Active
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2019
- 2019-03-04 US US16/291,784 patent/US11000554B2/en active Active
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