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ES2552226T3 - Reparación y regeneración de cartílago y hueso utilizando células derivadas posparto - Google Patents

Reparación y regeneración de cartílago y hueso utilizando células derivadas posparto Download PDF

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Publication number
ES2552226T3
ES2552226T3 ES10183911.6T ES10183911T ES2552226T3 ES 2552226 T3 ES2552226 T3 ES 2552226T3 ES 10183911 T ES10183911 T ES 10183911T ES 2552226 T3 ES2552226 T3 ES 2552226T3
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Spain
Prior art keywords
cells
mitomycin
bone
treated
protein
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Expired - Lifetime
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ES10183911.6T
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English (en)
Inventor
Anthony J. Kihm
Agnieszka Seyda
Sridevi Dhanaraj
Ziwei Wang
Alexander M. Harmon
Ian Ross Harris
Darin J. Messina
Sanjay Mistry
Chin-Feng Yi
Anna Gosiewska
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DePuy Synthes Products Inc
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DePuy Synthes Products Inc
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Publication of ES2552226T3 publication Critical patent/ES2552226T3/es
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Abstract

Población homogénea aislada de células derivadas de tejido de cordón umbilical humano, para su uso en el tratamiento de una afección ósea o cartilaginosa, en la que dicha población celular puede obtenerse mediante digestión enzimática de tejido de cordón umbilical humano exento de sangre con una metaloproteasa de matriz, una proteasa neutra y una enzima mucolítica, y en la que dicha población celular es capaz de autorrenovación y de proliferación en cultivo, tiene el potencial de diferenciarse a células de otros fenotipos, y en la que la población de células: a) produce cada uno de CD10, CD13, CD44, CD73, CD90, PDGFr-alfa y HLA-A,B,C, según se detecta mediante citometría de flujo; y b) no produce ninguno de CD31, CD34, CD45, CD117, CD141 o HLA-DR,DP,DQ, según se detecta mediante citometría de flujo.

Description

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reintroducen los factores de crecimiento, y, posteriormente, llevar a cabo el mismo número de duplicaciones que las células equivalentes que se han desarrollado continuamente. Asimismo, cuando las células se congelan en nitrógeno líquido después de diversos números de duplicaciones de la población y, a continuación, se descongelan y cultivan, experimentan sustancialmente el mismo número de duplicaciones que las células mantenidas no congeladas en cultivo. Las células senescentes no son células muertas o moribundas; en realidad son resistentes a la muerte celular programada (apoptosis), y se han mantenido en su estado de no división durante hasta tres años. Estas células están realmente vivas y son metabólicamente activas, pero no se dividen. Aún no se ha descubierto que el estado de no división de las células senescentes sea reversible mediante ningún agente biológico, químico o el viral.
Tal como se utiliza en el presente documento, la expresión Medio de crecimiento se refiere a un medio de cultivo suficiente para que proliferen las células derivadas posparto. El medio de cultivo del Medio de crecimiento contiene preferentemente medio esencial modificado de Dulbecco (DMEM). Más preferentemente, el Medio de crecimiento contiene glucosa. El Medio de crecimiento contiene preferentemente DMEM con bajo contenido de glucosa (DMEM-LG) (Invitrogen, Carlsbad, CA). El Medio de crecimiento contiene preferentemente suero a aproximadamente el 15% (v/v) (por ejemplo, suero bovino fetal, suero bovino definido). El Medio de crecimiento contiene preferentemente al menos un antibiótico y/o un antimicótico (por ejemplo, penicilina, estreptomicina, anfotericina B, gentamicina, nistatina, preferentemente, 50 unidades/mililitro de penicilina G sódica 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 con bajo contenido de glucosa, suero, 2-mercaptoetanol y un antibiótico.
Tal como se utiliza en el presente documento, condiciones de crecimiento normales se refiere a las condiciones atmosféricas normales que comprenden CO2 a aproximadamente el 5%, una temperatura de aproximadamente 35ºC-39ºC, más preferentemente 37ºC, y una humedad relativa de aproximadamente el 100%.
El término aislado se refiere a una célula, a un componente celular o a una molécula que se ha sacado de su entorno original.
El término aproximadamente se refiere a una aproximación de un valor indicado dentro de un intervalo de ±10%.
Afección (o lesión o enfermedad) ósea es una expresión incluyente que abarca afecciones, trastornos o enfermedades óseas agudas y crónicas, y metabólicas y no metabólicas. La expresión abarca las afecciones debidas a enfermedad o traumatismo o a la incapacidad del tejido para desarrollarse normalmente. Los ejemplos de afecciones óseas incluyen, pero no se limitan a, defectos óseos congénitos, fracturas óseas, lesiones o defectos del menisco, deformación ósea/vertebral, osteosarcoma, mieloma, displasia ósea y escoliosis, osteoporosis, enfermedad periodontal, pérdida ósea dental, osteomalacia, raquitismo, osteítis fibrosa, osteodistrofia renal, fusión vertebral, reconstrucción o extracción de disco vertebral, y la enfermedad ósea de Paget.
Afección (o lesión o enfermedad) cartilaginosa es una expresión incluyente que abarca afecciones, trastornos o enfermedades, agudas y crónicas, del cartílago. La expresión abarca afecciones que incluyen, pero no se limitan a, defectos congénitos, lesiones de menisco, artritis reumatoide, osteoartritis, o una lesión traumática o quirúrgica en el cartílago.
La expresión tratar (o tratamiento de) una afección ósea o cartilaginosa se refiere a mejorar los efectos de,
o retrasar, detener o revertir la evolución de, o retrasar o prevenir la aparición de, una afección ósea o cartilaginosa tal como se define en el presente documento.
La expresión cantidad eficaz se refiere a una concentración de un reactivo o composición farmacéutica, tal como un factor de crecimiento, un agente de diferenciación, un factor trófico, una población celular u otro agente, que es eficaz para producir un resultado deseado, incluido el crecimiento celular y/o la diferenciación in vitro o in vivo, o el tratamiento de una afección ósea o cartilaginosa tal como se describe en el presente documento. Con respecto a los factores de crecimiento, una cantidad eficaz puede variar de aproximadamente 1 nanogramo/mililitro a aproximadamente 1 microgramo/mililitro. Con respecto a las PPDC como se administran a un paciente in vivo, una cantidad eficaz puede variar de tan solo varios cientos o menos, hasta varios millones o más. En formas de realización específicas, una cantidad eficaz puede variar de 103-1011 . Se entenderá que el número de células a administrar variará dependiendo de las características específicas del trastorno a tratar, incluidas, pero no limitadas al tamaño o volumen/superficie total a tratar, así como la proximidad del sitio de administración a la situación de la zona a tratar, entre otros factores familiares para el biólogo médico.
Las expresiones período (o tiempo) eficaz y condiciones eficaces se refieren a un período de tiempo o de otras condiciones controlables (por ejemplo, temperatura, humedad para los métodos in vitro), necesarias o preferentes para que un agente o composición farmacéutica consiga su resultado previsto.
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IMAGE:4179671, proteína hipotética DKFZp564F013, regulada por disminución en el cáncer de ovario 1 y clon DKFZp547K1113. En las PPDC en las que la expresión, con respecto a una célula humana que es un fibroblasto, una célula madre mesenquimal o una célula de médula ósea de cresta ilíaca, está aumentada para al menos uno de miembro A2 de la superfamilia de lectinas tipo C, tumor de Wilms 1, miembro A2 de la familia de aldehído deshidrogenasa 1, renina, receptor 1 de lipoproteína de baja densidad oxidada, proteína quinasa C zeta, clon IMAGE:4179671, proteína hipotética DKFZp564F013, regulada por disminución en el cáncer de ovario 1 y clon DKFZp547K1113, preferentemente no hay niveles relativos aumentados de al menos uno de interleucina 8; reticulón 1; ligando 1 de quimiocina (motivo C-X-C) (actividad estimuladora del crecimiento de melanoma, alfa); ligando 6 de quimiocina (motivo C-X-C) (proteína 2 quimiotáctica para granulocitos); ligando 3 de quimiocina (motivo C-X-C); y factor de necrosis tumoral, proteína 3 inducida por alfa.
Las PPDC pueden tener expresión que, con respecto a una célula humana que es un fibroblasto, una célula madre mesenquimal o una célula de médula ósea de cresta ilíaca, está reducida para al menos uno de: 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 y rico en cisteínas; gen 1 de translocación de linfocitos B, antiproliferativa; colesterol 25hidroxilasa; 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; el experto en la materia entenderá que la expresión de una gran diversidad de genes se caracteriza convenientemente en una matriz génica, por ejemplo en un GENECHIP de Affymetrix.
Las PPDC pueden secretar diversos factores bioquímicamente activos, tales como factores de crecimiento, quimiocinas, citocinas y similares. Las células preferentes de la divulgación secretan al menos uno de MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES y TIMP1. Las PPDC pueden caracterizarse por su ausencia de secreción de al menos uno de TGF-beta2, ANG2, PDGF-bb, MIP1b, I309, MDC y VEGF, como se detecta mediante ELISA. Se dispone de estas y otras características para identificar y caracterizar las células, y distinguir las células de la invención de otras conocidas en la técnica.
En las formas de realización preferentes, la célula comprende dos o más de las características anteriormente indicadas. Resultan más preferentes las células que comprenden, tres, cuatro o cinco o más de las características. Resultan aún más preferentes las células derivadas posparto que comprenden seis, siete u ocho o más de las características. Actualmente resultan aún más preferentes las células que comprenden las nueve características que se reivindican.
También resultan preferentes actualmente células que producen al menos dos de entre GCP-2, NOGO-A, factor tisular, vimentina y alfa actina de músculo liso. Resultan más preferentes las células que producen tres, cuatro
o cinco de estas proteínas.
El experto en la materia entenderá que los marcadores celulares están sujetos cierta variación en condiciones de crecimiento muy diferentes, y que en general en el presente documento se describen caracterizaciones en Medio de Crecimiento, o variaciones del mismo. Las células derivadas posparto que producen al menos uno, dos, tres o cuatro de entre CD10, CD13, CD44, CD73, CD90, PDGFr-alfa, PD-L2 y HLA-A,B,C son células preferentes de la divulgación. Resultan más preferentes las células que producen cinco, seis, o siete de estos marcadores de superficie celular. Resultan aún más preferentes las células derivadas posparto que pueden producir ocho, nueve o diez de las proteínas marcadoras de superficie celular anteriormente indicadas.
Las PPDC que no producen 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, según se detecta mediante citometría de flujo, son células preferentes de la divulgación. Resultan preferentes las PPDC que no producen al menos cinco, seis, siete u ocho o más de estos marcadores. Resultan más preferentes las células que no producen al menos nueve o diez de
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que necesite tratamiento, por ejemplo, para una afección ósea o cartilaginosa tal como se describe en el presente documento. Como alternativa, en situaciones en las que sea apropiada o deseable la transferencia autóloga, las poblaciones celulares cocultivadas pueden separarse físicamente en cultivo, lo que permite extraer las células autólogas para administrarlas al paciente.
Medio acondicionado de PPDC
Otra forma de realización de la divulgación se ocupa del uso de las PPDC para la producción de un medio acondicionado, ya sea de PPDC indiferenciadas o de PPDC incubadas en condiciones que estimulan la diferenciación a un linaje osteogénico o condrogénico. Tales medios acondicionados se contemplan para su uso en el cultivo de células in vitro o ex vivo, por ejemplo, células madre o progenitoras, incluidas, pero no limitadas a, células de médula ósea, osteoblastos, osteocitos, osteoclastos, células de revestimiento óseo, condroblastos y condrocitos, o in vivo para sustentar a células trasplantadas que comprenden poblaciones homogéneas o heterogéneas de PPDC, osteocitos, osteoblastos, osteoclastos, células de revestimiento óseo, condrocitos, condroblastos y células de médula ósea, por ejemplo.
Aplicaciones terapéuticas de las PPDC
Las PPDC de la invención pueden utilizarse para tratar a pacientes que requieren la reparación o sustitución de tejido óseo o cartilaginoso resultado de una enfermedad o traumatismo o de la incapacidad del tejido para desarrollarse normalmente, o para proporcionar una función cosmética, tal como para aumentar los rasgos faciales u otros rasgos corporales. El tratamiento puede implicar el uso de las células de la invención para producir nuevo tejido cartilaginoso o tejido óseo. Por ejemplo, las células inducidas a diferenciarse condrogénicas o indiferenciadas de la invención pueden utilizarse para tratar una afección cartilaginosa, por ejemplo, la artritis reumatoide o la osteoartritis o una lesión traumática o quirúrgica del cartílago. Como otro ejemplo, las células inducidas a diferenciarse osteogénicas o indiferenciadas de la invención pueden utilizarse para tratar afecciones óseas, incluidas enfermedades óseas metabólicas y no metabólicas. Los ejemplos de afecciones óseas incluyen desgarros meniscales, fusión vertebral, extracción de disco vertebral, reconstrucción vertebral, fracturas óseas, deformación ósea/vertebral, osteosarcoma, mieloma, displasia ósea, escoliosis, osteoporosis, enfermedad periodontal, pérdida de hueso dental, osteomalacia, raquitismo, osteítis fibrosa, osteodistrofia renal y enfermedad ósea de Paget.
Las células de la invención pueden administrarse en solitario o en forma de mezclas con otras células. Las células que pueden administrarse junto con las PPDC incluyen, pero no se limitan a, otras células multipotentes o pluripotentes o condrocitos, condroblastos, osteocitos, osteoblastos, osteoclastos, células de revestimiento óseo o células de médula ósea. Las células de diferentes tipos pueden mezclarse con las PPDC inmediatamente o poco antes de la administración, o pueden cocultivarse juntas durante un período de tiempo antes de la administración.
Las PPDC pueden administrarse con otras moléculas biológicas o fármacos beneficiosos (factores de crecimiento, factores tróficos). Cuando las PPDC se administran con otros agentes, pueden administrarse juntas en una única composición farmacéutica o en composiciones farmacéuticas separadas, simultánea o secuencialmente con los demás agentes (ya sea antes o después de la administración de los demás agentes). Los factores bioactivos que pueden administrarse conjuntamente incluyen agentes antiapoptóticos (por ejemplo, EPO, mimeticuerpo de la EPO, TPO, IGF-I e IGF-II, HGF, inhibidores de caspasas); 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, TOLMENTIN, SUPROFEN); inmunosupresores/inmunomoduladores (por ejemplo, inhibidores de calcineurina, tales como ciclosporina, tacrolimus; inhibidores de mTOR (por ejemplo, SIROLIMUS, EVEROLIMUS); antiproliferativos (por ejemplo, azatioprina, micofenolato de mofetilo); corticosteroides (por ejemplo, prednisolona, hidrocortisona); anticuerpos tales como anticuerpos monoclonales contra IL-2Ralfa (por ejemplo, basiliximab, daclizumab), anticuerpos policlonales contra linfocitos T (por ejemplo, globulina anti-timocitos (ATG); globulina anti-linfocitos (ALG); anticuerpo monoclonal contra linfocitos T, OKT3)); antitrombogénicos (por ejemplo, heparina, derivados de heparina, uroquinasa, PPack (dextrofenilalanina prolina arginina clorometilcetona), compuestos antitrombina, antagonistas de receptores plaquetarios, anticuerpos antitrombina, anticuerpos contra receptores plaquetarios, aspirina, dipiridamol, protamina, hirudina, inhibidores de prostaglandinas e inhibidores plaquetarios); y antioxidantes (por ejemplo, probucol, vitamina A, ácido ascórbico, tocoferol, coenzima Q-10, glutatión, L-cisteína, N-acetilcisteína), así como anestésicos locales. Como otro ejemplo, las células pueden administrarse conjuntamente con un factor inhibidor de cicatrices como se describe en la patente de Estados Unidos Nº 5.827.735.
En una forma de realización, las PPDC se administran como células indiferenciadas, es decir, tal como se cultivan en el Medio de Crecimiento. Como alternativa, en una forma de realización de la divulgación, las PPDC pueden administrarse después de exponerlas en cultivo a condiciones que estimulan la diferenciación hacia un fenotipo deseado, por ejemplo, un fenotipo condrogénico u osteogénico.
Las células de la invención pueden implantarse quirúrgicamente, inyectarse, introducirse (por ejemplo, por medio de un catéter o jeringa), o administrarse de otra manera, directa o indirectamente, al sitio que necesita la
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5
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25
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45
55
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
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
48
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
Las Tablas 8-6, 8-7 y 8-8 muestran el aumento de la expresión de los genes en los fibroblastos humanos (Tabla 8-6), las células ICBM (Tabla 8-7) y las MSC (Tabla 8-8).
15
25
35

Tabla 8-6. Genes que demostraron tener una expresión aumentada en los fibroblastos en comparación con las demás líneas celulares ensayadas.
Genes aumentados en fibroblastos
Fosfatasa 2 de doble especificidad
Proteína KIAA0527
ADNc de Homo sapiens: FLJ23224 fis, clon ADSU02206
Dineína, citoplasmática, polipéptido intermedio 1
Anquirina 3, nódulo de Ranvier (anquirina G)
Inhibina, beta A (activina A, activina AB, polipéptido alfa)
Ectonucleótido pirofosfatasa/fosfodiesterasa 4 (función supuesta)
Proteína KIAA1053
Proteína 1A asociada a microtúbulos
Proteína con dedos de zinc 41
Proteína HSPC019
ADNc de Homo sapiens: FLJ23564 fis, clon LNG10773
ARNm de Homo sapiens; ADNc DKFZp564A072 (del clon DKFZp564A072)
Proteína LIM (similar a enigma de unión a proteína quinasa C de rata)
Inhibidor del potenciador del gen del polipéptido de cadena ligera kappa en linfocitos B, proteína asociada al complejo quinasa
Proteína hipotética FLJ22004
Secuencia de ARNm humano (clon CTG-A4)
ESTs, Moderadamente similar al factor 2 similar al receptor de citocinas; precursor CRL2 del receptor de citocinas [Homo sapiens]
Factor de crecimiento transformante, beta 2
Proteína hipotética MGC29643
Antígeno identificado por el anticuerpo monoclonal MRC OX-2
Tabla 8-7. Genes que demostraron tener una expresión aumentada en las células derivadas de ICBM en comparación con las demás líneas celulares ensayadas.
45
55
Genes aumentados en células ICBM
Proteína de repetición de anquirina cardiaca
ORF de la región MHC de clase I
Integrina, alfa 10
Proteína hipotética FLJ22362
UDP-N-acetil-alfa-D-galactosamina:polipéptido N-acetilgalactosaminiltransferasa 3 (GalNAc-T3)
Proteína 44 inducida por interferón
SRY (región determinante del sexo del cromosoma Y)-caja 9 (displasia campomélica, reversión sexual autosómica)
Proteína asociada a queratina 1-1
Tipo hipocalcina 1
Jagged 1 (síndrome de Alagille)
Proteoglicano 1, gránulo de secreción

Tabla 8-8. Genes que demostraron tener una expresión aumentada en las células MSC en comparación con las demás líneas celulares ensayadas.
Genes aumentados en las células MSC
interleucina 26 maltasa-glucoamilasa (alfa-glucosidasa)
49
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alogénico de control positivo. Los seis donantes de sangre restantes se seleccionaron como receptores. El donante alogénico de control positivo y líneas celulares derivadas de la placenta se trataron con mitomicina C y se cultivaron en una reacción linfocitaria mixta con los seis receptores alogénicos individuales. Las reacciones se realizaron por triplicado utilizando dos placas de cultivo celular con tres receptores por placa (Tabla 11-2). El índice medio de estimulación varió entre 1,3 (placa 2) y 3 (placa 1) y los donantes alogénicos de control positivo variaron entre 46,25 (placa 2) y 279 (placa 1) (Tabla 11-3).
15
25
35
45
55

Tabla 11-2. Datos de la reacción linfocitaria mixta -Línea celular B (placenta)
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
57
15
25
35
45
55
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
58
5
15
25
35
45
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)
5310 1 4283 9 4828 3 48074, 3 5134,1 8 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)
2355 4 1052 3 2896 5 21014, 0 9479,7 4 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
Donante alogénico de MLR IM03-7768 (tratadas con mitomicina C)
2889 3 3249 3 3474 6 32044, 0 2952,2 2 9,2
MLR con línea celular (célula tipo B tratada con mitomicina C)
a a a a a a
SI (donante)
35
SI (línea celular)
a

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 linfocitaria mixta -cordón umbilical. Se sometieron a criba seis donantes de sangre voluntarios humanos para identificar un solo donante alogénico que presentara una fuerte respuesta de proliferación en una reacción linfocitaria mixta con los otros cinco donantes de sangre. Este donante se seleccionó como el donante alogénico de control positivo. Los cinco donantes de sangre restantes se seleccionaron como receptores. El donante alogénico de control positivo y las líneas celulares derivadas de cordón umbilical se trataron con mitomicina C y se cultivaron en una reacción linfocitaria mixta con los cinco receptores alogénicos individuales. Las reacciones
65 se realizaron por triplicado utilizando dos placas de cultivo celular con tres receptores por placa (Tabla 11-4). El
59
índice medio de estimulación varió entre 6,5 (placa 1) y 9 (placa 2) y los donantes alogénicos de control positivo variaron entre 42,75 (placa 1) y 70 (placa 2) (Tabla 11-5).
5
15
25
35
45
55

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
60
5
15
25
35
45
55
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
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ES10183911.6T 2003-06-27 2004-06-25 Reparación y regeneración de cartílago y hueso utilizando células derivadas posparto Expired - Lifetime ES2552226T3 (es)

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