ES2597837T3 - Células posparto derivadas de tejido de la placenta, y métodos de fabricación y utilización de los mismos - Google Patents
Células posparto derivadas de tejido de la placenta, y métodos de fabricación y utilización de los mismos Download PDFInfo
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- ES2597837T3 ES2597837T3 ES04777231.4T ES04777231T ES2597837T3 ES 2597837 T3 ES2597837 T3 ES 2597837T3 ES 04777231 T ES04777231 T ES 04777231T ES 2597837 T3 ES2597837 T3 ES 2597837T3
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- Orthopedic Medicine & Surgery (AREA)
Abstract
Un aislado de células derivadas de placenta obtenible a partir de tejido de la placenta después del parto humano sustancialmente libre de sangre, en el que dicha célula se auto-renueva y se expande en cultivo, es multipotente, requiere L-valina para el crecimiento, crece en oxígeno de aproximadamente 5% a aproximadamente 20%, y tiene las siguientes características adicionales: a) producción de vimentina y actina de músculo liso alfa; b) expresión de la proteína quimiotáctica de granulocitos-2 (GCP-2) como se detecta por citometría de flujo; c) falta de producción de GRO-alfa y receptor de lipoproteína de baja densidad oxidada, tal como se detecta por citometría de flujo; d) producción de CD10, CD13, CD44, CD73, CD90, PDGFr-alfa, PD-L2 y HLA-A, B, C, tal como se detecta por citometría de flujo; e) falta de producción de CD31, CD45, CD80, CD86, CD117, CD141, CD 178, B7-H2, HLA-G, y HLA-DP, DQ, DR, tal como se detecta por citometría de flujo; f) expresión, la cual es relativa a una célula humana que es un fibroblasto, una célula madre mesenquimal, o una célula de médula ósea de cresta ilíaca, se reduce para homeobox de baja estatura 2; proteína 27kDa de choque térmico 2; quimiocina (C-X-C con motivos) ligando 12 (estromal el factor 1 derivado de células); elastina; ADNc DKFZp586M2022 (a partir del clon DKFZp586M2022); homeobox de mesenquimio 2; homólogo de homeobox sine oculis 1; cristalina, alfa B; activador asociado desgreñado de la morfogénesis 2; proteína DKFZP586B2420; similar a neuralina 1; tetranectina; src homólogía tres (SH3) y el dominio rico en cisteína; gen de translocación de células B 1, anti-proliferativa; colesterol 25-hidroxilasa; factor de transcripción relacionado con runt-3; proteína hipotética FLJ23191; receptor de interleucina 11, alfa; procolágeno promotor de C-endopepti- dase; homólogo rizado 7; gen hipotético BC008967; colágeno, tipo VIII, alfa 1; tenascina C; homeobox de proteínas Iroquois 5; hefaestina; integrina, beta 8; glicoproteína vesícula sináptica 2; ADNc FLJ12280 fis, Clon de MAMMA1001744; citoquina del receptor del factor-1 como; potasio intermedio/pequeña conductancia de calcio canal activado, subfamilia N, miembro 4; integrina, alfa 7; proteína de DKFZP586L151; co-activador transcripcional con motivo de unión PDZ (TAZ); homólogo de homeobox sine oculis 2; proteína KIAA1034; respuesta de crecimiento temprano 3; homeobox no distal 5; proteína hipotética FLJ20373; familia de reductasa aldo-ceto 1, miembro de C3 (deshidrogenasa de hidroxiesteroide 3-alfa, tipo II); biglicano; fibronectina 1; proencefalina; integrina beta tipo 1 (con EGF igual que los dominios de repetición); clon de ADNc EUROIMAGE 1968422; EphA3; proteína KIAA0367; receptor del péptido natriurético C/guanilato ciclasa C (receptor de péptido atrionatriurético C); proteína hipotética FLJ14054; ADNc DKFZp564B222 (a partir de DKFZp564B222 clon); proteína de membrana asociada a vesículas 5; fibulina que contiene EGF como la proteína de la matriz extracelular 1; BCL2/adenovirus E1B 19kDa proteína de interacción tipo 3; proteína de unión AE 1; citocromo c oxidasa subunidad polipeptídica VIIa 1 (músculo); neuroblastoma, supresión de tumorigenicidad 1; y proteína de unión de factor de crecimiento similar a la insulina 2, 36 kDa como se ensayó por microensayo; g) secreción de la proteína quimiotáctica de monocitos 1 (MCP-1), interleucina-6 (IL-6), quimiotáctica de granulocitos en proteínas 2 (GCP-2), factor de crecimiento de hepatocitos (HGF), factor de crecimiento de queratinocitos (KGF), heparina de unión al factor de crecimiento epidérmico (HB-EGF), inhibidor tisular de la metaloproteinasa de matriz 1 (TIMP1), trombopoyetina (TPO), Rantes (reguladas en la activación, células T normales expresadas y secretadas) y el timo y quimiocina regulada por activación (TARC), como detectada por ELISA; h) falta de secreción de factor de crecimiento de fibroblastos (FGF), angiopoyetina 2 (ANG2), factor de crecimiento derivado de plaquetas (PDGF-BB), factor de crecimiento transformante beta 2 (TGFbeta2), proteína inflamatoria de macrófagos 1beta (MIP1b), 1309, y quimioquina derivada de macrófagos (MDC), como se detecta por ELISA; i) tiene la capacidad de someterse a al menos 40 duplicaciones de la población en cultivo; y j) la expresión, que en relación con una célula humana que es un fibroblasto, una célula madre mesenquimal, o una célula de médula ósea de cresta ilíaca, se incrementa para miembro de superfamilia de lectina de tipo C A2, tumor de Wilms 1, deshidrogenasa de aldehído miembro 1 de la familia A2, renina, receptor de lipoproteínas oxidado de baja densidad 1, quinasa de proteína C zeta, clon IM-EDAD: 4179671, proteína hipotética DKFZp564F013, subregulado en el cáncer de ovario 1, y el clon DKFZp547K1113 como se ensayó mediante microensayos.
Description
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reparación del tejido. En algunas realizaciones de la invención, la condición a ser tratada es una condición de los tejidos blandos (por ejemplo, piel, músculo, vasos, tendones, ligamentos, vejiga, fascia, suelo pélvico), hueso, páncreas, riñón, hígado, sistema nervioso, ojo, corazón, o cartílago.
[0038] Los métodos de la invención incluyen además métodos para producir una población de células de la placenta-derivados por expansión de una célula de la invención en cultivo.
[0039] Otras características y ventajas de la invención serán evidentes a partir de la descripción detallada y los ejemplos que siguen.
DESCRIPCIÓN DETALLADA DE LAS REALIZACIONES ILUSTRATIVAS
Definiciones
[0040] Varios términos utilizados en la especificación y reivindicaciones se definen como se indica a continuación.
[0041] Las células madre son células no diferenciadas definidas por su capacidad a nivel de células individuales tanto para auto-renovar como diferenciarse para producir células de progenie, incluyendo progenitores de autorenovación, progenitores no-renovables de células y células terminales diferenciadas. Las células madre también se caracterizan por su capacidad para diferenciarse in vitro en células funcionales de diversos linajes celulares de múltiples capas germinales (endodermo, mesodermo y ectodermo), así como para dar lugar a los tejidos de múltiples capas germinales después del trasplante y para contribuir sustancialmente a la mayoría, si no a todos, los tejidos después de la inyección en blastocistos.
[0042] Las células madre se clasifican por su potencial de desarrollo como: (1) totipotente -capaces de dar lugar a todos los tipos de células embrionarias y extraembriónico; (2) pluripotentes -capaz de dar lugar a todos los tipos de células embrionarias; (3) multipotentes -capaz de dar lugar a un subconjunto de los linajes de células, pero todos dentro de un tejido, órgano o sistema fisiológico particular (por ejemplo, las células madre hematopoyéticas (HSC) pueden producir una progenie que incluyen HSC (auto-renovación), de glóbulos restringidos progenitores oligopotentes, y todos los tipos de células y elementos (por ejemplo, plaquetas) que son componentes normales de la sangre); (4) oligopotente -capaz de dar lugar a un subconjunto más restringido de linajes de células que las células madre multipotentes; y (5) unipotente -capaz de dar lugar a un único linaje de células (por ejemplo, células madre de espermatogénesis).
[0043] Las células madre también se clasifican sobre la base de la fuente de la que se pueden obtener. Una célula madre adulta es generalmente una célula indiferenciada multipotente que se encuentra en el tejido que comprende múltiples tipos de células diferenciadas. La célula madre adulta puede renovarse y, en circunstancias normales, diferenciarse para producir los tipos especializados de células del tejido del que se originó, y posiblemente otros tipos de tejidos. Una célula madre embrionaria es una célula pluripotente de la masa celular inteARN de un embrión en fase de blastocisto. Una célula madre fetal es una que se origina a partir de tejidos o membranas fetales. Una célula madre es una célula de posparto multipotente o pluripotente que se origina sustancialmente a partir de tejido extraembrionario disponible después del nacimiento, a saber, la placenta y del cordón umbilical. Se ha encontrado que estas células poseen rasgos característicos de las células madre pluripotentes, incluyendo la rápida proliferación y el potencial de diferenciación en muchos linajes celulares. Células madre posparto pueden ser derivadas de la sangre (por ejemplo, como son los obtenidos a partir de sangre de cordón umbilical) o no derivadas del sangre (por ejemplo, tal como se obtiene de los tejidos no sanguíneos del cordón umbilical y la placenta).
[0044] Tejido embrionario se define típicamente como tejido de origen del embrión (que en los seres humanos se refiere al período de la fertilización a aproximadamente seis semanas de desarrollo. Tejido fetal se refiere al tejido procedente del feto, que en los seres humanos se refiere al período de alrededor de seis semanas de desarrollo a parto. Tejido extraembriónico es el tejido asociado con, pero que no se origina a partir del embrión o el feto. Tejidos extraembriónicos incluyen membranas extraembriónicas (corion, amnios, saco vitelino y alantoides), cordón umbilical y la placenta (que a su vez se forma a partir del corion y la decidua basal materna).
[0045] La diferenciación es el proceso por el cual una célula ("no comprometida") no especializada o menos especializada adquiere las características de una célula especializada, como una célula nerviosa o una célula muscular, por ejemplo. Una célula diferenciada o la diferenciación inducida es una que ha adquirido una posición más especializada ("comprometida") dentro del linaje de una célula. El término cometido, cuando se aplica al proceso de diferenciación, se refiere a una célula que ha procedido en la vía de diferenciación a un punto en el que, en circunstancias normales, continuará para diferenciarse en un tipo celular específico o un subconjunto de tipos de células, y no puede, en circunstancias normales, diferenciarse en un tipo de célula diferente o volver a un tipo de células menos diferenciadas. De-diferenciación se refiere al proceso por el cual una célula vuelve a una posición menos especializada (o comprometida) dentro del linaje de una célula. En la presente memoria, el linaje de una célula define la herencia de la célula, es decir, de dónde las células venían y qué células se pueden. El linaje de una célula coloca la célula dentro de un esquema hereditario de desarrollo y diferenciación. Un marcador de linaje específico se refiere a una característica asociada específicamente con el fenotipo de las células de un linaje de
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[0107] Las células de la invención pueden modificarse por ingeniería genética para expresar una proteína terapéutica utilizando cualquiera de una variedad de vectores, incluyendo, pero no limitado a, la integración de vectores virales, por ejemplo, vector de retrovirus o vectores virales adeno-asociados; no integración de replicar vectores que, por ejemplo, vectores de virus papiloma, vectores de SV40, vectores adenovirales; o vectores víricos de replicación defectuosa. Otros métodos de introducción de ADN en las células incluyen el uso de liposomas, la electroporación, un disparador de partículas, o por inyección directa del ADN.
[0108] Células huésped se transforman o se transfectan con ADN controlado por, o en asociación operativa con, uno
o más elementos de control de expresión apropiado tales como secuencias de promotor o de potenciador, terminadores de transcripción, sitios de poliadenilación, entre otros, y un marcador seleccionable de preferencia.
[0109] Después de la introducción del ADN extraño, las células manipuladas pueden dejarse crecer en medio enriquecido y luego cambiarse a medio selectivo. El marcador seleccionable en ADN extraño confiere resistencia a la selección y permite que las células integren de forma estable el ADN extraño como, por ejemplo, en un plásmido, en sus cromosomas y se crecen para formar focos que, a su vez, se pueden clonar y expandir en líneas de célula.
[0110] Este método se puede utilizar ventajosamente para diseñar líneas celulares que expresan el producto del gen.
[0111] Cualquier promotor puede utilizarse para conducir la expresión del gen insertado. Por ejemplo, los promotores virales incluyen, pero no se limitan al promotor/potenciador de CMV, SV40, virus del papiloma, virus de Epstein-Barr o elastina promotor del gen. Preferiblemente, los elementos de control utilizados para controlar la expresión del gen de interés deben permitir la expresión regulada del gen de modo que el producto se sintetiza sólo cuando sea necesario in vivo. Si se desea la expresión transitoria, los promotores constitutivos se utilizan preferiblemente en un vector no integrante y/o de replicación defectuosa. AlteARNtivamente, los promotores inducibles se podrían utilizar para conducir la expresión del gen insertado cuando sea necesario.
[0112] Los promotores inducibles incluyen, pero no se limitan a, aquellos asociados con proteínas de metalotioneína y de choque térmico.
[0113] Ejemplos de regiones de control de la transcripción que exhiben especificidad de tejido que se han descrito y se pueden utilizar incluyen pero no se limitan a: región de control del gen de elastasa I, que es activa en células acinares pancreáticas (Swift et al, 1984, Cell 38: 639; Ornitz et al, 1985, Cold Spring Harbor Symp Quant Biol 50: 399; MacDonald, 1987, Hepatology 7: 425-515); región de control del gen de la insulina, que es activa en células beta pancreáticas (Hanahan, 1985, Nature 315: 115); región de control del gen de proteína básica de mielina, activa en células de oligodendrocitos en el cerebro (Readhead et al, 1987, Cell 48: 703); cadena ligera de miosina-2 región de control de genes, que es activa en el músculo esquelético (Shani, 1985, Nature 314: 283); y región de control del gen de la hormona de liberación gonadotrópica, que es activa en el hipotálamo (Mason et al, 1986, Science 234: 1372).
[0114] Las células de la invención pueden ser modificadas genéticamente para "knock out" o "knock down" de factores de expresión que promueven la inflamación o rechazo en la zona del implante. Técnicas moduladoras negativas para la reducción de los niveles de expresión del gen diana o niveles de actividad del producto del gen diana se discuten a continuación. "Modulación negativa", como se usa aquí, se refiere a una reducción en el nivel y/o actividad de gen diana producto en relación con el nivel y/o actividad del producto del gen diana en ausencia del tratamiento modulador. La expresión de un gen nativo para una célula puede reducir o eliminar mediante la utilización de un número de técnicas que incluyen, por ejemplo, la inhibición de la expresión mediante la inactivación del gen completo (comúnmente denominado "knockout") utilizando la técnica de recombinación homóloga. Por lo general, un exón que codifica una región importante de la proteína (o un exon 5’ para dicha región) es interrumpida por un marcador seleccionable positivo, por ejemplo, neo, la prevención de la producción de ARNm normal a partir del gen diana y que resulta en la inactivación del gen. Un gen también puede ser inactivado mediante la creación de una deleción en parte de un gen o mediante la supresión del gen entero. Mediante el uso de una construcción con dos regiones de homólogoía al gen diana que son muy separadas en el genoma, las secuencias que intervienen las dos regiones se pueden eliminar (Mombaerts et al, 1991, Proc. Nat. Acad. Sci. EE.UU. 88: 3084).
[0115] Antisentido, ADNzimas, ARN de interferencia pequeña, y moléculas de ribozima que inhiben la expresión del gen diana también pueden ser utilizados de acuerdo con la invención para reducir el nivel de actividad del gen diana. Por ejemplo, las moléculas de ARN antisentido que inhiben la expresión de los principales complejos de genes de histocompatibilidad (HLA) han mostrado ser más versátiles con respecto a las respuestas inmunes. Aún más, las moléculas de triple hélice pueden ser utilizadas en la reducción del nivel de actividad del gen diana.
[0116] Estas técnicas se describen en detalle por L.G. Davis et al. (eds), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2ª ed., Appleton & Lange, Norwalk, Conn.
[0117] El uso de cualquiera de las técnicas anteriores, por ejemplo, la expresión de IL-1 puede ser eliminada o derribada en las células de la invención para reducir la producción de mediadores inflamatorios por las células de la
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células criopreservadas se almacenaron. Tabla 1-1. El aislamiento y la expansión del cultivo de células de la placenta en condiciones diferentes:
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- Condición
- Medio FBS BME Gelatina O2 Factores de crecimiento
- 1
- DMEM-Lg 15% Y Y 20% N
- 2
- DMEM-Lg 15% Y Y 5% N
- 3
- DMEM-Lg 15% Y N 20% N
- 4
- DMEM-Lg 15% Y N 5% N
- 5
- DMEM-Lg 2% Y N (Laminina) 20% EGF/FGF (20 ng/ml)
- 6
- DMEM-Lg 2% Y N (Laminina) 5% EGF/FGF (20 ng/ml)
- 7
- DMEM-Lg 2% Y N (Fibronectina) 20% PDGF/VEGF
- 8
- DMEM-Lg 2% Y N (Fibronectina) 5% PDGF/VEGF
- 9
- DMEM-Lg 15% N Y 20% N
- 10
- DMEM-Lg 15% N Y 5% N
- 11
- DMEM-Lg 15% N N 20% N
- 12
- DMEM-Lg 15% N N 5% N
- 13
- DMEM-Lg 2% N N (Laminina) 20% EGF/FGF (20 ng/ml)
- 14
- DMEM-Lg 2% N N (Laminina) 5% EGF/FGF (20 ng/ml)
- 15
- DMEM-Lg 2% N N (Fibronectina) 20% PDGF/VEGF
- 16
- DMEM-Lg 2% N N (Fibronectina) 5% PDGF/VEGF
- Clave: Lg: Glucosa baja; N: Ninguno; Y: Sí; BME: beta-mercaptoetlianol; FGF: Factor de crecimiento de fibroblasto; EGF: Crecimiento epidérmico factor; PDGF: Factor de crecimiento derivado de plaqueta; VEGF: Factor de crecimiento vascular endotelial.
30 Resultados
[0200] El aislamiento de PDC usando diferentes condiciones de crecimiento. En todas las condiciones establecidas en la Tabla 1-1, las células se unieron y ampliaron bien entre el paso entre 0 y 1. Las casillas de la condición 5 a 8 y 13 a 16 se demostraron a proliferar así hasta al menos cuatro pasajes después de la siembra.
35 [0201] El aislamiento de las células de placenta utilizando diferentes combinaciones de enzimas. La digestión del tejido con colagenasa: dispasa y colagenasa: dispasa: hialuronidasa como resultado del aislamiento de poblaciones de células de tejidos de la placenta que se expandieron fácilmente.
40 [0202] Resumen. PDC se pueden aislar utilizando una combinación de una metaloproteasa de matriz y proteasa neutra, tal como, pero no limitado a una combinación de colagenasa y dispasa. PDC se aíslan preferiblemente usando una combinación de enzimas de una metaloproteasa de matriz, una proteasa neutra, y una enzima mucolítica que degrada el ácido hialurónico, tales como, pero no limitado a una combinación de colagenasa, dispasa, y hialuronidasa o una combinación de LIBERASETM (Boehringer Mannheim Corp., Indianapolis, IN) y
45 hialuronidasa. Blendcima 3, que es la colagenasa (4 unidades Wunsch/g) y termolisina (1714 unidades de caseína/g) se puede usar junto con hialuronidasa para aislar células.
EJEMPLO 2
50 Evaluación de Medios de Crecimiento de las Células Derivadas del Placenta
[0203] Se evaluaron varios medios de cultivo celular por su capacidad para apoyar el crecimiento de células derivadas de la placenta. El crecimiento de las células derivadas de la placenta en oxígeno normal (20%) y bajo (5%) se evaluó después de 3 días utilizando el ensayo colorimétrico MTS.
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Métodos y materiales
[0204] Las Células derivadas de la placenta en el paso 8 (P8) se sembraron a 1 x 103 células/pocillo en placas de 96 pocillos en medio de crecimiento (DMEM de baja glucosa (Gibco, Carlsbad CA), 15% (v/v) de bovino fetal suero 60 (Cat. #SH30070.03; Hyclone, Logan, UT), 0,001% (v/v) betamercaptoetanol (Sigma, St. Louis, MO), penicilina 50 unidades/mililitro, 50 microgramos/mililitro estreptococos micina (Gibco). Después de 8 horas el medio se cambió al descrito en la Tabla 2-1 y las células se incubaron en condiciones normales (20%, v/v) o bajo (5%, v/v) de oxígeno a 37°C, 5% de CO2 durante 48 horas. MTS se añadió al medio de cultivo (una solución acuosa CELLTITER96® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI) durante 3 horas y la absorbancia medida a
65 490 nanómetros (Molecular Devices, Sunnyvale CA).
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Table 8-4. Los genes que se muestran han aumentado específicamente la expresión en las células del cordón umbilical derivadas en comparación con las otras líneas celulares ensayadas.
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- Genes aumentados en células derivadas de cordon umbilical
- Identificación de conjunto de sondas
- Nombre de gen Nombre de gen
- 202859_x_at
- interleucina 8 NM_000584
- 211506_s_at
- interleucina 8 AF043337
- 210222_s_at
- reticulon 1 BC000314
- 204470_at
- quemoquina (C-X-C motivo) ligando 1 (melanoma actividad de estimulación de crecimiento) NM_001511
- 206336_at
- quemoquina (C-X-C motivo) ligando 6 (granulocito proteína quemotáctica 2) NM_002993
- 207850_at
- quemoquina (C-X-C motivo) ligando 3 NM_002090
- 203485_at
- reticulon 1 NM_021136
- 202644_s_at
- tumor factor necrosis, proteína inducida alfa 3 NM_006290
Table 8-5. Los genes que se muestran tener una disminución en la expresión de las células del cordón umbilical y la placenta en comparación con las otras líneas celulares ensayadas.
- Genes aumentados en células derivadas de cordon umbilical
- Identificación de conjunto de sondas
- Nombre de gen Nombre de gen
- 210135_s_at
- homeobox de baja estatura 2 AF022654.1
- 205824_at
- choque térmico 27kDa proteína 2 NM_001541.1
- 209687_at
- quemoquina (C-X-C motivo) ligando 12 (factor derivado de células estromales 1) U19495.1
- 203666_at
- quemoquina (C-X-C motivo) ligando 12 (factor derivado de células estromales 1) NM_000609.1
- 212670_at
- elastina (estenosis aortica supravalvular, síndrome Williams-Beuren) AA479278
- 213381_at
- Homo sapiens ARNm; ADNc DKFZp586M2022 (de clon DKFZp586M2022) N91149
- 206201_s_at
- homeobox de mesénquima 2 (homeobox específico de crecimiento detenido) NM_005924.1
- 205817_at
- sine oculis homeobox homólogo 1 (Drosophila) NM_005982.1
- 209283_at
- cristalina, alfa B AF007162.1
- 212793_at
- activador de morfogenesis asociado desgreñado 2 BF513244
- 213488_at
- DKFZP586B2420 proteína AL050143.1
- 209763_at
- similar a neuralina 1 AL049176
- 205200_at
- tetranectina (proteína de unión de plasminogen) NM_003278.1
- 205743_at
- src homología tres (SH3) y dominio rico en cisteína NM_003149.1
- 200921_s_at
- gen de traslocación de célula B 1, anti-proliferativo NM_001731.1
- 206932_at
- colesterol 25-hidroxilasa NM_003956.1
- 204198_s_at
- factor de transcripción relacionado a runt 3 AA541630
- 219747_at
- proteína hipotética FLJ23191 NM_024574.1
- 204773_at
- interleucina 11 receptor, alfa NM_004512.1
- 202465_at
- procolagen C-potenciador de endopeptidasa NM_002593.2
- 203706_s_at
- homólogo frizzled 7 (Drosophila) NM_003507.1
- 212736_at
- gen hipotético BC008967 BE299456
- 214587_at
- colagen, tipo VIII, alfa 1 BE877796
- 201645_at
- tenascina C (hexabrachion) NM_002160.1
- 210239_at
- iroquois homeobox proteína 5 U90304.1
- 203903_s_at
- hefaestina NM_014799.1
- 205816_at
- integrina, beta 8 NM_002214.1
- 203069_at
- glicoproteína de vesículo sináptico 2 NM_014849.1
- 213909_at
- Homo sapiens ADNc FLJ12280 fis, clone MAMMA1001744 AU147799
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- 206315_at
- citoquina similar al receptor del factor 1 NM_004750.1
- 204401_at
- potasio intermedia / pequeña conductancia de calcio canal activado, 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
- co-activador transcripcional con motivo de unión PDZ (TAZ) AA081084
- 206511_s_at
- homeobox de homólogo sine oculis 2 (Drosophila) NM_016932.1
- 213435_at
- proteína KIAA1034 AB028957.1
- 206115_at
- respuesta de crecimiento temprano 3 NM_004430.1
- 213707_s_at
- homeobox no distal 5 NM_005221.3
- 218181_s_at
- proteína hipotética FLJ20373 NM_017792.1
- 209160_at
- aldo-ceto reductasa familia 1, miembro de C3 (3-alfa hidroxiesteroide deshidrogenasa, tipo II) AB018580.1
- 213905_x_at
- biglicano AA845258
- 201261_x_at
- biglicano BC002416.1
- 202132_at
- co-activador transcripcional con motivo de unión PDZ (TAZ) AA081084
- 214701_s_at
- fibronectina 1 AJ276395.1
- 213791_at
- proencefalina NM_006211.1
- 205422_s_at
- integrina beta tipo 1 (con dominios de recepción similares a EGF) NM_004791.1
- 214927_at
- Homo sapiens ARNm de longitud completa inserción ADNc clon Euroimage 1968422 AL359052.1
- 206070_s_at
- EphA3 AF213459.1
- 212805_at
- proteína KIAA0367 AB002365.1
- 219789_at
- natriurético receptor del péptido C/guanilato ciclasa C (péptido atrionatriuretic receptor C) AI628360
- 219054_at
- hipotética proteína FLJ14054 NM_024563.1
- 213429_at
- Homo sapiens ARNm; ADNc DKFZp564B222 (a partir del clon DKFZp564B222) AW025579
- 204929_s_at
- proteína de membrana asociada a vesículas 5 (myobrevin) NM_006634.1
- 201843_s_at
- EGF que contiene proteína de la matriz extracelular fibulin tipo 1 NM_004105.2
- 221478_at
- interacción de proteínas BCL2 / adenovirus E1B de 19 kDa 3-como AL132665.1
- 201792_at
- proteína de unión a AE 1 N_001129.2
- 204570_at
- citocromo c oxidasa subunidad polipeptídica VIIa 1 (músculo) NM_001864.1
- 201621_at
- neuroblastoma, supresión de tumorigenicidad 1 NM_005380.1
- 202718_at
- proteína de unión a factor de crecimiento similar a la insulina 2, 36 kDa NM_000597.1
5
10
15
20
42
[0268] Tablas 8-6, 8-7, y 8-8 muestran la expresión de genes aumentada en los fibroblastos humanos (Tabla 8-6), las células de ICBM (Tabla 8-7), y MSC (Tabla 8-8).
Tabla 8-6. Los genes se han demostrado aumentar la expresión en fibroblastos en comparación con las otras líneas celulares ensayadas.
10
15
20
25
30
35
- Genes aumentados en fibroblastos
- fosfatasa de doble especificidad 2
- proteína KIAA0527
- Homo sapiens ADNc: 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)
- pirofosfatasa ectonucleótido/fosfodiesterasa 4 (función putativa)
- proteína KIAA1053
- proteína asociada a microtúbulos 1A
- proteína dedos de zinc 41
- proteína HSPC019
- Homo sapiens ADNc: FLJ23564 fis, clon LNG10773
- ARNm de Homo sapiens; ADNc DKFZp564A072 (del clon DKFZp564A072)
- proteína LIM (similar al enigma de quinasa de proteína C de rata vinculante)
- inhibidor de potenciador del gen de polipéptido ligero kappa en las células B, proteína quinasa de complejo asociado
- proteína hipotética FLJ22004
- (Clon CTG-A4) secuencia de ARNm humano
- EST, moderadamente similares a citoquina factor de 2 similar al receptor; CRL2 precursor del receptor de citoquinas [Homo sapiens]
- factor de crecimiento transformante, beta 2
- proteína hipotética MGC29643
- antígeno identificado por el anticuerpo monoclonal MRC OX-2
Tabla 8-7. Los genes que se han mostrado tener una mayor expresión en las células derivadas de ICBM en 40 comparación con las otras líneas celulares ensayadas.
45
50
55
60
- Genes aumentados en las células ICBM
- proteína con repeticiones de anquirina cardiaca
- MHC de clase I región ORF
- integrina, alfa 10
- proteína hipotética FLJ22362
- UDP-N-acetil-alfa-D-galactosamina: polipéptido N-acetilgalactosaminiltransferasa 3 (GalNAc-T3)
- proteína inducida por interferón 44
- SRY (región determinante del sexo Y)-box 9 (displasia campomélica, autosómica reversión sexual)
- proteína de queratina asociada 1-1
- hipocalcina tipo 1
- dentado 1 (síndrome de Alagille)
- proteoglicano 1, gránulo secretor
43
[0306] Reacción mixta de linfocitos. Viales criopreservadas de PDC derivadas de placenta de pasaje 11 placenta etiquetadas como línea de células B se enviaron en hielo seco a CTBR (Senneville, Quebec) para llevar a cabo una reacción de linfocitos mixta utilizando CTBR SOP no. CAC-031. Se recogieron las células mononucleares de sangre periférica (PBMC) de donantes voluntarios femeninos y masculinos múltiples. Estimulador (donante) alogénico
5 PBMC, PBMC autólogas, y las líneas celulares derivadas de la placenta se trataron con mitomicina C. Células estimuladoras tratadas C de autólogo y mitomicina se añadieron para responder (receptores) PBMC y se cultivaron durante 4 días. Después de la incubación, [3H]-timidina se añadió a cada muestra y se cultivó durante 18 horas. Después de la cosecha de las células, se extrajo el ADN radiomarcado, y [3H]-timidina se midió utilizando un contador de centelleo.
10 [0307] El índice de estimulación para el donante alogénico (SIAD) se calculó como la proliferación media del receptor más donante alogénico de mitomicina C-tratada dividida por la proliferación de línea de base del receptor. El índice de estimulación de la célula derivada de placenta se calculó como la media de proliferación del receptor de la línea celular derivada de placenta C-tratada más mitomicina dividida por la proliferación de línea de base del
15 receptor.
Resultados
[0308] Reacción de linfocitos mixtos-Placenta. Siete donantes de sangre voluntarios humanos fueron
20 examinados para identificar un único donante alogénico que exhibiría una robusta respuesta de proliferación en una reacción de linfocitos mezclados con los otros seis donantes de sangre. Este donante fue seleccionado como el donante de control alogénico positivo. Los seis donantes de sangre restantes fueron seleccionados como receptores. El donante de control positivo alogénico y las líneas células de placenta fueron tratados con mitomicina C y se cultivaron en una reacción de linfocitos mezclados con los seis receptores alogénicos individuales. Las
25 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 de 1.3 (placa de 2) a 3 (placa 1) y los controles positivos de donante alogénico oscilaron entre 46,25 (placa de 2) a 279 (Placa 1) (Tabla 11-3).
Tabla 11-2. Reacción de datos de linfocito mixtos -línea de células B (placenta) 30
- DPM para ensayo de proliferación
- Placa de identificación: Placa1
- Analítico
- Cultivo Replicados
- número
- Sistema 1 2 3 Medio SD CV
- IM03-7769
- Línea base de proliferación del receptor 79 119 138 112,0 30,12 26,9
- El control de autoestimulación (células autólogas tratadas por mitomicina C)
- 241 272 175 229,3 49,54 21,6
- Donante alogénico MLR IM037768 (tratadas con mitomicina C)
- 23971 22352 20921 22414,7 1525,97 6,8
- MLR con línea celular (tipo celular B tratada por mitomicina C)
- 664 559 1090 771,0 281,21 36,5
- SI (donante)
- 200
- SI (línea celular)
- 7
- IM03-7770
- Proliferación de línea de base de receptor 206 134 262 200,7 64,17 32,0
- El control de autoestimulación (células autólogas tratadas por mitomicina C)
- 1091 602 524 739,0 307,33 41,6
- MLR alogénica IM03-7768 donante (tratadas con mitomicina C)
- 45005 43729 44071 44268,3 660,49 1,5
- MLR con la línea celular (mitomicina C tipo celular tratado
- 533 2582 2376 1830,3 1128,24 61,6
50
- B)
- DPM para ensayo de proliferación
- Placa ID: Placa1
- Analítico
- Cultivo Replicados
- número
- Sistema 1 2 3 Medio SD CV
- SI (donante)
- 221
- SI (línea celular)
- 9
- IM03-7771
- Proliferación de línea de base de receptor 157 87 128 124.0 35.17 28.4
- El control de autoestimulación (células autólogas tratadas por mitomicina C)
- 293 138 508 313.0 185.81 59.4
- MLR alogénica IM037768 donante (tratadas con mitomicina C)
- 24497 34348 31388 30077.7 5054.53 16.8
- MLR con la línea celular (mitomicina C tipo celular tratado B)
- 601 643 a 622.0 29.70 4.8
- SI (donante)
- 243
- SI (línea celular)
- 5
- IM03-7772
- Proliferación de línea de base de receptor 56 98 51 68.3 25.81 37.8
- El control de autoestimulación (células autólogas tratadas por mitomicina C)
- 133 120 213 155.3 50.36 32.4
- MLR alogénica IM037768 donante (tratadas con mitomicina C)
- 14222 20076 22168 18822.0 4118.75 21.9
- MLR con la línea celular (mitomicina C tipo celular tratado B)
- a a a a a a
- SI (donante)
- 275
- SI (línea celular)
- a
- IM03-7768 (donante alogénico)
- Proliferación de línea de base de receptor 84 242 208 178.0 83.16 46.7
- El control de autoestimulación (células autólogas tratadas por mitomicina)
- 361 617 304 427.3 166.71 39.0
- DPM para ensayo de proliferación
- Placa ID: Placa1
- Analítico
- Cultivo Replicados
51
- número
- Sistema 1 2 3 Medio SD CV
- Línea de células tipo B
- Proliferación de línea de base de receptor 126 124 143 131.0 10.44 8.0
- El control de autoestimulación (células autólogas tratadas por mitomicina)
- 822 1075 487 794.7 294.95 37.1
- Placa ID: Placa2
- Número analítico
- Sistema de cultivo Replicados 123 Medio SD CV
- IM03-7773
- Proliferación de línea de base de receptor El control de autoestimulación (células autólogas tratadas por mitomicina C) Donante alogénico MLR IM037768 (tratada con Mitomicina C) MLR con línea celular (Tipo celular B tratado con Mitomicina C) 908 181 330 269 405 572 29151 28691 567 732 905 28315 473,0 415,3 28719,0 734,7 384,02 151,76 418,70 169,02 81.2 36.5 1.5 23.0
- SI (donante) SI (línea celular)
- 61 2
- IM03-7774
- Proliferación de línea de base de receptor El control de autoestimulación (células autólogas tratadas por mitomicina C) Donante alogénico MLR IM037768 (tratada con Mitomicina C) MLR con línea celular (Tipo celular B tratado con Mitomicina C) 893 1376 185 261 381 568 53101 42839 515 789 294 48283 818,0 403,3 48074,3 532,7 599,03 154,71 5134,18 247,97 73.2 38.4 10.7 46.6
- SI (donante) SI (línea celular)
- 59 1
- Placa ID: Placa 2
- Número analítico
- Sistema de cultivo Replicados 123 Medio SD CV
- IM03-7775
- Proliferación de línea de base de receptor El control de autoestimulación (células autólogas tratadas por mitomicina C) Donante alogénico MLR IM03-7768 (tratada con Mitomicina C) MLR con línea celular (Tipo celular B tratado con Mitomicina C) 1272 300 544 232 199 484 23554 10523 768 924 563 28965 705,3 305,0 21014,0 751,7 505,69 155,89 9479,74 181,05 71.7 51.1 45.1 24.1
- SI (donante) SI (línea celular)
- 30 1
- IM03-7776
- Proliferación de línea de base de receptor El control de autoestimulación (células autólogas tratadas por mitomicina C) Donante alogénico MLR IM03-7768 (tratada con Mitomicina C) MLR con línea celular (Tipo celular B tratado con Mitomicina C) 1530 137 1046 420 218 394 28893 32493 aaa 34746 904,3 344,0 32044,0 a 707,22 109,89 2952,22 a 78.2 31.9 9.2 a
- SI (donante) SI (línea celular)
- 35 a
Tabla 11-3. Índice de estimulación medio de células de la placenta y un donante alogénico en una reacción mixta de linfocitos con seis receptores alogénicos individuales.
5
- Índice medio de estimulación
- Receptor
- Placenta
- Placa 1 (Receptores 1-3)
- 279 3
- Placa 2 (Receptores 4-6)
- 46,25 1,3
10
52
Tabla 22-2. Resumen de las condiciones para protocolo de diferenciación de dos etapas
- A
- B
- COND. #
- PRE-DIFERENCIACIÓN DIFF DE 2A ETAPA
- 1
- NPE + F + E NPE + SHH (200 nanogramo/mililitro) + F8 (100 nanogramo/mililitro)
- 2
- NPE + F + E NPE + SHH (200 nanogramo/mililitro) + F8 (100 nanogramo/mililitro) + RA (1 micromolar)
- 3
- NPE + F + E NPE + RA (1 micromolar)
- 4
- NPE + F + E NPE + F (20 nanogramo/mililitro) + E (20 nanogramo/mililitro)
- 5
- NPE + F + E Medio de Crecimiento
- 6
- NPE + F + E Condición 1B + rhGDF-5 (20 nanogramo/mililitro)
- 7
- NPE + F + E Condición 1B + BMP7 (20 nanogramo/mililitro)
- 8
- NPE + F + E Condición 1B + GDNF (20 nanogramo/mililitro)
- 9
- NPE + F + E Condición 2B + rhGDF-5 (20 nanogramo/mililitro)
- 10
- NPE + F + E Condición 2B + BMP7 (20 nanogramo/mililitro)
- 11
- NPE + F + E Condición 2B + GDNF (20 nanogramo/mililitro)
- 12
- NPE + F + E Condición 3B + rhGDF-5 (20 nanogramo/mililitro)
- 13
- NPE + F + E Condición 3B + BMP7 (20 nanogramo/mililitro)
- 14
- NPE + F + E Condición 3B + GDNF (20 nanogramo/mililitro)
- 15
- NPE + F + E NPE + rhGDF-5 (20 nanogramo/mililitro)
- 16
- NPE + F + E NPE + BMP7 (20 nanogramo/mililitro)
- 17
- NPE + F + E NPE + GDNF (20 nanogramo/mililitro)
[0412] Protocolo de Co-cultivo de Progenitor Neural. Progenitores adulto de hipocampo de rata (062603) se cultivaron como esferas neurológicas o células individuales (10.000 células/pocillo) en placas de 24 pocillos recubiertas con laminina (BD Biosciences) en NPE + F (20 nano gramos/mililitro) + E (20 nanogramos /mililitro).
[0413] Por otra parte, células derivadas de la placenta de (022803) P11 se descongelaron y expandidas en cultivo en NPE + F (20 nanogramos/mililitro) + E (20 nanogramos/mililitro) a 5.000 células/cm2 durante un periodo de 48 horas. Las células fueron tripsinizadas y se sembraron a 2.500 células/pocillo en cultivos existentes de progenitores neurales. En ese momento, el medio existente se cambió por medio fresco. Cuatro días más tarde, los cultivos se fijaron con helado de 4% de paraformaldehído (Sigma) durante 10 minutos a temperatura ambiente, y se tiñeron para la proteína nuclear humana (hNuc; Chemicon) (v/w) (Tabla 221-1 anteriormente) para identificar PPDCs.
[0414] Inmunocitoquímica. La inmunocitoquímica se llevó a cabo utilizando los anticuerpos enumerados en la Tabla 22-1. Los cultivos se lavaron con solución salina tamponada con fosfato (PBS) y se expusieron a una solución de bloqueo de proteína que contiene PBS, 4% (v/v) de suero de cabra (Chemicon, Temecula, CA), y 0,3% (v/v) de Triton (Triton X-100; Sigma) durante 30 minutos para acceder a antígenos intracelulares. Los anticuerpos primarios, se diluyó en solución de bloqueo, se aplicaron a los cultivos durante un período de 1 hora a temperatura ambiente. A continuación, las soluciones de anticuerpos primarios fueron removidos y cultivos lavaron con PBS antes de la aplicación de las soluciones de anticuerpo secundario (1 hora a temperatura ambiente) que contiene la solución de bloqueo, junto con anticuerpo de cabra anti-IgG de ratón -Texas Red (1: 250; Molecular Probes, Eugene, OR) y de cabra anti-IgG de conejo -Alexa 488 (1: 250; Molecular Probes). Después, los cultivos se lavaron y 10 micromolar DAPI (Molecular Probes) aplicaron durante 10 minutos para visualizar los núcleos celulares.
[0415] Después de la inmunotinción, la fluorescencia se visualizó usando el filtro de fluorescencia correspondiente en un microscopio invertido Olympus epi-fluorescencia (Olympus, Melville, NY). En todos los casos, la tinción positiva representó la señal de fluorescencia por encima de la tinción de control donde todo el procedimiento descrito anteriormente fue seguido con la excepción de la aplicación de una solución de anticuerpo primario. Imágenes representativas fueron capturadas utilizando un software ImagePro® (Media Cybernetics, Carlsbad, CA) y cámara de vídeo digital en color. Para las muestras de triple manchado, cada imagen fue tomada utilizando un solo filtro de emisión a la vez. montajes en capas se prepararon a continuación, utilizando el software Adobe Photoshop® (Adobe, San Jose, CA).
Resultados
[0416] Protocolo Woodbury-Black. (A) Tras la incubación en esta composición de inducción neural, todos los tipos de células transformadas en células con morfologías bipolares y procesos extendidos. También se observaron otras morfologías no bipolares más grandes. Además, las poblaciones de células inducidas se tiñeron positivamente para
70
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