ES2565582T3 - 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 PDFInfo
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- ES2565582T3 ES2565582T3 ES04809466.8T ES04809466T ES2565582T3 ES 2565582 T3 ES2565582 T3 ES 2565582T3 ES 04809466 T ES04809466 T ES 04809466T ES 2565582 T3 ES2565582 T3 ES 2565582T3
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Abstract
Un método ex vivo para inducir la diferenciación de células que se pueden obtener de tejido del cordón umbilical humano sustancialmente libre de sangre, en donde las células son capaces de autorrenovación y diferenciación, y en donde la expresión por dichas células de genes que codifican interleucina 8; reticulón 1; ligando 1 de quimiocina (motivo C-X-C); ligando 3 de quimiocina (motivo C-X-C); ligando 6 de quimiocina (motivo C-X-C) y proteína 3 inducida por el factor de necrosis tumoral alfa está aumentada en relación a una célula humana que es un fibroblasto, una célula madre mesenquimal o una célula madre ósea de cresta iliaca como se caracteriza en un conjunto de genes, a un fenotipo condrogénico que comprende exponer dichas células a uno o más agentes inductores de diferenciación condrogénica en donde dichas células comprenden además las siguientes características: (i) potencial de experimentar al menos 40 duplicaciones en el cultivo; (ii) 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; (iii) producción de vimentina y actina del músculo liso alfa; (iv) producción de cada uno de CD10, CD13, CD44, CD73, CD90, PDGFr-alfa y HLA-A,B,C, como se detectan por citometría de flujo; (v). falta de expresión de CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G y HLADR, DP,DQ como se detectan por citometría de flujo; (vi) secreción de MCP-1, IL-6, GCP-2, IL-8, TIMP1, TPO, KGF, HGF, FGF, HBEGF y BDNF como se detectan potr ensayo ELISA; (vii) falta de secreción de SDF-1 alfa, VEGF, TGF-beta2, ANG2 y PDGFbb como se detectan por ensayo ELISA; (viii). crecimiento en alrededor del 5% a alrededor del 20% de oxígeno (ix). requieren L-valina para el crecimiento; (x) expresión de PD-L2 y factor tisular como se miden por citometría de flujo; (xi) una disminución en la expresión de los siguientes genes en relación a una célula humana que es un fibroblasto, una célula madre mesenquimal, o una célula madre ósea de cresta iliaca como se caracteriza en un conjunto de 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; y (xii) producción de GCP-2 y NOGO-A como se ensayan por inmunocitoquímica.
Description
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Tabla 5-2: Características de crecimiento para diferentes poblaciones celulares utilizando proliferación del crecimiento a baja densidad desde el pase 10
- Tipo celular
- Senescencia Duplicaciones totales de la población Rendimiento celular total
- Fibroblastos (P10)
- 80 días 43,68 2.59 E11
- Células derivadas de cordón umbilical (P10)
- 80 días 53,6 1.25 E14
EJEMPLO 6
Análisis del cariotipo de las PPDC
Las líneas celulares utilizadas en terapia celular son preferentemente homogéneas y carecen de cualquier tipo de células contaminantes. Las células humanas utilizadas en terapia celular deben tener una estructura y número de cromosomas (46) normales. Para identificar las líneas celulares de placenta y cordón umbilical derivadas posparto que son homogéneas y carecen de células de origen tisular no posparto, se analizaron los cariotipos de muestras celulares.
Materiales y métodos
Se cultivaron PPDC de tejido posparto de un neonato varón en Medio de crecimiento (DMEM con bajo contenido de glucosa (Gibco Carlsbad, CA), suero bovino fetal al 15% (v/v) (FBS) (Hyclone, Logan, UT), betamercaptoetanol al 0,001% (v/v) (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 varón (X,Y) para permitir la distinción entre las células de origen neonatal y las células de origen materno (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 hicieron proliferar hasta aproximadamente el 80% de confluencia. Se llenó hasta el cuello con Medio de crecimiento un matraz T25 que contenía las células. Las muestras se enviaron a un laboratorio de citogenética clínica por mensajería (el tiempo estimado de transporte de laboratorio a laboratorio es de una hora). El análisis cromosómico fue realizado por el Center for Human & Molecular Genetics de la New Jersey Medical School, Newark, NJ. Las células se analizaron durante la metafase, momento en el que los cromosomas se visualizan mejor. De veinte células en metafase contadas, se analizaron cinco para determinar un número de cariotipo homogéneo normal (dos). Una muestra de células se caracterizaba como homogénea si se observaban dos cariotipos. Una muestra de células se caracterizaba como heterogénea si se observaban más de dos cariotipos. Se realizó el recuento de las células en metafase adicionales y se analizaron cuando se identificaba un número de cariotipo heterogéneo (cuatro).
Resultados
El personal del laboratorio de citogenética interpretó que todas las muestras celulares enviadas para el análisis cromosómico presentaban una apariencia normal. Tres de las dieciséis líneas celulares analizadas presentaron un fenotipo heterogéneo (XX y XY) que indicaba la presencia de células derivadas de ambos orígenes neonatal y materno (Tabla 6-1). Se aislaron células derivadas del tejido Placenta-N del aspecto neonatal de la placenta. En el pase cero, esta línea celular parecía XY homogénea. Sin embargo, en el pase nueve, la línea celular era heterogénea (XX/XY), lo que indicaba una presencia anterior no detectada de células de origen materno.
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- 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
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- 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).
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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.
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- 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)
53
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
61
- 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
62
- 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 se realizaron por triplicado utilizando dos placas de cultivo celular con tres receptores por placa (Tabla 11-4). El
63
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).
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
64
- 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
65
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. Las células derivadas de cordón umbilical secretaron una cantidad significativamente mayor de factores tróficos que las células derivadas de la placenta y los fibroblastos. Algunos de estos factores tróficos, tales como HGF, bFGF, MCP-1 e IL-8, desempeñan papeles importantes en la angiogénesis. Otros factores tróficos, tales como BDNF e IL-6, tienen papeles importantes en la regeneración neural. En estas condiciones, la expresión de algunos factores se limitó a las células derivadas de cordón umbilical, tales como MIP1b, Rantes, I309 y FGF.
Referencias
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, J Laterra, Polverini PJ, Rubin JS, Goldberg ID. (1.997) 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 Bio1.258; 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 de plasma
La terapia celular puede inyectarse sistémicamente para determinadas aplicaciones en las que las células sean capaces de dirigirse al sitio de acción. Es importante que las células inyectadas no provoquen trombosis, que puede ser fatal. El factor tisular, una glicoproteína 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 tisular también desempeña un papel importante en la formación de los vasos embrionarios, por ejemplo, en la formación de la pared vascular primitiva (Brodsky et al. (2002) Exp. Nephrol. 10:299-306). Para determinar el potencial de las PPDC para iniciar la coagulación, se evaluaron PPDC derivadas de cordón umbilical y de la placenta en cuanto a la expresión de factor tisular y su capacidad para iniciar la coagulación plasmática.
68
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. Las PPDC derivadas de la placenta y de cordón umbilical expresan factor tisular, que puede inducir la coagulación. La adición de un anticuerpo contra el factor tisular puede inhibir el factor tisular. El factor tisular se encuentra normalmente en las células en una conformación que es inactiva, pero que se activa mediante estrés mecánico o químico (por ejemplo, LPS) (Sakariassen et al. (2001) Thromb. Res. 104:149-74; Engstad et al. (2002) Int. Immunopharmacol. 2:1585-1597). Por lo tanto, la minimización del estrés durante el proceso de preparación de las PPDC puede evitar la activación del factor tisular. Además de la actividad trombogénica, el factor tisular se ha asociado con la actividad angiogénica. Por lo tanto, la actividad del factor tisular puede ser beneficiosa cuando las PPDC derivadas de cordón umbilical o de la placenta se trasplantan en tejido, pero debe inhibirse cuando las PPDC se inyectan por vía intravenosa.
Referencias
Doshi y Marmur, Critical Care Med., 30:5241-5250 (2002). Moll y Ortel, Ann. Intern. Med., 127:177-185 (1997).
EJEMPLO 14
Diferenciación de las PPDC a un fenotipo osteogénico
Las células madre mesenquimales (MSC) derivadas de médula ósea pueden diferenciarse a células tipo osteoblasto que mineralizan y expresan fosfatasa alcalina. También se han utilizado marcadores adicionales expresados por los osteoblastos, tales como la osteocalcina y la sialoproteína ósea, para demostrar la diferenciación a una célula tipo osteoblasto. Se realizó una determinación en cuanto a si las células derivadas posparto también pueden diferenciarse a un fenotipo osteogénico mediante cultivo en un medio osteogénico y en presencia de proteínas morfogénicas óseas (BMP) -2 (Rickard et al., 1994) ó -4, y factor de crecimiento transformante beta 1.
Métodos y material
Cultivo celular. Antes del inicio de la osteogénesis, se cultivaron células madre mesenquimales (MSC) en medio Mesenchymal Stem Cell Growth Medium BulletKit (MSCGM, Cambrex, Walkerville, MD). Otras células se cultivaron en Medio de crecimiento (DMEM con bajo contenido de glucosa (Gibco, Carlsbad, CA), suero bovino fetal al 15% (v/v) (SH30070.03; Hyclone, Logan, UT), betamercaptoetanol al 0,001% (v/v) (Sigma, St. Louis, MO), penicilina/estreptomicina (Gibco)), en un matraz T75 recubierto con gelatina y se lavaron con solución salina tamponada con fosfato (PBS).
Se cultivaron osteoblastos (9F1721; Cambrex) en medio de crecimiento para osteoblastos (Cambrex) y se extrajo el ARN como se describe más adelante.
70
Protocolo 2. Se observó diferenciación osteogénica, como se muestra mediante tinción de von Kossa positiva para la mineralización, con células derivadas de la placenta P4 e ICBM (070203) P3 incubadas con medio osteogénico complementado con BMP2 ó 4, y MSC (092903) P3 incubadas con medio osteogénico complementado con BMP 4 (Tabla 14-1). Ninguna de las demás células se diferenció al fenotipo osteogénico ni se tiñó con von Kossa. Para asegurar que la tinción de von Kossa se relacionaba con la célula y no con la matriz extracelular, se realizó la contratinción de las células con rojo nuclear rápido. Se observaron grandes gotitas de lípidos en algunas MSC, coherentes con un fenotipo de adipocito. Esto sugiere que las MSC no se diferencian específicamente a un fenotipo osteogénico en estas condiciones. Además, la adipogénesis aumentó cuando las MSC se incubaron en medio osteogénico complementado con BMP2 o con BMP4.
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), células madre mesenquimales (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
Resumen. Se ha demostrado que las MSC derivadas de la médula ósea (Kadiyala et al., 1997) así como células derivadas de otros tejidos tales como el adiposo (Halvorsen et al., 2001) se diferencian a células tipo osteoblasto. Las MSC también han demostrado diferenciarse a adipocitos o a osteoblastos en respuesta a las BMP (Chen et al., 1998) debido a las diferentes funciones del receptor tipo IB y IA de la proteína morfogenética ósea (BMP).
Las MSC y células derivadas de la placenta de origen neonatal presentaron mineralización así como inducción de osteocalcina y sialoproteína ósea. En las condiciones utilizadas, las células derivadas de cordón umbilical no presentaron mineralización ni inducción de genes de osteoblasto. Las células derivadas de la placenta maternas pueden necesitar la adición de BMP-4 o TGF al medio osteogénico para que se produzca la mineralización. La edad gestacional de la muestra también puede ser un factor en la capacidad de las células derivadas de tejidos posparto para diferenciarse.
Referencias:
Kadiyala S, Young RG, Thiede MA, Bruder SP. (1997) Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant.6:125-34.
Chen D, Ji X, Harris MA, Feng JQ, Karsenty G, Celeste AJ, Rosen V, Mundy GR, Harris SE. (1998) Differential roles for bone morphogenic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages. J Cell Biol. 142:295-305.
Halvorsen YD, Franklin D, Bond AL, Hitt DC, Auchter C, Boskey AL, Paschalis EP, Wilkison WO, Gimble JM (2001) Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng. 7:729-41.
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ES04777287.6T Expired - Lifetime 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 |
ES04809466.8T Expired - Lifetime ES2565582T3 (es) | 2003-06-27 | 2004-06-25 | Reparación y regeneración de cartílago y hueso utilizando células derivadas posparto |
ES04756395.2T Expired - Lifetime 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 |
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ES04777235.5T Expired - Lifetime ES2568463T3 (es) | 2003-06-27 | 2004-06-25 | Regeneración y reparación de tejido neural usando células del postparto derivadas del cordon umbilical |
ES10184221.9T Expired - Lifetime ES2541604T3 (es) | 2003-06-27 | 2004-06-25 | Reparación y regeneración de tejido ocular usando células derivadas del cordón umbilical post parto |
ES10183053.7T Expired - Lifetime 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 |
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ES10184221.9T Expired - Lifetime ES2541604T3 (es) | 2003-06-27 | 2004-06-25 | Reparación y regeneración de tejido ocular usando células derivadas del cordón umbilical post parto |
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