ES2554343T3 - Células posparto derivadas de tejido del cordón umbilical y métodos de preparación y uso de las mismas - Google Patents

Células posparto derivadas de tejido del cordón umbilical y métodos de preparación y uso de las mismas Download PDF

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ES2554343T3
ES2554343T3 ES10184306.8T ES10184306T ES2554343T3 ES 2554343 T3 ES2554343 T3 ES 2554343T3 ES 10184306 T ES10184306 T ES 10184306T ES 2554343 T3 ES2554343 T3 ES 2554343T3
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cells
umbilical cord
protein
cell
npe
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Ian Ross Harris
Alexander M. Harmon
Anthony J. Kihm
Darin J. Messina
Sanjay Mistry
Agnieszka Seyda
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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Abstract

Una población de células derivada de tejido del cordón umbilical humano, en la que dicha población de células es capaz de auto-renovación y expansión en cultivo, tiene el potencial de diferenciarse en células de otros fenotipos, es obtenible aislando en presencia de metaloproteasas, proteasas neutras y actividades de enzima mucolítica de tejido del cordón umbilical humano libre de sangre, y en la que las células: a) producen cada uno de CD10, CD13, CD44, CD73, CD90, PDGFr-alfa y HLA-A,B,C, como se detecta por citometría de flujo; y b) no producen cada uno de CD31, CD34, CD45, CD117, CD141 o HLA-DR,DP,DQ, como se detecta por citometría de flujo.

Description

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una secuencia siguiendo el uso de otras enzimas, ya que pueden degradar las otras enzimas que se usan. La temperatura y tiempo de contacto con las serina proteasas debe monitorizarse. Las serina proteasas pueden inhibirse con alfa 2 microglobulina en suero y, por tanto, el medio usado para la digestión está preferentemente libre de suero. Comúnmente se usan EDTA y DNasa y pueden mejorar los rendimientos o eficiencias. Métodos preferidos implican el tratamiento enzimático con, por ejemplo, colagenasa y dispasa, o colagenasa, dispasa e hialuronidasa, y se proporcionan métodos tales en los que en ciertas realizaciones preferidas una mezcla de colagenasa y la proteasa neutra dispasa se usan en la etapa de disociación. Son más preferidos aquellos métodos que emplean digestión en presencia de al menos una colagenasa de Clostridium histolyticum, y cualquiera de las actividades de proteasa, dispasa y termolisina. Son todavía más preferidos métodos que emplean digestión con tanto actividades de enzima colagenasa como dispasa. También se prefieren métodos que incluyen la digestión con una actividad de hialuronidasa, además de actividades de colagenasa y de dispasa. El experto apreciará que muchos de tales tratamientos con enzimas se conocen en la técnica para aislar células de diversas fuentes de tejido. Por ejemplo, es muy útil la serie LIBERASE Blendzyme (Roche) de combinaciones de enzimas y puede usarse en los presentes métodos. Se conocen otras fuentes de enzimas, y el experto también puede obtener tales enzimas directamente de sus fuentes naturales. El experto también está bien equipado para evaluar enzimas nuevas, o adicionales, o combinaciones de enzimas, para su utilidad en aislar las células de la invención. Tratamientos con enzima preferidos durante 0,5, 1, 1,5 o 2 horas o más. En otras realizaciones preferidas, el tejido se incuba a 37 ºC durante el tratamiento con enzima de la etapa de disociación. Diluir el digesto también puede mejorar el rendimiento de las células, ya que las células pueden estar atrapadas dentro de un digesto viscoso.
Aunque se prefiere actualmente el uso de actividades de enzima, no se quiere para métodos de aislamiento como se describe en el presente documento. Los métodos basados en la separación mecánica sola pueden ser satisfactorios en aislar las presentes células del cordón umbilical como se trata anteriormente.
Las células pueden resuspenderse después de disociarse el tejido en cualquier medio de cultivo como se trata en el presente documento anteriormente. Las células pueden resuspenderse tras una etapa de centrifugación para separar las células del tejido u otros residuos. La resuspensión puede implicar métodos mecánicos de resuspensión,
o simplemente la adición de medio de cultivo a las células.
El proporcionar las condiciones de crecimiento permite un amplio intervalo de opciones en cuanto al medio de cultivo, enriquecimientos, condiciones atmosféricas y humedad relativa para las células. Una temperatura preferida es 37 ºC, sin embargo, la temperatura puede oscilar de aproximadamente 35 ºC a 39 ºC dependiendo de las otras condiciones de cultivo y el uso deseado de las células o cultivo.
Actualmente se prefieren métodos que proporcionan células que no requieren factores de crecimiento exógenos, excepto como están disponibles en el suero enriquecido proporcionado con el medio de crecimiento. También en el presente documento se proporcionan métodos de derivación de células umbilicales capaces de expansión en ausencia de factores de crecimiento particulares. Los métodos son similares al método anterior; sin embargo, requieren que los factores de crecimiento particulares (para los que las células no tienen requisito) estén ausentes en el medio de cultivo en el que las células van a resuspenderse y crecerán en él por último lugar. En este sentido, el método es selectivo para aquellas células capaces de división en ausencia de factores de crecimiento particulares. Células preferidas en algunas realizaciones son capaces de crecimiento y expansión en medios de crecimiento químicamente definidos sin suero añadido. En tales casos, las células pueden requerir ciertos factores de crecimiento, que pueden añadirse al medio para soportar y sostener las células. Los factores actualmente preferidos que van a añadirse para el crecimiento sobre medios libre de suero incluyen uno o más de FGF, EGF, IGF y PDGF. En realizaciones más preferidas, dos, tres o los cuatro de los factores se añaden a medios libres de suero o químicamente definidos. En otras realizaciones, LIF se añade al medio sin suero para soportar o mejorar el crecimiento de las células.
También se describen métodos en los que las células pueden expandirse en presencia de aproximadamente el 5 % a aproximadamente el 20 % de oxígeno en su atmósfera. Métodos para obtener células que requieren L-valina requieren que esas células se cultiven en presencia de L-valina. Después de obtenerse una célula, su necesidad de L-valina puede probarse y confirmarse cultivando sobre medio que contiene D-valina que carece del isómero L.
Se describen métodos en los que las células pueden someterse a al menos 25, 30, 35 o 40 duplicaciones antes de alcanzar un estado senescente. Se describen métodos para derivar células capaces de duplicación para alcanzar 1014 células o más. Se prefieren aquellos métodos que derivan células que pueden duplicarse suficientemente para
1014 1011 1016 1017
producir al menos aproximadamente , , o o más células cuando se siembran a de aproximadamente 103 a aproximadamente 106 células/cm2 en cultivo. Preferentemente, estos números de células se producen dentro de 80, 70 o 60 días o menos.
En el presente documento también se proporcionan células derivadas de tejido del cordón umbilical humano aisladas derivadas por el método anterior. Las células mantienen un cariotipo normal coherente a pesar de someterse a pases repetidos en ciertas realizaciones. También se proporcionan cultivos de células derivadas de tejido del cordón umbilical humano derivadas por el método anterior, en los que los cultivos están libres de células maternas. Se proporcionan cultivos terapéuticos que se obtienen por los métodos de la divulgación.
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Aislamiento de células usando combinaciones de enzimas y condiciones de crecimiento diferentes. Las células se unieron y se expandieron bien entre el pase 0 y 1 bajo todas las condiciones probadas para la digestión y crecimiento de enzimas (Tabla 1-2). Las células en las condiciones experimentales 5-8 y 13-16 proliferaron bien hasta 4 pases después de la siembra, momento en el que se criopreservaron. Todas las células se criopreservaron para análisis posterior.
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Tabla 1-2: Aislamiento y expansión del cultivo de células posparto bajo condiciones variables:
Condición
Medio 15% FBS BME Gelatina 20% O2 Factores de crecimiento
1
DMEM-Lg Y Y Y Y N
2
DMEM-Lg Y Y Y N (5%) N
3
DMEM-Lg Y Y N Y N
4
DMEM-Lg Y Y N N (5%) N
5
DMEM-Lg N (2%) Y N (Laminina) Y EGF/FGF (20 ng/ml)
6
DMEM-Lg N (2%) Y N (Laminina) N (5%) EGF/FGF (20 ng/ml)
7
DMEM-Lg N (2%) Y N (Fibronectina) Y PDGF/VEGF
8
DMEM-Lg N (2%) Y N (Fibronectina) N (5%) PDGF/VEGF
9
DMEM-Lg Y N Y Y N
10
DMEM-Lg Y N Y N (5%) N
11
DMEM-Lg Y N N Y N
12
DMEM-Lg Y N N N (5%) N
13
DMEM-Lg N (2%) N N (Laminina) Y EGF/FGF (20 ng/ml)
14
DMEM-Lg N (2%) N N (Laminina) N (5%) EGF/FGF (20 ng/ml)
15
DMEM-Lg N (2%) N N (Fibronectina) Y PDGF/VEGF
16
DMEM-Lg N (2%) N N (Fibronectina) N (5%) PDGF/VEGF
Aislamiento de células de sangre residual en los cordones. Las células nucleadas se unieron y crecieron rápidamente. Estas células se analizaron por citometría de flujo y fueron similares a las células obtenidas por digestión con enzimas.
Aislamiento de células de sangre del cordón. Las preparaciones contuvieron glóbulos rojos y plaquetas. No se unieron células nucleadas y se dividieron durante las 3 primeras semanas. El medio se cambió 3 semanas después de la siembra y no se observó que se unieran y crecieran células.
45 Resumen. Podrían aislarse poblaciones de células de tejido umbilical usando eficazmente la combinación de enzimas colagenasa (una metaloproteasa), dispasa (proteasa neutra) e hialuronidasa (enzima mucolítica que degrada ácido hialurónico). También puede usarse LIBERASE, que es una mezcla de colagenasa y una proteasa neutra. También se usó Blendzyme 3, que es colagenasa (4 unidades de Wunsch/gramo) y termolisina (1714 unidades de caseína/gramo), junto con hialuronidasa para aislar células. Estas células se expandieron fácilmente durante muchos pases cuando se cultivaron en medio de crecimiento de expansión sobre plástico recubierto de gelatina.
También se aislaron células de sangre residual en los cordones, pero no sangre del cordón. La presencia de células
55 en coágulos de sangre lavados del tejido, que se adhieren y crecen en las condiciones usadas, puede ser debida a células que se liberan durante el proceso de disección.
Referencia
1. Ho, Tony, W., et al., WO2003025149 A2 “CELL POPULATIONS WHICH CO-EXPRESS CD49C AND CD90” NEURONYX, INC. Solicitud Nº US0229971 US, presentada 20020920, A2 publicada 20030327, A3 publicada 20031218.
EJEMPLO 2
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Características de crecimiento de células derivadas del cordón umbilical
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estadística, Universidad de Stanford (www-stat.stanford.edu/~tibs/SAM/).
Resultados
Se analizaron catorce poblaciones diferentes de células en este estudio. Las células junto con la información de los pases, sustrato de cultivo y medios de cultivo se enumeran en la Tabla 6-1.
Tabla 6-1. Células analizadas por el estudio de micromatrices. Las líneas de células se enumeran por su código de
identificación junto con el pase en el momento del análisis, sustrato de crecimiento celular y medio de crecimiento.
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25
30
Populación células
Paso Substrato Media
Umbilical (022803)
2 Gelatina DMEM,15%FBS,βME
Umbilical (042103)
3 Gelatina DMEM,15%FBS,βME
Umbilical (071003)
4 Gelatina DMEM,15%FBS,βME
Placenta (042203)
12 Gelatina DMEM,15%FBS,βME
Placenta (042903)
4 Gelatina DMEM,15%FBS,βME
Placenta (071003)
3 Plástico DMEM,15%FBS,βME
ICBM (070203) (5% O2)
3 Plástico MEM 10% FBS
ICBM (062703) (std O2)
5 Plástico MEM 10% FBS
ICBM (062703)(5% O2)
5 Plástico MEM 10% FBS
hMSC (Lot 2F1655)
3 Plástico MSCGM
hMSC (Lot 2F1656)
3 Plástico MSCGM
hMSC (Lot 2F1657)
3 Plástico MSCGM
hFibroblasto (9F0844)
9 Plástico DMEM-F12, 10% FBS
hFibroblasto (CCD39SK)
4 Plástico FBS DMEM-F12, 10% FBS
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Los datos se evaluaron por Análisis de Componentes Principales con el software SAM como se ha descrito anteriormente. Los análisis revelaron 290 genes que se expresaron en diferentes cantidades relativas en las células probadas. Este análisis proporcionó comparaciones relativas entre las poblaciones.
40 La Tabla 6-2 muestra las distancias euclídeas que se calcularon para la comparación de los pares de células. Las distancias euclídeas se basaron en la comparación de las células basándose en los 290 genes que se expresaron diferencialmente entre los tipos de células. La distancia euclídea es inversamente proporcional a la similitud entre la expresión de los 290 genes.
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Tabla 6-2. Distancias euclídeas para los pares de células. La distancia euclídea se calculó para los tipos de células usando los 290 genes que se expresaron diferencialmente entre los tipos de células. La similitud entre las células es inversamente proporcional a la distancia euclídea.
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Par de células
ICBM-hMSC
Placenta-umbilical
ICBM-Fibroblasto
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ICBM-placenta
Fibroblasto-MSC
ICBM-Umbilical
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Fibroblasto-Umbilical
MSC-Placenta
MSC-Umbilical
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ICBM-placenta
Distancia Euclidea
24.71
25.52
36.44
37.09
39.63
40.15
41.59
42.84
46.86
48.41
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Las Tablas 6-3, 6-4 y 6-5 muestran la expresión de genes elevada en células derivadas de la placenta (Tabla 6-3), elevada en células derivadas del cordón umbilical (Tabla 6-4) y reducida en células derivadas del cordón umbilical y de la placenta (Tabla 6-5).
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Tabla 6-3. Genes que tienen específicamente expresión elevada en las células derivadas de la placenta en comparación con las otras líneas celulares ensayadas.
Genes incrementados en células derivadas de Placenta
ID conjunto pruebas
Nombre de Gen NCBI Número de acceso
209732_at
C-type (calcium dependent, carbohydrate-recognition domain) lectin, superfamily member 2 (activationinduced) AF070642
206067_s_at
Wilms tumor 1 NM_024426
207016_s_at
aldehyde dehydrogenase 1 family, member A2 AB015228
206367_at
Renin NM_000537
210004_at
oxidized low density lipoprotein (lectin-like) receptor 1 AF035776
214993_at
Homo sapiens, clone IMAGE:4179671, mRNA, partial cds AF070642
202178_at
protein kinase C, zeta NM_002744
209780_at
hypothetical protein DKFZp564F013 AL136883
204135_at
downregulated in ovarian cancer 1 NM_014890
213542_at
Homo sapiens mRNA; cDNA DKFZp547K1113 (from clone DKFZp547K1113) AI246730
Tabla 6-4. Genes que se tienen específicamente expresión elevada en células derivadas del cordón umbilical en comparación con las otras líneas celulares ensayadas.
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40
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50
55
Genes incrementados en células derivadas de Umbilical
ID conjunto pruebas
ID conjunto pruebas
ID conjunto pruebas
202859_x_at
Interleukin 8 NM_000584
211506_s_at
Interleukin 8 AF043337
210222_s_at
reticulon 1 BC000314
204470_at
chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity NM_001511
206336_at
chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) NM_002993
207850_at
Chemokine (C-X-C motif) ligand 3 NM_002090
203485_at
reticulon 1 NM_021136
202644_s_at
tumor necrosis factor, alpha-induced protein 3 NM_006290
31
5
10
15
20
25
30
35
40
45
50
55
60

Tabla 6-5. Genes que tienen expresión reducida en células del cordón umbilical y de la placenta en comparación con las otras líneas celulares ensayadas.
Genes incrementados en células derivadas de Placenta y de Umbilical
ID conjunto pruebas
Nombre de Gen NCBI Número de acceso
210135_s_at
short stature homeobox 2 AF022654.1
205824_at
heat shock 27kDa protein 2 NM_001541.1
209687_at
chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) U19495.1
203666_at
chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) NM_000609.1
212670_at
elastin (supravalvular aortic stenosis, Williams-Beuren syndrome) AA479278
213381_at
Homo sapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022) N91149
206201_s_at
mesenchyme homeobox 2 (growth arrest-specific homeobox) NM_005924.1
205817_at
Sine oculis homeobox homolog 1 (Drosophila) NM_005982.1
209283_at
crystallin, alpha B AF007162.1
212793_at
dishevelled associated activator of morphogenesis 2 BF513244
213488_at
DKFZP586B2420 protein AL050143.1
209763_at
similar to neuralin 1 AL049176
205200_at
Tetranectin (plasminogen binding protein) NM_003278.1
205743_at
src homology three (SH3) and cysteine rich domain NM_003149.1
200921_s_at
B-cell translocation gene 1, anti-proliferative NM_001731.1
206932_at
cholesterol 25-hydroxylase NM_003956.1
204198_s_at
runt-related transcription factor 3 AA541630
219747_at
hypothetical protein FLJ23191 NM_024574.1
204773_at
Interleukin 11 receptor, alpha NM_004512.1
202465_at
Procollagen C-endopeptidase enhancer NM_002593.2
203706_s_at
Frizzled homolog 7 (Drosophila) NM_003507.1
212736_at
hypothetical gene BC008967 BE299456
214587_at
Collagen, type VIII, alpha 1 BE877796
201645_at
Tenascin C (hexabrachion) NM_002160.1
210239_at
iroquois homeobox protein 5 U90304.1
203903_s_at
Hephaestin NM_014799.1
205816_at
integrin, beta 8 NM_002214.1
203069_at
synaptic vesicle glycoprotein 2 NM_014849.1
213909_at
Homo sapiens cDNA FLJ12280 fis, clone MAMMA1001744 AU147799
206315_at
cytokine receptor-like factor 1 NM_004750.1
32
5
10
15
20
25
30
35
40
45
50
55
60
65
Genes incrementados en células derivadas de Placenta y de Umbilical
ID conjunto pruebas
Nombre de Gen NCBI Número de acceso
204401_at
potassium intermediate/small conductance calciumactivated channel, subfamily N, member 4 NM_002250.1
216331_at
integrin, alpha 7 AK022548.1
209663_s_at
integrin, alpha 7 AF072132.1
213125_at
DKFZP586L151 protein AWO07573
202133_at
transcriptional co-activator with PDZ-binding motif (TAZ) AA081084
206511_s_at
Sine oculis homeobox homolog 2 (Drosophila) NM_016932.1
213435_at
KIAA1034 protein AB028957.1
206115_at
early growth response 3 NM_004430.1
213707_s_at
distal-less homeobox 5 NM_005221.3
218181_s_at
hypothetical protein FLJ20373 NM_017792.1
209160_at
aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II) AB018580.1
213905_x_at
Biglycan AA845258
201261_x_at
Biglycan BC002416.1
202132_at
transcriptional co-activator with PDZ-binding motif (TAZ) AA081084
214701_s_at
fibronectin 1 AJ276395.1
213791_at
Proenkephalin NM_006211.1
205422_s_at
Integrin, beta-like 1 (with EGF-like repeat domains) NM_004791.1
214927_at
Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422 AL359052.1
206070_s_at
EphA3 AF213459.1
212805_at
KIAA0367 protein AB002365.1
219789_at
natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide receptor C) AI628360
219054_at
hypothetical protein FLJ14054 NM_024563.1
213429_at
Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp564B222) AWO25579
204929_s_at
vesicle-associated membrane protein 5 (myobrevin) NM_006634.1
201843_s_at
EGF-containing fibulin-like extracellular matrix protein 1 NM_004105.2
221478_at
BCL2/adenovirus E1B 19kDa interacting protein 3-like AL132665.1
201792_at
AE binding protein 1 NM_001129.2
204570_at
cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) NM_001864.1
201621_at
neuroblastoma, suppression of tumorigenicity 1 NM_005380.1
202718_at
Insulin-like growth factor binding protein 2, 36kDa NM_000597.1
33
Las Tablas 6-6, 6-7 y 6-8 muestran la elevada expresión de genes en fibroblastos humanos (Tabla 6-6), células ICBM (Tabla 6-7) y MSC (Tabla 6-8).
10
15
20
25
30
35
40

Tabla 6-6. Genes que tienen expresión elevada en fibroblastos en comparación con las otras líneas celulares ensayadas.
Genes incrementados en Fibroblastos
dual specificity phosphatase 2
KIAA0527 protein
Homo sapiens cDNA: FLJ23224 fis, clone ADSU02206
dynein, cytoplasmic, intermediate polypeptide 1
ankyrin 3, node of Ranvier (ankyrin G)
inhibin, beta A (activin A, activin AB alpha polypeptide)
ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function)
KIAA1053 protein
microtubule-associated protein 1A
zinc finger protein 41
HSPC019 protein
Hobo sapiens cDNA: FLJ23564 fis, clone LNG10773
Homo sapiens mRNA; cDNA DKFZp564A072 (from clone DKFZp564A072)
LIM protein (similar to rat protein kinase C-binding enigma)
inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein
hypothetical protein FLJ22004
Human (clone CTG-A4) mRNA sequence
ESTs, Moderately similar to cytokine receptor-like factor 2; cytokine receptor CRL2 precursor [Homo sapiens]
transforming growth factor, beta 2
hypothetical protein MGC29643
antigen identified by monoclonal antibody MRC OX-2
putative X-linked retinopathy protein
Tabla 6-7. Genes que tienen expresión elevada en las células derivadas de ICBM en comparación con las otras líneas celulares ensayadas.
Genes Incrementados en Células ICBM
50
•
cardiac ankyrin repeat protein
•
MHC class I region ORF
•
integrin, alpha 10
•
hypothetical protein FLJ22362 55 •UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3)
•
interferon-induced protein 44
•
SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal)
•
keratin associated protein 1-1
•
hippocalcin-like 1 60 •jagged 1 (Alagille syndrome)
•proteoglycan 1, secretory granule
34
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5
10
15
20
25
30
35
40
45
50
55
60
65

Tabla 10-2. Datos de la reacción de linfocitos mixtos -Línea celular A (cordón umbilical) DPM para el ensayo de proliferación en placa ID: Placa 1
Número Analítico
Sistema de Cultura Proliferación de línea de base de receptor Control de la Replicas 1 2 3 Media SD CV
1074 406 391
623.7 390.07 62.5
autoestimulación (mitomicina C tratada células autólogas)
672 510 1402 861.3 475.19 55.2
IM04-2478
MLR alogénico IM04-2477
donante (mitomicina C tratada) MLR con la línea celular
43777 48391 38231 43466.3 5087.12 11.7
(mitomicina C tratada tipo de célula A)
2914 5622 6109 4881.7 1721.36 35.3
SI (donante) SI (línea de célula)
70 8
Proliferación de línea de base de receptor Control de la
530 508 527 521.7 11.93 2.3
autoestimulación (mitomicina C tratada células autólogas)
701 567 1111 793.0 283.43 35.7
IM04-2479
MLR alogénico IM04-2477
donante (mitomicina C tratada) MLR con la línea celular
25593 24732 22707 24344.0 1481.61 6.1
(mitomicina C tratada tipo de célula A)
5086 3932 1497 3505.0 1832.21 52.3
SI (donante) SI (línea de célula)
47 7
Proliferación de línea de base de receptor Control de la
1192 854 1330 1125.3 244.90 21.8
autoestimulación (mitomicina C tratada células autólogas)
2963 993 2197 2051.0 993.08 48.4
IM04-2480
MLR alogénico IM04-2477
donante (mitomicina C tratada) MLR con la línea celular
25416 29721 23757 26298.0 3078.27 11.7
(mitomicina C tratada tipo de célula A)
2596 5076 3426 3699.3 1262.39 34.1
SI (donante) SI (línea de célula)
23 3
43
5
10
15
20
25
30
35
40
45
50
55
60
Número Analítico
Sistema de Cultura Proliferación de línea de base de receptor Control de la autoestimulación (mitomicina C tratada células autólogas) MLR alogénico IM04-2477 donante (mitomicina C tratada) MLR con la línea celular (mitomicina C tratada tipo de célula A) Réplicas 1 2 3 Media SD CV
IM04-2481
695 451 738 1252 13177 24885 4495 3671 555 464 15444 4674 567.0 818.0 17835.3 4280.0 122.44 400.04 6209.52 534.95 21.6 48.9 34.8 12.5
SI (donante) SI (línea de célula)
31 8
ID Plato: Plato 2
Número Analítico
Sistema de Cultura Proliferación de línea de base de receptor Control de la autoestimulación (mitomicina C tratada células autólogas) MLR alogénico IM04-2477 donante (mitomicina C tratada) MLR con la línea celular (mitomicina C tratada tipo de célula A) Réplicas 1 2 3 Media SD CV
IM04-2482
432 533 1459 633 24286 30823 2762 1502 274 598 31346 6723 413.0 896.7 28818.3 3662.3 130.54 487.31 3933.82 2724.46 31.6 54.3 13.7 74.4
SI (donante) SI (línea de célula)
70 9
IM04-2477 (donante alogénico)
Proliferación de línea de base de receptor Control de la autoestimulación (mitomicina C tratada células autólogas) 312 419 567 604 349 374 360.0 515.0 54.34 123.50 15.1 24.0
Linea de célula tipo A
Proliferación de línea de base de receptor Control de la autoestimulación (mitomicina C tratada células autólogas) 5101 3735 1924 4570 2973 2153 3936.3 2882.3 1078.19 1466.04 27.4 50.9
44
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anticuerpo anti-factor de tejido, las células se incubaron con 20 microgramos/mililitro de CNTO 859 (Centocor, Malvern, PA) durante 30 minutos. Se añadió cloruro de calcio (30 microlitros) a cada pocillo. La placa se sembró inmediatamente en un lector de microplacas de temperatura controlada y se midió la absorbancia a 405 nanómetros a intervalos de 40 segundos durante 30 minutos.
5 Tinción de anticuerpos. Las células se lavaron en PBS y se desprendieron del matraz con tripsina/EDTA (Gibco Carlsbad, CA). Las células se recogieron, se centrifugaron y se resuspendieron en 3 % (v/v) de FBS en PBS a una concentración de células de 1x107 por mililitro. Se añadió anticuerpo a 100 microlitros de suspensión de células según las especificaciones del fabricante. Las células se incubaron en la oscuridad durante 30 minutos a 4 ºC.
10 Después de la incubación, las células se lavaron con PBS, a continuación se centrifugaron a 150 x g durante 5 minutos para eliminar el anticuerpo sin unir. Las células se resuspendieron en 100 microlitros de 3 % de FBS y se añadió anticuerpo secundario según las instrucciones del fabricante. Las células se incubaron en la oscuridad durante 30 minutos a 4 ºC. Después de la incubación, las células se lavaron con PBS y se centrifugaron para eliminar el anticuerpo secundario no unido. Las células lavadas se resuspendieron en 500 microlitros de PBS y se
15 analizaron por citometría de flujo.
Análisis de citometría de flujo. El análisis de citometría de flujo se realizó con un instrumento FACSCalibur (Becton Dickinson, San Jose, CA).
20 Resultados
El análisis de citometría de flujo reveló que las células posparto derivadas del cordón umbilical son menos activas en promover la coagulación del plasma que las células J82. Aunque un ensayo de coagulación del plasma demostró que el factor de tejido presente en las células derivadas del cordón umbilical era activo, la coagulación duró más que
25 con las células J-82, como se prueba por el mayor tiempo hasta la absorbancia al 50 % (T½ hasta el máx; Tabla 111). El T ½ hasta el máx es inversamente proporcional al número de células J-82. Las células derivadas del cordón umbilical disminuyeron la tasa de coagulación como se indica por el T ½ hasta el máx. La coagulación se observó con tanto células sometidas a pases tempranos (P5) como tardíos (P 18). La preincubación de células umbilicales con CNTO 859, un anticuerpo para factor de tejido, inhibió la reacción de coagulación estableciendo que el factor de
30 tejido era responsable de la coagulación.
35
40
45
50
55
60
65

Tabla 11-1. El efecto del factor de tejido humano (Simplastin®) y las células derivadas del cordón umbilical (Umb) sobre la coagulación del plasma. El tiempo hasta la absorbancia al 50 % (T½ hasta el máx.) en la meseta en segundos se usó como unidad de medición.
Estandar (Disolución 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
Umb P5
50,000
833
Umb P18
50,000
443
46
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15
25

Tabla 14-2. Resumen de condiciones para el protocolo de diferenciación de dos etapas
A
B
COND. #
PRE-DIFERENCIACIÓN 2ª ETAPA DIF.
1
NPE + F (20 ng/ml) + E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml)
2
NPE + F (20 ng/ml) + E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) + RA (1 micromolar)
3
NPE + F (20 ng/ml) + E (20 ng/ml) NPE + RA (1 micromolar)
4
NPE + F (20 ng/ml) + E (20 ng/ml) NPE + F (20 ng/ml) + E (20 ng/ml)
5
NPE + F (20 ng/ml) + E (20 ng/ml) Crecimiento Medio
6
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 1B + rhGDF-5 (20 ng/ml)
7
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 1B + BMP7 (20 ng/ml)
8
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 1B + GDNF (20 ng/ml)
9
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 2B + rhGDF-5 (20 ng/ml)
10
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 2B + BMP7 (20 ng/ml)
11
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 2B + GDNF (20 ng/ml)
12
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 3B + rhGDF-5 (20 ng/ml)
13
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 3B + BMP7 (20 ng/ml)
14
NPE + F (20 ng/ml) + E (20 ng/ml) Condición 3B + GDNF (20 ng/ml)
15
NPE + F (20 ng/ml) + E (20 ng/ml) NPE + rhGDF-5 (20 ng/ml)
16
NPE + F (20 ng/ml) + E (20 ng/ml) NPE + BMP7 (20 ng/ml)
17
NPE + F (20 ng/ml) + E (20 ng/ml) NPE + GDNF (20 ng/ml)
35 Protocolo de inducción de múltiples factores de crecimiento. Se descongelaron PPDC derivadas del cordón umbilical (P11) y el cultivo se expandió en medio de crecimiento a 5.000 células/cm2 hasta que se alcanzó la subconfluencia (75 %). A continuación, las células se tripsinaron y se sembraron a 2.000 células/cm2, sobre placas de 24 pocillos recubiertas de laminina (BD Biosciences) en presencia de NPE + F (20 nanogramos/mililitro) + E (20 nanogramos/mililitro). Además, algunos pocillos contuvieron NPE + F + E + 2 % de FBS o 10 % de FBS. Después de cuatro días de condiciones de “pre-diferenciación”, se extrajeron todos los medios y las muestras se cambiaron a medio NPE enriquecido con erizo sónico (SHH; 200 nanogramos/mililitro; Sigma, St. Louis, MO), FGF8 (100 nanogramos/mililitro; Peprotech), BDNF (40 nanogramos/mililitro; Sigma), GDNF (20 nanogramos/mililitro; Sigma) y ácido retinoico (1 micromolar; Sigma). Siete días después del cambio de medio, los cultivos se fijaron con 4 %
45 (peso/volumen) de paraformaldehído frío en hielo (4 ºC) (Sigma) durante 10 minutos a temperatura ambiente, y se tiñeron para la expresión de nestina humana, GFAP, TuJ1, desmina y alfa-actina de músculo liso.
Protocolo de co-cultivo de progenitores neurales. Se sembraron progenitores hipocámpicos de rata adulta (062603) como neuroesferas o células individuales (10.000 células/pocillo) sobre placas de 24 pocillos recubiertas de laminina (BD Biosciences) en NPE + F (20 nanogramos/mililitro) + E (20 nanogramos/mililitro).
Se descongelaron las PPDC derivadas del cordón umbilical (P11) y el cultivo se expandió en NPE + F (20 nanogramos/mililitro) + E (20 nanogramos/mililitro) a 5.000 células/cm2 durante un periodo de 48 horas. A continuación, las células se tripsinaron y se sembraron a 2.500 células/pocillo sobre cultivos existentes de
55 progenitores neurales. El medio existente se intercambió por medio fresco. Cuatro días después, los cultivos se fijaron con 4 % (peso/volumen) de paraformaldehído frío en hielo (4 ºC) (Sigma) durante 10 minutos a temperatura ambiente, y se tiñeron para proteína nuclear humana (hNuc, Chemicon) (Tabla 14-1 anteriormente) para identificar PPDC.
Inmunocitoquímica. Se realizó inmunocitoquímica usando los anticuerpos enumerados en la Tabla 14-1. Los cultivos se lavaron con solución salina tamponada con fosfato (PBS) y se expusieron a una disolución de bloqueo de proteína que contenía PBS, 4 % (v/v) de suero de cabra (Chemicon, Temecula, CA) y 0,3 % (v/v) de Triton (Triton X100; Sigma) durante 30 minutos para acceder a los antígenos intracelulares. Los anticuerpos primarios, diluidos en disolución de bloqueo, se aplicaron a continuación a los cultivos durante un periodo de 1 hora a temperatura
65 ambiente. Se eliminaron las disoluciones de anticuerpo primario y los cultivos se lavaron con PBS antes de la aplicación de disoluciones de anticuerpo secundario (1 hora a temperatura ambiente) que contenían disolución de
52
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25
35
45
55
65

Tabla 14-3. Resultados de la tinción para nestina humana, GFAP y TuJ1, respectivamente en el experimento de diferenciación de dos etapas. Obsérvese que + significa que al menos una porción (> 0 %) de las células fueron positivas para la tinción indicada. Nestina humana: citoblastos y células progenitoras neurales inmaduros; GFAP: astrocitos; TuJ1: neuronas inmaduras y maduras.
CONDICIÓN
Fibroblastos Umbilical PPDCs Progenitores Neurales
1
-/-/ -/-/ +/+/+
2
-/-/ -/-/ +/+/+
3
-/-/ -/-/ +/+/+
4
-/-/ -/-/ +/+/+
5
-/-/ -/-/ +/+/+
6
-/-/ -/-/ +/+/+
7
-/-/ -/-/ +/+/+
8
-/-/ -/-/ +/+/+
9
-/-/ -/-/ +/+/+
10
-/-/ -/-/ +/+/+
11
-/-/ -/-/ +/+/+
12
-/-/ -/-/ +/+/+
13
-/-/ -/-/ +/+/+
14
-/-/ -/-/ +/+/+
15
-/-/ -/-/ +/+/+
16
-/-/ -/-/ +/+/+
17
-/-/ -/-/ +/+/+
Resultados de la inducción de múltiples factores de crecimiento. Tras una exposición de una semana a una variedad de agentes de diferenciación neural, las células se tiñeron para marcadores indicativos de progenitores neurales (nestina humana), neuronas (TuJ1) y astrocitos (GFAP). Las células cultivadas en la primera etapa en medio que no contenía suero tuvieron morfologías diferentes de aquellas células en medio que contenía suero (2 %
o 10 %), que indica posible diferenciación neural. Específicamente, tras un procedimiento de dos etapas de exposición de PPDC umbilicales a EGF y bFGF, seguido de SHH, FGF8, GDNF, BDNF y ácido retinoico, las células mostraron procesos extendidos largos similares a las morfología de astrocitos cultivados. Cuando se incluyeron 2 % de FBS o 10 % de FBS en la primera etapa de diferenciación, aumentó el número de células y la morfología de la célula no cambió de cultivos de control a alta densidad. La posible diferenciación neural no se demostró por análisis inmunocitoquímico para nestina humana, TuJ1 o GFAP.
Procedimientos de co-cultivo de progenitores neurales y PPDC. Se sembraron células derivadas del cordón umbilical sobre cultivos de progenitores neurales de rata dos días antes en condiciones de expansión neural (NPE + F + E). Aunque la confirmación visual del cordón umbilical en placa demostró que estas células se sembraron como células individuales, la tinción nuclear específica de ser humano (hNuc) 4 días después de la siembra (duración total del experimento de 6 días) mostró que tendieron a formar una pelota y evitar el contacto con los progenitores neurales. Además, si las células del cordón umbilical se unieron, estas células se extendieron y pareció que estaban inervadas por neuronas diferenciadas que fueron de origen de rata, sugiriendo que las células umbilicales pueden haberse diferenciado en células de músculo. Esta observación se basó en la morfología bajo microscopía de contraste de fases. Otra observación fue que cuerpos de células normalmente grandes (más grandes que los progenitores neurales) poseyeron morfologías que se parecieron a los progenitores neurales, con procesos delgados que abarcaban múltiples direcciones. La tinción con HNuc (encontrada en la mitad del núcleo de la célula) sugirió que en algunos casos estas células humanas pueden haberse fusionado con progenitores de rata y asumido su fenotipo. Los pocillos de control que contenían progenitores neurales solo tuvieron menos progenitores totales y células diferenciadas evidentes que los pocillos de co-cultivo que contenían cordón umbilical, que indica adicionalmente que las células derivadas del cordón umbilical influyeron en la diferenciación y comportamiento de progenitores neurales tanto por la liberación de quimiocinas y citocinas, como por efectos mediados por el contacto.
Resumen. Se realizaron múltiples protocolos para determinar el potencial a corto plazo de las PPDC derivadas del cordón umbilical de diferenciarse en células de linaje neural. Éstos incluyeron obtención de imágenes por contraste
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