ES2539105T3 - Células mesenquimatosas y osteoblastos procedentes de células madre embrionarias humanas - Google Patents

Células mesenquimatosas y osteoblastos procedentes de células madre embrionarias humanas Download PDF

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ES2539105T3
ES2539105T3 ES02756367.5T ES02756367T ES2539105T3 ES 2539105 T3 ES2539105 T3 ES 2539105T3 ES 02756367 T ES02756367 T ES 02756367T ES 2539105 T3 ES2539105 T3 ES 2539105T3
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cells
embryonic stem
human embryonic
mesenchymal
osteoblast
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Chunhui Xu
R. Scott Thies
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Asterias Biotherapeutics Inc
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Abstract

Un medio de cultivo para obtener osteoprogenitores y/u osteoblastos a partir de células madre embrionarias humanas (hES) o su progenie, que comprende BMP4, dexametasona y ácido ascórbico o uno de sus análogos.

Description

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E02756367
10-06-2015
Se transdujo una subpoblación para expresar la transcriptasa inversa de la telomerasa humana (hTERT). Esto se logró infectando con un constructo retrovírico pBABE puro hTERT, que contenía la secuencia codificante de hTERT controlada por MoLV LTR y el gen de resistencia a la puromicina controlado por el promotor temprano SV40. Se reemplazó el medio de crecimiento con una mezcla que contenía 5 mL de patrón retrovírico (1 x 106 pfu/mL) y 4 µg/mL de polybrene y se incubó a 37 ºC. Después de 8 h, se añadieron 5 mL adicionales de la mezcla retrovirus/polybrene y las células se incubaron a 37 ºC. Al día siguiente, la mezcla retrovirus/polybrene se retiró y se reemplazó con medio de crecimiento fresco. Al día siguiente se reemplazó el medio con medio de crecimiento suplementado con 0.5 microgramos/mL de puromicina. Se dividieron las células una vez a la semana con una relación de 1:4 durante 8 semanas en medio que contenía puromicina y se probaron para determinar su actividad telomerasa.
La Figura 2 (Cuadro A) muestra la morfología de la línea celular telomerizada HEF1. El cuadro B (debajo) muestra la actividad telomerasa, según se ha medido con el ensayo TRAP. Las células transducidas con el casete de expresión de hTERT mostraron una actividad telomerasa positiva 20 o 65 días después de la transducción. La línea celular sin transducir, o las células transducidas con el vector de control no mostraron actividad telomerasa. Tanto las células HEF1 transducidas con hTERT como las células transducidas con el vector de control, duplicaron su cantidad aproximadamente una vez cada 2 días, hasta el día 38, cuando las células de control cesaron de dividirse. Las células transfectadas con hTERT continuaron proliferando más allá de los 60 días (30 duplicaciones) con una velocidad de crecimiento constante.
El medio de crecimiento de las células ES se acondicionó como en el Ejemplo 8, utilizando células HEF1 irradiadas con 6000 rad, y se sembraron con una densidad de ~4.1 a 5.5 x 104 células cm-2. Se probó el medio para determinar su capacidad de favorecer el crecimiento de la línea celular hES H9 cultivada en un sustrato Matrigel®. Las células hES se mantuvieron utilizando el medio acondicionado para HEF1 durante más de 4 pases, mostraron morfología de células ES indiferenciadas y mantuvieron la expresión de hTERT y Oct-4.
Ejemplo de referencia 3: Diferenciación adicional en células similares a osteoblastos
Las células ES humanas (línea celular H1, pase 30) se mantuvieron en condiciones exentas de alimentadoras, como se ha descrito anteriormente. Para su uso en este experimento, las células hES se sembraron con una densidad de ~1 × 105 cm-2 en Matrigel® en medio acondicionado para mEF. Las células HEF1 telomerizadas se colocaron en placas con una densidad de 3.1 × 103 cm-2 en DMEM con un 10% de FBS, un 1% de aminoácidos no esenciales y Lglutamina 2 mM. Las células madre mesenquimatosas humanas (hMSC) normales se obtuvieron de BioWhittaker Inc., MD (una filial de Cambrex Co.). Se mantuvieron en medio de crecimiento para MSC (BioWhittaker Part n.º PT3001) de acuerdo con las indicaciones del proveedor. La línea celular de fibroblastos BJ5ta (Bodnar et al., Science 279:349, 1998) se mantuvo en un medio estándar constituido por un 10% de FBS en 1:3 M199/DMEM.
Dos días después del último pase, cada medio de cultivo se reemplazó por medio de inducción de osteoblastos (OIM) para inducir la diferenciación. La base del OIM fue el medio de crecimiento para MSC (ClonTech n.º de cat. PT-3238) (patente de los EE. UU. 5 486 359) suplementado con dexametasona 0.1 µM, ácido 2-fosfato ascórbico 5 µM, β-glicerofosfato 10 mM y 100 ng/mL de BMP-4. Las células se alimentaron con OIM fresco cada 2-3 días.
Después de 11 días en OIM, todas las células mostraron cambios en la morfología celular. Las células HEF1, hMSC y células BJ cambiaron de una forma en husillo a una forma cuboidal y algunas células se volvieron planas. Las células hES mostraron una morfología heterogénea que parecía ser una población diferenciada mixta.
Las células se fijaron con paraformaldehído al 2% en PBS durante 20 min, se lavaron con PBS y se analizaron para determinar los marcadores de osteoblastos. Se detectó la fosfatasa alcalina (AP) con sustrato Vector (Vector Laboratories, Inc., Burlingame, CA). La expresión de AP estuvo claramente ubicada en agrupaciones de células, células H1 diferenciadas así como también células HEF1, BJ y hMSC.
Las proteínas matriciales producidas por los osteoblastos, colágeno-1 y osteocalcina se detectaron mediante inmunotinción. Se permeabilizaron los cultivos mediante tratamiento con EtOH al 100% durante 2 min. Después de los lavados en PBS, se incubaron los cultivos con un 5% de suero de cabra normal en PBS durante 2 h y a continuación con anticuerpo de conejo primario contra el colágeno-1 (1:10, Monosan n.º de cat. P5041) u osteocalcina (1:50, Biomedical Technologies Inc. n.º de cat. 13T593). La tinción se reveló con el anticuerpo secundario anti-inmunoglobulina de conejo producido en cabra marcado con FITC (1:100, Southern Biotechnology Associates inc. n.º de cat. 4050-02).
La Figura 3 muestra los resultados. Los cuadros A y B muestran los resultados inmunocitoquímicos de los marcadores osteocalcina y colágeno-1. El cuadro C muestra la tinción de la actividad fosfatasa alcalina. Estos rasgos son característicos de las células del linaje de osteoblastos.
Estos datos son coherentes con la hipótesis de que tanto las células hES como las células HEF1 tienen la capacidad de generar osteoblastos cuando se someten a un protocolo de diferenciación apropiado in vitro.
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ES02756367.5T 2001-07-06 2002-07-03 Células mesenquimatosas y osteoblastos procedentes de células madre embrionarias humanas Expired - Lifetime ES2539105T3 (es)

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PCT/US2002/020998 WO2003004605A2 (en) 2001-07-06 2002-07-03 Mesenchymal cells and osteoblasts from human embryonic stem cell

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