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ES2579909T3 - Medio de cultivo para células madre epiteliales y organoides que comprenden dichas células madre - Google Patents

Medio de cultivo para células madre epiteliales y organoides que comprenden dichas células madre Download PDF

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ES2579909T3
ES2579909T3 ES10703131.2T ES10703131T ES2579909T3 ES 2579909 T3 ES2579909 T3 ES 2579909T3 ES 10703131 T ES10703131 T ES 10703131T ES 2579909 T3 ES2579909 T3 ES 2579909T3
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stem cells
crypts
small intestine
colon
epithelial stem
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Toshiro Sato
Johannes Carolus Clevers
Meritxell Huch Ortega
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Koninklijke Nederlandse Akademie van Wetenschappen
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Abstract

Método para cultivar células madre epiteliales de intestino delgado o de colon, o fragmentos de tejido que comprenden dichas células madre epiteliales, por ejemplo, criptas de colon o criptas de intestino delgado aisladas, comprendiendo el método proporcionar una matriz extracelular; incubar células madre epiteliales de intestino delgado o de colon, o fragmentos de tejido que comprenden dichas células madre epiteliales, por ejemplo, criptas de colon o criptas de intestino delgado aisladas, con la matriz extracelular; y cultivar las células madre epiteliales de intestino delgado o de colon, o los fragmentos de tejido que comprenden dichas células madre epiteliales, por ejemplo, criptas de colon o criptas de intestino delgado aisladas, en presencia de un medio de cultivo celular, que comprende un medio basal para células de animales o humanas al que se añade: un inhibidor de la proteína morfogenética ósea (BMP); y entre 5 ngram/ml y 500 ngram/ml de un factor de crecimiento mitogénico seleccionado de entre el factor de crecimiento epidérmico (EGF), el factor de crecimiento de transformación alfa, el factor de crecimiento de fibroblastos básico, el factor neurotrófico derivado del cerebro y el factor de crecimiento queratinocítico; y opcionalmente un agonista de Wnt.

Description

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sustituirse por "consistir esencialmente en" refiriéndose a que un producto tal como se define en el presente documento puede comprender otro(os) componente(s) adicional(es) además de los identificados específicamente, no modificando dicho(s) componente(s) adicional(es) la característica única de la invención. Además, un método como se define en el presente documento puede comprender otra(s) etapa(s) adicional(es) además de las identificadas específicamente, no modificando dicha(s) etapa(s) adicional(es) la característica única de la invención. Además, la referencia a un elemento mediante el artículo indefinido "un" o "una" no excluye la posibilidad de haya más de uno de los elementos, a menos que el contexto requiera claramente que haya uno y sólo uno de los elementos. Por lo tanto, el artículo indefinido "un" o "una" se refiere por lo general a "al menos uno/a". El término "aproximadamente" cuando se utiliza en asociación con un valor numérico (aproximadamente 10) se refiere preferentemente a que el valor puede ser el valor dado de 10 más o menos un 1% del valor.
Los siguientes ejemplos se ofrecen con fines ilustrativos solamente, y no pretenden limitar el alcance de la presente invención en modo alguno.
Descripción de las figuras
Figura 1. Necesidad de factores de crecimiento del cultivo de criptas.
a: Se sembraron 500 criptas con EGF (E; 0 ng/ml-50 ng/ml) y R-espondina 1 (R: 0 ng/ml-500 ng/ml) por triplicado; los organoides de cripta se contaron 7 días después de la siembra.
b: Se cultivaron 500 criptas/organoides de cripta con EGF (50 ng/ml) y R-espondina 1 (500 ng/ml) con las cantidades indicadas de nogina y, se siguieron durante 3 pases. Se contaron los organoides de cripta en cada pase. El experimento se repitió tres veces con resultados comparables.
Figura 2. Establecimiento del sistema de cultivo de las criptas intestinales.
a: Evolución temporal de una sola cripta aislada desarrollándose hasta formar un organoide. La imagen de contraste de interferencia diferencial pone de manifiesto células de Paneth que contienen gránulos en el fondo de las criptas (flechas). b, c: Las criptas aisladas individuales forman de manera eficaz organoides de cripta. A través de una repetida fisión de cripta, las estructuras generan numerosos organoides de cripta con forma de pulpo el día 14.
d: Imagen confocal reconstruida en 3D de un solo organoide después de un cultivo de 3 semanas. Las células madre Lgr5-GFP+ (gris claro) se localizan en la punta de los dominios de tipo cripta. Tinción de contraste para ADN: ToPro-3 (gris oscuro).
e: Representación esquemática de un organoide de cripta. El organoide consiste en una luz central revestida de epitelio de tipo vellosidad y varios dominios de tipo cripta circundantes. Las células gris oscuro en la punta del dominio cripta indican la posición de las células madre Lgr5+, que están presentes en cada dominio cripta. La barra de escala indica 50 µm.
Figura 3. Análisis por conglomerados de los perfiles de expresión génica. El análisis por conglomerados de los niveles de expresión utilizando criptas colónicas y de intestino delgado recién aisladas, así como organoides de intestino delgado mostró alto grado de similitud entre los organoides de intestino delgado y el tejido del que se derivaron, las criptas de intestino delgado. Las criptas colónicas se agrupaban en una rama separada, lo que indica un patrón de expresión génica diferente de este tejido estrechamente relacionado. Es de destacar que sólo el 1,2% de todos los genes expresados estaban significativamente enriquecidos en los organoides con respecto a las criptas de intestino delgado, mientras que viceversa -el 2% estaban enriquecidos en las criptas de intestino delgado. El análisis Ingenuity Pathway sobre estos genes diferenciales puso de manifiesto la presencia específica de una firma de linfocitos en las criptas recién aisladas, mientras que no pudo identificarse ninguna vía significativa en el pequeño número de genes enriquecidos en los organoides (no mostrado). Los inventores llegaron a la conclusión de que el último grupo representa el ruido biológico, mientras que la firma de linfocitos deriva de la contaminación por células inmunitarias intraepiteliales, perdidas tras el cultivo.
Figura 4. Los organoides de cripta conservan las características básicas de las criptas-vellosidades. a-e: El código de activación de Wnt se conserva en los dominios cripta, a: La β-catenina nuclear (gris oscuro, flechas) sólo se observó en los dominios cripta. Imagen de mayor resolución de la Fig. 5. Asterisco, matrigel; Lu, luz b: EphB2 (gris claro) se expresa en un gradiente en las células CBC y células TA. Adviértanse las células madre Lgr5-GFP+ como indica la flecha blanca, c: Células apoptóticas caspasa-3+ (gris oscuro, flechas) desprendiéndose hacia la luz central revestida de enterocitos, d: 40 cromosomas en una extensión de células de un cultivo de criptas de > 3 meses de antigüedad. e-g: Rastreo de linaje de células madre Lgr5+ in vitro, e: Se estimularon criptas de ratones reporteros Lgr5-EGFPires-CreERT2/Rosa26-lacZ mediante tamoxifeno in vitro durante 12 horas, y se cultivaron durante los días indicados. La tinción LacZ (gris oscuro) muestra que las células LacZ+ individuales dispersadas (día 1) generaban criptas LacZ+ enteras in vitro (día 2-14). Los recuadros muestran a mayor aumento los organoides de cripta teñidos, f: El análisis histológico muestra que un dominio cripta LacZ+ entero (gris oscuro/negro) se introduce en el dominio vellosidad, g: El porcentaje de organoides de cripta con células LacZ+ permaneció estable en el tiempo, lo que indica que las células Lgr5+ poseen actividad de célula madre a largo plazo. Se
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Claims (1)

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ES10703131.2T 2009-02-03 2010-02-03 Medio de cultivo para células madre epiteliales y organoides que comprenden dichas células madre Active ES2579909T3 (es)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US149622P 1999-08-16
US14962209P 2009-02-03 2009-02-03
EP09151970 2009-02-03
EP09151970 2009-02-03
EP09171831 2009-09-30
EP09171831 2009-09-30
PCT/NL2010/000017 WO2010090513A2 (en) 2009-02-03 2010-02-03 Culture medium for epithelial stem cells and organoids comprising said stem cells.

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ES2579909T3 true ES2579909T3 (es) 2016-08-17

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ES18182285T Active ES2948761T3 (es) 2009-02-03 2010-02-03 Combinaciones de inhibidores de la replicación del virus de la influenza

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JP (2) JP5458112B2 (es)
KR (1) KR101904224B1 (es)
CN (1) CN102439135B (es)
AU (1) AU2010211428B2 (es)
CA (1) CA2751332C (es)
DK (2) DK2393917T3 (es)
ES (2) ES2579909T3 (es)
HK (1) HK1163746A1 (es)
HR (2) HRP20230650T1 (es)
HU (2) HUE062459T2 (es)
IL (1) IL214381A (es)
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PL (3) PL3441458T3 (es)
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