Этот протокол описывает изоляции, обогащение, и поддержание медуллобластомой опухоли стволовые клетки, полученные из мутантных мышей с внематочной Соник деятельности путем ежа.
Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.
Мы описываем эффективный способ изолировать, обогащать и поддерживать медуллобластомой стволовых клеток из первичной опухоли ткани извлекаются из мутантных мышей с конститутивно Ежик сигнализации. Мы обнаружили, что один важный шаг в создании успешно здоровых стволовых клеток опух?…
The authors have nothing to disclose.
Это исследование было поддержано грантами Вандербильт-Инграм онкологический центр поддержки Грант (P30 CA068485), опухоли мозга у детей и Фонд Национальных институтов здравоохранения (NS042205).
Material Name | タイプ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Neurobasal medium | Invitrogen | 21103049 | ||
hEGF | Invitrogen | PHG0311 | 25ng/ml | |
bFGF | Invitrogen | PHG0023 | 25ng/ml | |
N2 | Invitrogen | 17502048 | 1X | |
B27(-RA) | Invitrogen | 12587010 | 1X | |
Accutase | Invitrogen | A1110501 | 50% | |
Gelatin | Sigma | G1393 | 0.1% | |
Glutamine | Invitrogen | 25030081 | 2mM | |
Primaria dishes | Fisher | 087724C | ||
Sox2 antibody | Millipore | MAB4343 | Mouse, 1:1000 | |
Nestin antibody | DSHB | rat401 | Mouse, 1:200 | |
YFP antibody | Molecular Probes | A11122 | Rabbit, 1:2000 | |
GFAP antibody | Neuromics | CH22102 | Chicken, 1:1000 | |
Tuj1 antibody | Sigma | T5076 | Mouse, 1:2000 | |
NeuN antibody | Millipore | MAB377 | Mouse, 1:2000 | |
CNPase antibody | Millipore | MAB326 | Mouse, 1:1000 |