अलगाव, संवर्धन, और medulloblastoma स्टेम सेल के रखरखाव

Summary

इस प्रोटोकॉल अलगाव, संवर्धन, और medulloblastoma ट्यूमर स्टेम अस्थानिक ध्वनि हाथी मार्ग गतिविधि के साथ उत्परिवर्ती चूहों से ली गई कोशिकाओं के रखरखाव का वर्णन करता है.

Abstract

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 Patched1^LacZ/+ 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.

Protocol

1. ट्यूमर असर सेरिबैलम के लघु विच्छेदन, ट्यूमर ऊतक और चढ़ाना के पृथक्करण ट्यूमर ऊतक के रिट्रीवल बीमार चूहों असर medulloblastoma अक्सर runted थे, hydrocephaly और पीछे पक्षाघात और जब पलट मुद्रा हासिल विफलता सहित विशिष्?…

Discussion

हम एक कुशल तरीका अलग, समृद्ध और medulloblastoma constitutively सक्रिय हाथी संकेतन के साथ उत्परिवर्ती चूहों से प्राप्त प्राथमिक ट्यूमर ऊतकों से स्टेम कोशिकाओं को बनाए रखने का वर्णन है. हमने पाया है कि सफलतापूर्वक एक स्वस?…

Materials

Material Name	Type	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