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人们用于评价肿瘤干细胞状态的实验中提供了许多方法，可以通过不同的方法、全方位的证实肿瘤干细胞的特点与状态，值得学习，需要掌握。
# K0 G/ h, `: V. L$ m 1. Self-renewal/Single Cell Clonal Analysis
8 W8 N% u- L' R$ ~% OSelf-renewal is recognized as one of the hallmarks of all stem cells, which enables a single cell to produce two daughter cells as they form spheroids and proliferate indefinitely . To generate a homogenous population, a single cell needs to be isolated and plated, for example, in 192 wells per experiment. After a week in culture we usually see in our laboratory that the majority (80%–90%) of the wells contain at least one tumor sphere and continued to expand after approximately 2 weeks.: C# i1 H" G' t7 B; E
6 X" I. U4 ~) x- B- O, e2 ?8 q Self-renewal needs to be assayed by serially passaging of spheres in cell culture dishes in vitro to justify that sphere-forming cells are able to reform spheres.
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6 ~0 [5 D+ E, Z6 d2 t; [0 Q/ ~/ ]2. Stem Cell Markers
' I+ a( y1 s7 l' O Q4 E To verify that stem cells generated from GBM patient-derived tissue express neural stem cell (NSC) markers, tumor spheres need to be cryosectioned and stained with NSC antibodies. Patient-derived GBM stem cells show usually strong expression of GFAP, Nestin, Sox-2, Musashi-1, Bmi-1, whereas no immuno-reactivity is observed with differentiated cell markers, such as Tuj1, NeuN, which are early and late neuronal markers, respectively, or Olig-1, which is specific for oligodendritic lineages.
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3. Differentiation
: E2 e4 M: o+ X' N The nature of NSCs is that they can differentiate and give rise to neuronal, astrocytic, and oligodendrocytic lineages. To prove this capacity in patient-derived tumor spheres, inducing cell differentiation in media containing FBS needs to be performed. Within a week after exposure to differentiation media, TSCs start to express GFAP, Tuj-1, beta-III-Tubulin, Olig-1 F and later the late neuronal marker NeuN. ! D9 g6 f6 d2 i
/ t, D6 m2 J$ e# T0 m4. Tumorigenicity of TSCs: In Vitro vs. In Vivo $ \, s6 U% i- u+ I9 a+ D" W
A method to determine whether patient driven TSCs adopt the invasive characteristic of cancer cells, GBM stem cells can be seeded along with normal human NSCs as negative control in soft agar. Normal neurospheres did not grow in soft agar until the third week but began forming colonies toward the end of fourth week. Usually, neural stem cells form small and few colonies anywhere between 4 and 7 weeks after they are implanted into soft agar whereas GBM stem cells start colony formation
& S( \% |. v# z+ t! V- E5 ^during the first week. & y# ]9 _ b# |6 `
To validate if GBM stem cells preserve their tumorigenic character TSCs need to be implanted subcutaneously and intracranially into animals (such as mice or rats), respectively. For instance, in our experience, we recorded the tumor volume over 10 weeks and 6 months for the subcutaneous and intracranial injections, respectively . In flank injections, mice receiving 1 × 10 6 cells per injection developed tumors as early as the fourth week and gradually progressed during the subsequent ten weeks. In the orthotopic injections, mice receiving 100,000 cells per injection showed tumor formation on MRI at 6 months . |
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发表于 2011-6-23 22:36
发表于 2011-6-23 23:30
