Lecture_5-_Not_all_ESCs_are_created_equal-1

Lecture_5-_Not_all_ESCs_are_created_equal-1 - Lets Recap...

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Question: Can ESC’s differentiate into neural type cells? 1) ESC’s can become NPC’s, which can become neurons or glia. 2) Neurons derived from ESC’s are active and ‘functional’. Let’s Recap… Immunofluorescent evidence. Electrophysiological evidence. Yes
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Not All ESC’s Are Created Equal
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HSF-1 Anterior cells HSF-6 Posterior cells H1 or H9 (plastic) Noggin Retinoic Acid (RA) Different human ES cells have different differentiation bias Wu, et al. Proc. Natl. Acad. Sci. USA, 2007; 104(34):13821-6 Observation: When differentiating ESC’s from two different lines into neurons, the neurons were different. hESC, San Francisco, line 1 hESC, San Francisco, line 6
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How do we confirm this observation? HSF 1hESCs are prone to production of forebrain neurons, but HSF 6, non forebrain Wu, et al. Proc. Natl. Acad. Sci. USA, 2007; 104(34):13821-6 Why not stain for markers known to be specific to the different neuronal populations (anterior vs. posterior)? Nestin NPC marker FoxG1B anterior neuron marker Hoechst Nuclear stain Tuj1 general neuron marker PAX2 posterior neuron marker
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Lets recap the steps in a typical immunofluorescent procedure: [Note: this is a summarized version, not all the steps are shown] Immunofluorescence 1) Fix (cross-link proteins) the cells. 2) Wash the tissue in the appropriate buffers to remove the excess fix (ex. PFA) and to remove lipid membranes (adding detergents)
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4) Add the 1 o (primary) antibody against the protein of interest. Variable region, specifically recognizes protein of interest. Constant region, the same in all the antibodies (from that specific class) produced by your animal of choice. Immunofluorescence
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This note was uploaded on 04/19/2011 for the course LS 2 taught by Professor Pires during the Spring '08 term at UCLA.

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Lecture_5-_Not_all_ESCs_are_created_equal-1 - Lets Recap...

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