direct conversion of neurons to fibroblasts
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- 1. CREDIT SEMINAR Direct conversion of fibroblasts to functional neurons by defined factors functional neurons by defined factors functional neurons by defined factors
- Major Advisor
- Dr. Gaya Prasad
- Syed Shafiq
- Date: May 17, 2010. 2009V166M
- Dept. of ABT
- 2. The research article has been published in NATURE journal dated feb.25, 2010 (vol 463) and is authored by: Thomas Vierbuchen, Austin Ostermeier, Zhiping P. Pang, Yuko Kokubu1, Thomas C. Sudhof & Marius Wernig
- 3. Introduction
- The diverse cell types present in the adult organism are produced during development by lineage-specific transcription factors that define and reinforce cell-type-specific gene expression patterns.
- Cellular phenotypes are further stabilized by epigenetic modifications.
- Forced expression of lineage-specific genes in somatic cells can induce traits of other cell types.
- For example, the basic helixloophelix (bHLH) transcription factor MyoD (also called Myod1) can induce muscle-specific properties in fibroblasts but not hepatocytes.
- 5. A screen for neuronal-fate-inducing factors
- 6. Text Transcription factors screened for neuron-inducing activity in MEFs A pool of lentiviruses containing all 19 genes (19F pool) was prepared to infect mouse embryonic fibroblasts (MEFs) from TauEGFP knock-in mice, which express EGFP specifically in neurons
- 7. Care was taken to exclude neural tissue from the MEF preparation, and the authors were unable to detect evidence for the presence of neurons or neural progenitor cells in these cultures using immunofluorescence , fluorescence activated cell sorting (FACS) and polymerase chain reaction with reverse transcription (RT-PCR) analyses.
- 8. Representative Tuj1-positive cells 13 days after infection with Ascl1 alone or in combination with the indicated genes. Note the increased complexity of the neurites in the Ascl1+Myt1l condition. Uninfected p3 TauEGFP MEFs 19F pool, post 32 days infection
- 9. The effect of 18 transcription factors in combination with Ascl1 on neuronal induction 13 days post infection 5F pool, 13 days post infection
- 10. 5F pool, 22 days post infection iNs derived from Balb/c MEFs
- 11. Characterization of 5-factor iN cells
- Patch-clamp recordings of TauEGFP-positive cells on days 8, 12 and 20 after infection, were performed.
- For the majority of the iN cells analysed (85.1%), action potentials could be elicited by depolarizing the membrane in current-clamp mode.
- Six cells (14.2%) showed spontaneous action potentials , some as early as 8 days after transduction.
- The resting membrane potentials ranged between -30 and -69 mV with an average of , -55 mV on day 20.
- Induced neuronal cells responded to exogenous application of GABA , and this response could be blocked by the GABA receptor antagonist picrotoxin.
- Thus, MEF-derived iN cells seem to exhibit the functional membrane properties of neurons and possess ligand-gated GABA receptors .
- 13. K-CURRENT CLAMP MODE; L-VOLTAGE CLAMP; M-SPONTANEOUS AP
- 14. 5F pool, 22 days post infection Characterization of neurotransmitter phenotype of iNs Some iN cells (9 out of ,500) contained the Ca2+binding protein calretinin , a marker for cortical interneurons and other neuronal subtypes. No expression of tyrosine hydroxylase, choline acetyltransferase or serotonin was detected The vast majority of iN cells were negative for peripherin , an intermediate filament characteristic of peripheral neurons
- 15. Functional neurons from tail fibroblasts
- Tail-tip fibroblasts (TTFs) from 3-day-old TauEGFP and Rosa26-rtTA mice were isolated.
- Pre-existing neurons, glia, or neural progenitor cells could not be detected in the cultures.
- Electrophysiological recordings 12 days after infection demonstrated an average resting membrane potential of ,-57 mV (range: -35 to -70 mV), firing of action potentials (81.8%) and expression of functional voltage-gated membrane channel proteins .
- Tyrosine hydroxylase, choline acetyltransferase, or serotonin expression was not detected.
- 17. 5F pool, post 13 days post infection 5F pool, 21 days post infection Nine of eleven cells recorded exhibited action potentials
- 18. Neuronal induction is fast and efficient
- The authors assessed the kinetics and efficiency of 5F iN conversion.
- Tuj1-positive cells with immature neuron-like morphology were found as early as 3 days after infection.
- After 5 days, neuronal cells with long, branching processes were readily detected, and over time increasingly complex morphologies were evident.
- TauEGFP expression was detected as early as day five .
- Electrophysiological parameters also showed signs of maturation over time.
- To estimate the conversion efficiency, they first determined how many of the MEF-derived iN cells divided after induction of the viral transgenes by treating the cells with 5-bromodeoxyuridine (BrdU) throughout the duration of the culture period and beginning 1 day after gene induction.
- Vast majority of iN cells became post mitotic by 24 h after transgene activation.
- The efficiency ranged from 1.8% to 7.7% in MEF and TTF iN cells presumably due to slight variations in titres of the viruses.
- 20. Cells were maintained at a potential of approximately -65 to -70 mV. Step current injection protocols were used from -50 to +70 pA
- 21. BrdU-positive iN cells after BrdU treatment from day 013 or day 113 after transgene induction. 5F pool, 5 days post infection 13 days post infection
- 22. iN cells form functional synapses
- Two independent methods were used.
- First, they determined whether iN cells were capable of synaptically integrating into pre-existing neural networks.
- TauEGFP-positive iN cells were FACS purified 78 days after infection of MEFs and plated on cortical neuronal cultures (7 days in vitro).
- One week after re-plating, patch-clamp recordings from TauEGFP-positive iN cells were performed.
- Spontaneous and rhythmic network activity typical of cortical neurons in culture, was observed.
- Both EPSCs and IPSCs could be evoked after electrical stimulation delivered from a c oncentric electrode placed 100150 mm away from the patched iN cell.
- In the pr
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