groundbreaking study

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

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

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

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

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 0–13 or day 1–13 after transgene induction. 5F pool, 5 days post infection 13 days post infection

22. iN cells form functional synapses

26. iNs from TTF

28. NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl- benzo[f]quinoxaline-2,3-dione) is an AMPA-receptor antagonist These data indicate that iN cells are capable of forming functional synapses with each other, and that the majority of iN cells exhibit an excitatory phenotype.

29. MEF-derived 5F-iN cells on glia express markers of glutamatergic neurons

30. Genes sufficient for neuronal conversion

33. 13 days post infection

34. 12 days post infection

35. 12 days post infection BAM pool, 22 days post infection

36. BAM pool, perinatal TTF derived iNs, 12d post infection BAM pool, 21d post infection

37. BAM pool, 22d post infection

38. BAM iN cells derived from TTF isolated from a six-week-old TauEGFP mouse

39. Patch-clamp recordings for BAM iNs showing synaptic formation 53% cells were Tbr1 +ve only 1% are GAD +ve

40. immature responses from Ascl alone Thus, it seems likely that Ascl1 alone is sufficient to induce some neuronal traits, such as expression of functional voltage-dependent channel proteins that are necessary for the generation of action potentials; however, co-infection of additional factors is necessary to facilitate neuronal conversion and maturation.

41. Discussion

45. THANKS