lec 13 Flashcards
What is direct reprogramming?
Instead of forming IPSCs and differentiating direct reprogramming aims to reprogramme cells directly to the desired type of cell.
What had to be first identified before direct reprogramming could be tested?
here were 14 candidates which were present in the anterior cardiac crescent. The alpha MHC gene is only present in cardiomyocytes and is not a TF so can be used as a marker to check for cardiomyocytes.
Why do the 14 TFs have to be narrowed down?
The 14 have to be narrowed down as:
- Dilutes effects of other TFs
- Costly to produce all 14 TFs every time
Some can be inhibitory
Why do we use a reporter system and which one?
By having a GFP-MHC reporter system we can tell when the cardio fibroblast has been turned into a cardio myocyte.
We use the same method as Yamanaka when he discovered IPSCs.
Which are the 3 TFs that we narrowed it down to and how?
Using the GFP-aMHC reporter system and cTNT expression the 4 were narrowed down further to 3 TFs.
- Gata4 - Mef2c - Tbx5
How do we check the Fibroblast has been reprogrammed into a CM?
The reprogrammed fibroblasts can be seen to have epigenetic resetting (CM promoter regions are reset and demethylated) Nppa and Myh6
iCM intracellular Ca2+ influx analysis showed characteristic neonatal CM also injection of GMT-iCMs into SCID mice to observe integration of the cells into the mice heart via GFP expression.
What method do we use to see the effects of in vivo direct reprogramming?
Mouse fibroblast was tagged using LacZ - periostin inducable by tamoxifen. The CMs do not express periostin therefore will not be tagged. So when the Fibroblasts undergo reprogramming into iCMs we can tell that the new iCMs are from the fibroblasts.
How was in vivo direct reprogramming carried out?
Reprogramming in vivo was tested by inducing cardiac injury via ligating artery. GMT was injected into the heart using retroviral transduction. Post 4 weeks there was a significant increase in CM compared to the control.
The iCMs in the mice expressed cardiac markers - alpha actin, tropomyosin, cardiac MHC and cTNT and all Cardio fibroblast markers were absent.
Which model did we use to show crossing lineage using DR
fibroblasts to neurons
mesodermal to ectodermal
Which TFs were identifed for neuronal differentiation from fibroblast?
19 candidate factors were identified for neurons, a GFP-tau reporter system was made.
Lentiviral transduction of the TFs showed GFP and Tuj1 expression indicative of neuronal expression.
The TFs were narrowed down to Ascl1, Brn2, Myt1l, zic1 and olig2
Ascl1 was identified as the most important TF as it would by itself result in simple neural morphology
The transduction TFs for iN are called BAM (brn2, Ascl1 and myt1l)
How do we tell if it a neuronal cell?
iNs express pan neuronal markers (MAP2) and membrane potential properties of neurons as seen by the patch clamp recording of cells and they can form functional synapses.
What is the problem with neuronal differentiation from the same lineage??
However, not all cells can be reprogrammed into iNs using BAM. Keratinocytes which are from the same lineage could not be directly reprogrammed using BAM.
This was due to the action of Ascl1, it requires trivalent binding site which was seen to be different in keratinocytes, therefore no action of Ascl1, if we demethylated one of the trivalent structures on the fibroblasts we would also see a negative result.
