The infographic assignment was really fun.
What we did:
we choose a technique
Made a poster
had in-class poster session
One of my group mate is an engineer so he advocated for our infographic to be more application-based rather than an experimental technique.
Everyone else did the drawing (mostly the engineer) because I’m not a really good artist.
I hope everyone who came to check out our infographic had as much fun as we did. I really enjoyed discussing the potential of this technique with my classmates.infographic poster
The following is our notes. Feel free to read through them and comment to generate discussion.
goal: In vivo cell programming
nano-transfection by applying electric field that nanoporates cell membranes and moves DNA of interest (Tissue Nano-Transfection, TNT)
goal was reprogramming skin cells into induced endothelial cells (iECs)
reprogramming factors (all genes): Etv2, Foxc2 and Fli1 (referred to usually as EFF)
“Co-transfection of these three genes into the dorsal skin of C57BL/6 mice resulted in marked stroma reprogramming within a week”
“successful anastomosis [interconnected blood vessels] of the newly formed blood vessels with local functional cutaneous arteries.” happens due to new iCEs
the TNT technique is then used to recover mice limbs suffering tissue necrosis due to low access to blood
[Work to be done (and by who):
Talk about transformation technique (TNT) -> giulio
Talking about iECs made by reprogramming -> Ida
Talking about new blood vessels and how that stops necrosis (the main application of this tech) -> Katrine
Talking about applications, the future of the tech, potential harms/sideffects and more things to test out -> Rosalie
Put things together -> Giulio]
Summary of chip: (by giulio)
This chip has nanoscopic (500nm) holes etched onto its surface with lasers, just like most commercial electronic chips.
These holes are the only exit of a reservoir filled with an electrolyte solution full of the target plasmid (that has EFF genes).
When pulses of voltage are applied between this chip (negative pole) and the inserted needle (positive pole), the electrolyte and target DNA are shot out of the chip and into the skin.
Because the holes are so small, the electric fields (the path of electric charges) are very concentrated on the top of the membrane of the topical cells, so they are extremely likely to get transfected, but also not have too many holes in their membranes.
Because the current going through the tissue is smaller than in BEP (bulk electrophoresis), it is much less likely to cause inflamation.
Bullet points:
nano holes
focused electric fields
more transfection
less unnecessary membrane damage and inflamation
In vivo cell reprogramming and induced endothelial cells
In vivo cell reprogramming circumvents the need for isolation and induced pluripotency by using already available cell sources. To try out the transfection method, the researchers first used the previously established ABM (Asl1/Brn2/Myt1) reprogramming factors. ABM is known to reprogram fibroblasts into neurons in vitro. Reprogramming into neurons was confirmed by measuring Tuj1 expression and electrophysical activity. To confirm the origin of the reprogrammed cells, K14-Cre reporter system was used, as well as eGFP tagging of Col1A1 and the results suggested fibroblastic origin.
They also wanted to reprogram skin cells into induced endothelial cells, and therefore identified a set of reprogramming factors: Etv2, Foxc2 and Fli1 (EFF). Co-transfection of EFF was sufficient to reprogram fibroblasts into iECs in less than a week. This was confirmed by Pecam-1 and vWF expression and enhanced proliferative activity. K14-Cre and Col1A1-eGFP reporter systems showed that the iECs had dermal origin.
Restoration of blood flow (full text):
In relation to promoting reprogramming of somatic cells to iECs (induced endothelial cells) with EFF (a set of reprogramming factors to promote somatic cells –> iECs) TNT-mediated treatment, the research group study whether it also can lead to functional reperfusion (restoration of blood flow) of ischaemic tissue and whole body limb rescue. In both experiments laser speckle monitoring showed higher blood perfusion in EFF treated compared to control (limiting the tissue damage), whereas the control showed more pronounced signs of tissue necrosis compared to EFF treated.
Test of whole limb rescue: Transection of the femoral artery in mouse model, laser speckle monitoring recorded a significant reduction in blood flow to the limb immediately after surgery. EFF TNT showed improved perfusion 7 days after TNT-treatment (conducted on the inner thigh skin, 3 days post-surgery)
In the whole limb rescue an immunofluorescence analysis revealed revascularization far beyond the treatment area and inducing angiogenesis (formation of new blood vessels) in distal location within the limb (gastrocnemius muscle/calf muscle). By injecting EVs (extracellular vesicles), isolated from EFF TNT treated skin, directly into gastrocnemius muscle in a hindlimb ischaemia mouse model it also had the potential to induce blood vessel formation. In the article they propose/suggest that EV’s derived from EFF TNT treated skin might also play a role in spreading pro-angiogenic signals (preloaded with pro-angiogenic vascular endothelial growth factor) within the first hours after treatment besides its expected mechanism for propagating of EFF signals the target tissue.
Necrosis vs. apoptosis (simple explanation):
Apoptosis: programmed cellular death (tightly controlled and regulated)
Necrosis: (unprogrammed) cell death that is triggered by external factors/diseases e.g. infection. In this case low access to oxygen.
Keypoints:
EFF TNT-mediated treatment –> restoration of blood flow in treated tissue/whole limb rescue and induced angiogenesis in distal location within the limb in mouse model.
Pronounced signs of tissue necrosis in control tissue/mouse model compared to EFF TNT-treated.
Proposed model of EV’s spreading pro-angiogenic signals after treatment.
Application( by Rosalie [author of this blog])
In this paper, two different cell re-programmed demonstrated
From epithelial cells to neurons and from epithelial cells to endothelial tissue (blood vessel)
-Cell- reprogramming has been used before, this chip is unique because
Its application is non-viral
When delivery system is viral, there is a chance of introducing mutations into the genome
b) this approach is non-invasive and it is topical while rapid
c) unlike other transplant surgeries where tissue is taken from donor, this tissue is endogenous and there is no risk of tissue/organ rejection( because surface proteins of an individual)
Some potentials:
In-vivo correction treatment for degenerative disease
-when neurons are degenerative (as in alzheimer’s), the induction of epithelial cells into neurons to replace dying neurons help control disease
In-vivo regeneration of nerves damaged
-regrow short nerves to continue neural transmission when injury damaged PNS
Rapid treatment for Burn victim
-the reprogramming back to fibroblast to encourage tissue growth, direct cell growth to repair epithelia and increase perfusion by reprogramming cells into endothelial
Combination of Crispr/Cas9 or genetic engineering to treat genetic defects(cystic fibrosis )
-reprogram tissues (lung epithelia ) to fibroblast
-deliver gene editing technique ( mindful of off-target activity that may make new mutations → cancer) fix CFTR mutation
-re-differentiate cell that can expression functional copy of CFTR