Absence of p21 Expression Sensitizes iPSCs to iCaspase9 Induced Apoptosis While Not Impacting Ability to Participate in Exogenous Cartilage Repair

Larijani, Leila

Absence of p21 Expression Sensitizes iPSCs to iCaspase9 Induced Apoptosis While Not Impacting Ability to Participate in Exogenous Cartilage Repair

dc.contributor.advisor	Rancourt, Derrick
dc.contributor.advisor	Krawetz, Roman
dc.contributor.author	Larijani, Leila
dc.contributor.committeemember	Childs, Sarah J.
dc.contributor.committeemember	Bob, Argiropoulos
dc.date	2023-02
dc.date.accessioned	2022-11-03T15:12:23Z
dc.date.available	2022-11-03T15:12:23Z
dc.date.issued	2022-11-01
dc.description.abstract	p21 is a multifunctional protein that is critical to the control cellular proliferation and plays a number of other functions in the cell. In the nucleus, p21 functions as a tumour suppressor, while in the cytoplasm, phosphorylated p21 functions as a proto-oncogene and can also suppress apoptosis. Because p21 deficiency has been linked to increased tissue regeneration, in this thesis, I sought to investigate if p21 mutations can improve the regenerative capacity of induced pluripotent stem cells in a cellular therapy approach for cartilage injuries in mice. However, because p21 is a tumor suppressor and its loss can result in tumorigenesis, I employed the inducible Caspase9 cell suicide system to purge iPSCs through forced apoptosis. In vitro, iCaspase9-mediated cell death resulted in a statistically significant increase in the apoptosis rate in p21-/- iPSCs compared to p21+/+ iPSCs (wild-type). RNA sequencing was undertaken to determine which pathways are involved in p21 mediated apoptosis. Increased expression of 41 apoptotic and 6 healing-related genes were observed in p21-/- iPSCs compared to wildtype. In vitro chondrogenesis of p21-/- iPSCs showed an increase in chondrogenic genes relative to the wild-type cells. When these iPSCs were transplanted into a focal cartilage injury in mice, ectopic cartilage formation was observed. Neither tumors, nor ectopic cartilage formation was observed in mice that were treated with CID drug to induce iCasp9 mediated apoptosis. Finally, I performed a drug screen to discover compounds that promote apoptosis with iCasp9. In addition to identifying several epigenetic drugs that promoted iCasp9 function in vitro, I also demonstrated that p21 inhibitors could phenocopy the enhanced iCasp9 mediate cell killing seen previously in the p21 mutant iPSCs. Overall, my findings indicate that p21 does play a role in protecting iPSCs from apoptosis and its downregulation can be exploited to increase cell-suicide approach effectiveness. I was also able to demonstrate that iPSCs can induce robust cartilage repair in mice regardless of the expression of p21. Therefore, modulation of p21 warrants further research to determine if exploiting this protein can be used in iPSC clinical trials safely in the future.	en_US
dc.identifier.citation	Larijani, L. (2022). Absence of p21 expression sensitizes iPSCs to iCaspase9 induced apoptosis while not impacting ability to participate in exogenous cartilage repair (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.	en_US
dc.identifier.uri	http://hdl.handle.net/1880/115401
dc.identifier.uri	https://dx.doi.org/10.11575/PRISM/40382
dc.language.iso	eng	en_US
dc.publisher.faculty	Cumming School of Medicine	en_US
dc.publisher.institution	University of Calgary	en
dc.rights	University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.	en_US
dc.subject	iPSC	en_US
dc.subject	iCaspas9	en_US
dc.subject	Suicide gene	en_US
dc.subject	Chondrogenesis	en_US
dc.subject	Cartilage	en_US
dc.subject	Drug screening	en_US
dc.subject	Epigenetics	en_US
dc.subject.classification	Biology--Cell	en_US
dc.subject.classification	Genetics	en_US
dc.subject.classification	Biology--Molecular	en_US
dc.title	Absence of p21 Expression Sensitizes iPSCs to iCaspase9 Induced Apoptosis While Not Impacting Ability to Participate in Exogenous Cartilage Repair	en_US
dc.type	doctoral thesis	en_US
thesis.degree.discipline	Medicine – Medical Sciences	en_US
thesis.degree.grantor	University of Calgary	en_US
thesis.degree.name	Doctor of Philosophy (PhD)	en_US
ucalgary.item.requestcopy	true	en_US