Link to the University of Pittsburgh Homepage
Link to the University Library System Homepage Link to the Contact Us Form

Inhibition of the Canonical WNT Signaling Pathway Promotes the Proliferation of PRX1+ Stem Cells Resident in the Calvarial Sutures

Alafandi, MAzat (2023) Inhibition of the Canonical WNT Signaling Pathway Promotes the Proliferation of PRX1+ Stem Cells Resident in the Calvarial Sutures. Master's Thesis, University of Pittsburgh. (Unpublished)

Primary Text
Restricted to University of Pittsburgh users only until 31 January 2025.

Download (1MB) | Request a Copy


Craniofacial bone regeneration is one of the most challenging medical interventions due to its great complexity. Stem cells residing within the cranial sutures are known as calvarial Skeletal Stem Cells (cSSCs) and present a strong regenerative and migratory capacity. PRX1 is a transcription factor found in the cranial sutures and serves as a marker of cSSCs. PRX1 expressing cells (PRX1+) are multipotent stem cells that undergo differentiation to restore calvarial bone tissues; however, their number declines with aging, limiting their regenerative capacity.
In this study, we investigate the effect of WNT signaling inhibition on PRX1+ cells to understand the mechanism responsible for their proliferation and osteoblastic differentiation. We hypothesized that the inhibition of the canonical WNT signaling pathway promotes PRX1+ cells to undergo proliferation and self-renewal. We began by testing WNT signaling inhibitors including Niclosamide, Quercetin, and Pyrvinium. We utilized an in-vitro cell viability assay (MTT) to survey the efficacy of these inhibitors in enhancing the proliferation of PRX1+ cells. Using RT-PCR, we evaluated the expression of genes associated with osteoblastic differentiation (RUNX2, SP7, OCN) and WNT-Signaling (AXIN2, TCF7, β-Catenin), arguing that treatment with WNT inhibitors would decrease their expression. We followed an ex-vivo approach by isolating calvarial sutures from a PRX1-creER-EGFP+ mouse model and treating them with Niclosamide for 2 weeks. Then, we assessed the efficacy of WNT inhibition in inducing the proliferation of the suture resident GFP-expressing cells using flow cytometry.
Our cell viability studies revealed that WNT signaling inhibition by 0.2 µM Niclosamide induced a significant increase of 60% in the proliferation of PRX1+ cells after 48 hours. Our gene-expression analysis showed that osteoblastic differentiation genes and WNT-signaling-associated genes were significantly downregulated in Niclosamide-treated PRX1+ cells. Finally, our ex-vivo flow cytometry analysis of the sutures confirmed the efficacy of Niclosamide in inducing the proliferation of PRX1+ cells; we observed a two-fold increase in the number of GFP/PRX1+ cells isolated from the sutures only after two weeks of treatment with 2.0 µM Niclosamide. In conclusion, we demonstrated that inhibition of the canonical WNT signaling pathway by Niclosamide induces cellular proliferation of PRX1+ cells and temporarily inhibits their differentiation.


Social Networking:
Share |


Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Alafandi, MAzatmaa386@pitt.edumaa3860000-0001-6741-4839
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorIntini, Giuseppegii5@pitt.edugii50000-0002-6862-7040
Committee ChairIntini, Giuseppegii5@pitt.edugii50000-0002-6862-7040
Committee MemberBeniash, Eliaebeniash@pitt.eduebeniash0000-0001-9019-5160
Committee MemberClark, Danieldaniel.clark@pitt.edudaniel.clark0000-0003-3453-6498
Date: 31 January 2023
Date Type: Publication
Defense Date: 8 December 2022
Approval Date: 31 January 2023
Submission Date: 6 December 2022
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 66
Institution: University of Pittsburgh
Schools and Programs: School of Dental Medicine > Dental Science
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: PRX1, WNT Signaling Inhibition, WNT Inhibitors, Niclosamide, Calvarial Skeletal Stem Cells, Osteoblastic Differentiation, Cellular Proliferation
Date Deposited: 31 Jan 2023 17:47
Last Modified: 31 Jan 2023 17:47


Monthly Views for the past 3 years

Plum Analytics

Actions (login required)

View Item View Item