Understanding the role of skeletal stem cells in craniofacial and skeletal bone disease
Abstract/Contents
- Abstract
- Craniofacial and skeletal bone disease, injury and impaired healing effects millions of individuals world-wide and represents a significant biomedical burden in the United States. Craniofacial disease can manifest in significant developmental aberrancies impacting cognitive ability, facial structure and function resulting in significant reduction in quality of life if not treated. Likewise, non-union facture healing of the skeletal bone in the context of disease such as diabetes, arthritis and other various conditions, particularly in an aging population, represents an enormous clinical challenge. Both clinical and therapeutic advancements of bone regeneration have been impaired due to the lack of understanding of the cellular the molecular players driving development and regeneration in the craniofacial and skeletal bone compartments. The need for cellular and molecular therapeutics is ever more apparent in our current population. Skeletal stem cells are a subset of cells found within the craniofacial and skeletal bone compartments. These multipotent cell types reside in these compartments with the purpose of coordinating development and healing of bony tissue. Skeletal stem cells are multipotent in nature, with the potential to differentiate into bone, cartilage and supporting stromal tissue. Since the initial concept of a skeletal stem cells was proposed, several groups have identified various multipotent, self-renewing cell types in bone compartments, gaining the title of skeletal stem cells, yet much is to be debated on the identity of a skeletal stem cells. Nevertheless, several groups have suggested the critical role of skeletal stem cells populations in craniofacial and skeletal bone development, disease and regeneration. With this motivation, we sought to better understand from a cellular and molecular perspective the role of skeletal stem cells in the craniofacial disease, Craniosynostosis and the skeletal long bone disease Marfan syndrome. Chapter I presents a comparative analysis between mouse and human calvarial vault. The vertebrate calvarial vault is an ancient and highly conserved structure across species, however the mechanisms governing osteogenesis of the calvarial vault and how they might be conserved across mammalian species remains unclear. The aim of chapter I is to determine if regional differences in osteogenic potential of the calvarial vault, first described in mice, extend to human. Human frontal and parietal osteoblasts were derived from fetal calvarial tissue, demonstrating enhanced osteogenic potential both in-vitro and in-vivo of human frontal derived osteoblasts compared to parietal derived osteoblasts. Furthermore, we found shared differential signaling patterns in the canonical WNT, TGF-β, BMP and FGF pathways previously described in the mouse to govern these regional differences in osteogenic potential. Taken together, our findings unveil evolutionary conserved similarities both at a functional and molecular level between the mouse and human calvarial bones, providing further support that studies employing mouse models, are suitable for translational studies to human. Chapter II presents a study which investigates whether the Mouse Skeletal Stem Cell (mSSC) is resident in the cranial sutures. Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis, one of the more common craniofacial developmental diseases. Prospective isolation by FACS identified mSSCs within the cranial sutures with a significant difference in spatio-temporal representation between fusing and patent sutures. Transcriptomic analysis highlighted a distinct signature in mSSCs-derived from the physiological closing PF suture, and single cell RNA sequencing identifies transcriptional heterogeneity among the PF, SAG and COR sutures, suggestive of mSSC subpopulations transcriptionally primed for bone or cartilage fate. Activation of canonical Wnt signaling increases mSSCs representation in the sutures, conversely, inhibition decreases the mSSC representation within the cranial suture. With this in mind, crossing an Axin2LacZ/+ mouse, endowed with enhanced Wnt activation, to a Twist1+/− mouse model of coronal craniosynostosis, enriches mSSC population resident in the coronal suture, in effect restoring patency. Finally, co-transplantation of mSSCs with recombinant Wnt3a prevents resynostosis following suturectomy in Twist1+/− mice. This study reveals that a decrease and/or imbalance of skeletal stem cells representation within the cranial sutures may underlie craniosynostosis providing translational implications toward therapeutic approaches for craniosynostosis. Chapter III presents a study describing a cranial suture explant culture method that can be applied wildly to study the molecular and cellular mechanisms of cranial suture biology. Craniofacial research approaches multiple facets of basic science spanning from molecular regulation of craniofacial development, cell biology/signaling and ultimately translational craniofacial biology. Cranial sutures coordinate development of the skull, and the premature fusion of one or more, leads to craniosynostosis. With limited access to human samples and particular the lack of non-disease controls, animal models provide significant contributions toward craniofacial biology and many clinical/surgical treatments of craniofacial disorders. However, studies employing mouse models are costly and time consuming due to housing, breeding and variability of the in-vivo context. Here, we present the establishment of a cranial suture explant 2-D culture method that has shown fidelity in comparison to in-vivo isolations procedures, allowing for isolation of high yields of skeletal stem and progenitor cells from small number of mice. Moreover, this method allows the opportunity for specific genetic manipulations, phenocoping models of craniosynostosis in-vitro. Additionally, this model allows for in-vitro tamoxifen-induction of Cre based reporter systems, an example being the ActinCreERT2; R26Rainbow mouse often utilized to trace clonal expansion. This versatile method tackles needs of large number of mice to perform cranial suture research. Finally, Chapter IV represents a study on Marfan Syndrome (MFS), an autosomal dominant disease affecting approximately 10,000 individuals worldwide. MFS manifests in 3 different systems: skeletal, cardiovascular, and ocular. Chapter IV presents a study focused on the effects of MFS on the skeletal system, commonly characterized by overgrowth of the endochondral long bones using a mouse model. The Fibrillin-1+\- mouse is a model of MFS displaying some of the hallmark characteristics of over-growth and reduced bone volume in the endochondral long bones. Given the shared skeletal phenotype in MFS human patients and Fbn1+/- mouse, we hypothesized that these skeletal abnormalities would correlate to perturbed skeletal stem cells function, through dysregulation of the TGFB pathway. Here we demonstrate that mouse skeletal stem cell (mSSC) derived from Fbn-1+/- mice growth plates have increased activation of TGFB resulting in impaired differentiation capacity, producing smaller bone nodules and significantly reduced mineralization in-vitro. These findings suggest that the targeted inhibition of TGFB signaling in mSSCs might provide potential therapeutic approach to treating the skeletal effects of MFS. The findings stemming from this study not only provide potential insight into mitigation for improper bone development and healing in MFS patients but also in several bone pathologies related to reduced bone volume and/or mineralization
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2022; ©2022 |
| Publication date | 2022; 2022 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Menon, Siddharth |
|---|---|
| Degree supervisor | Longaker, Michael T |
| Degree supervisor | Wan, Derrick |
| Thesis advisor | Longaker, Michael T |
| Thesis advisor | Wan, Derrick |
| Thesis advisor | Chan, Charles K. F. (Charles Kwok Fai), 1975-2024 |
| Thesis advisor | Roncarolo, Maria-Grazia |
| Degree committee member | Chan, Charles K. F. (Charles Kwok Fai), 1975-2024 |
| Degree committee member | Roncarolo, Maria-Grazia |
| Associated with | Stanford University, Institute for Stem Cell Biology and Regenerative Medicine |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Siddharth Menon |
|---|---|
| Note | Submitted to the Institute for Stem Cell Biology and Regenerative Medicine |
| Thesis | Thesis Ph.D. Stanford University 2022 |
| Location | https://purl.stanford.edu/rx145ym5863 |
Access conditions
- Copyright
- © 2022 by Siddharth Menon
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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