VASCULAR INJURY IN Col3a1+/- MICE MODEL OF VASCULAR EHLER-DANLOS SYNDROME

Vascular type of Ehlers-Danlos Syndrome (vEDS) is an inherited cardiovascular disease affecting the middle to large sized arteries, with an incidence rate of 1/5000. vEDS patients also show a significant phenotype of easily bruised skin, indicating aberrant wound healing and injury repair ability. Over 70% of the patients carry a glycine mutation located in their COL3A1 gene, which encodes the propeptide of type III collagen. Mutations in glycine residues lead to a disruption in the assembly and maturation of type III collagen. The goal and significance of the current study was to investigate the potential role of COL3A1 haploinsufficiency in the development of vEDS and develop new potential therapies for vEDS patients. Carotid ligation was applied to the Col3a1+/- mouse as an injury model, and the results confirm that Col3a1+/- mice have aberrant arterial injury repair. Arteries from the injured Col3a1+/- mice showed increased cell proliferation, inflammation, and neovessel formation. In vitro, fibroblasts explanted from Col3a1+/- mice have persistent myofibroblast status after treatment with TGF-β1, which validates the in vivo findings. Finally, two treatments were tested on Col3a1+/- mice after carotid ligation: bone marrow transplantation and celiprolol. Transplantation of Col3a1+/+ bone marrow to Col3a1+/- mice corrects the post-injury phenotypes, suggesting that bone marrow derived fibrocytes can be differentiated into myofibroblasts and produce sufficient type III collagen for successful wound healing. Celiprolol treatment on the Col3a1+/- mice also corrects the wound healing impairment by decreasing inflammation and cell proliferation. Therefore, this study validates a novel paradigm for vEDS that decreased supply of mature type III collagen fibrils affects fibroblasts in arterial wound healing and also provides evidence for bone marrow transplantation and celiprolol as potential new therapeutic approaches to the treatment of vEDS patients

Skin cell heterogeneity and dynamics during morphogenesis, tissue homeostasis, and regeneration

Skin is our protective barrier against various environmental harms. For the skin to fulfill its crucial function, it relies on multiple cell types working in concert; but most importantly it relies on skin-resident epithelial stem cells. These cells ensure an intact barrier through constant replacement of the epidermis and they ensure proper hair production through cyclical regeneration of hair follicles. This combination of constant and cyclical renewal within one tissue makes skin a prime model system for the study of adult tissue stem cells. The overall aim of this thesis was to transcriptionally dissect this well-established model system in a systematic and unbiased way. The majority of data presented in this thesis is based on the combination of single-cell RNA sequencing and in situ stainings of mRNA. This combination allows us to appreciate the genome-wide transcriptional heterogeneity while still being able to place the identified cell populations in their spatial tissue context. In Paper I, the first whole-transcriptome study of skin at the single-cell level, we examined the vectors describing cellular heterogeneity within the epidermal compartment of mouse skin during its resting stage (telogen). In Paper II, we expanded on this analysis by including full-thickness skin during rest (telogen) and growth (anagen). This allowed for an unbiased census of all major cell types contained in the skin, and it furthermore enabled us to study how skin achieves and accommodates hair growth. In Paper III, we studied the role of dermal fibroblasts in early embryonic skin development. We uncovered unexpected heterogeneity among embryonic fibroblasts and explored their supportive functions for skin maturation. Moreover, we identified novel keratinocyte subpopulations and closely analyzed epidermal fate decisions. In Paper IV, we monitored transcriptional adaptations of two distinct epidermal stem cell populations during their contribution to wound healing. This allowed us to answer fundamental questions about stem cell plasticity and the dynamics of cell adaptations following injury. In sum, this thesis uncovers the dynamic and heterogeneous nature of mouse skin during adult tissue homeostasis, embryonic development, and tissue regeneration after injury. Most importantly, we provide new insights into how stem cell identity is shaped and how developmental as well as regenerative processes are orchestrated

Therapeutic Potential of Vasculogenesis and Osteogenesis Promoted by Peripheral Blood CD34-Positive Cells for Functional Bone Healing

Failures in fracture healing are mainly caused by a lack of vascularization. Adult human circulating CD34(+) cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to differentiate into osteoblasts in vitro; however, the therapeutic potential of CD34(+) cells for fracture healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that functional fracture healing is supported by vasculogenesis and osteogenesis via regenerative plasticity of CD34(+) cells. Peripheral blood CD34(+) cells, isolated from total mononuclear cells of adult human volunteers, showed gene expression of osteocalcin in 4 of 20 freshly isolated cells by single cell reverse transcriptase-polymerase chain reaction analysis. Phosphate-buffered saline, mononuclear cells, or CD34(+) cells were intravenously transplanted after producing nonhealing femoral fractures in nude rats. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining at the peri-fracture site demonstrated molecular and histological expression of human-specific markers for endothelial cells and osteoblasts at week 2. Functional bone healing assessed by biomechanical as well as radiological and histological examinations was significantly enhanced by CD34(+) cell transplantation compared with the other groups. Our data suggest circulating human CD34(+) cells have therapeutic potential to promote an environment conducive to neovascularization and osteogenesis in damaged skeletal tissue, allowing the complete healing of fractures