Non-bone marrow CD34+ stem cells are crucial for endothelial repair of injured arteries

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Background: Endothelial cells play a critical role in multiple cardiovascular diseases. Circulating CD34+ cells are believed to be endothelial progenitors and have been used to treat cardiovascular disease, but achieved distinct clinical outcomes. The lack of long-term benefits and the insufficiency of CD34 antibody-coating stents have halted further application of CD34+ cell therapy. To fulfill the translational use of CD34+ cells, the exact identity and role of CD34+ cells in vascular regeneration are urged to be addressed.

Objective: We sought to elucidate the roles of CD34+ stem/ progenitor cells from different origins in neointimal formation and to understand the underlying mechanisms.

Methods: (1) A single-cell atlas of wild type mouse femoral artery was depicted by the single-cell RNA sequencing technology (scRNA-seq) and Cd34 expression, distribution and biological functions were examined. (2) Using a genetic Cd34-lineage tracing mouse model (Cd34-CreERT2;R26-tdTomato), we combined histological examination and scRNA-seq analysis of mouse femoral artery under physiological and pathological (guide-wire injury) conditions. By tracing the expression of lineage marker tdTomato, we achieved the fate-mapping of CD34+ cells during the process of vascular regeneration and neointimal formation. (3) Bone marrow transplantation between wild type and Cd34-lineage tracing mouse was performed to identify the different contributions of bone marrow and non-bone marrow origins of lesional CD34-lineage cells. (4) CD34+ cell depletion models (Cd34-CreERT2;R26-DTA/tdTomato and Cd34-CreERT2;R26-DTR/tdTomato) was used to specifically ablate CD34+ cells due to the Cre induced cytotoxic effect of diphtheria toxin, and to delineate contribution of CD34+ cell to neointimal formation. (5) Vessel wall CD34+ cells were isolated and cultivated in vitro to study the process of CD34+ cell differentiating into endothelial cells. Pathway enrichment analysis and transcription factors prediction were used to investigate underlying mechanisms, and further validated by traditional molecular biology experiments.

Results: (1) The single-cell atlas of normal mouse femoral artery was described, which included mesenchymal cells, smooth muscle cells (SMC), endothelial cells (EC) and immune cells. A heterogeneous population of CD34+ cells was identified including most mesenchymal cells and minor endothelial cells, within which a stem/progenitor sub-population was found with angiogenesis capacity. (2) Using a guide-wire induced endothelial denudation model on the inducible lineage tracing Cd34-CreERT2;R26-tdTomato mouse, we showed that CD34+ cells acquired both luminal and adventitia microvessel endothelial cell fate in the injured femoral artery, and also differentiate into immune cells within the lesion and in the perivascular area, but not contribute to SMC hyperplasia. (3) Combining the lineage tracing mouse model and bone marrow transplantation experiments, we proved that non-bone marrow rather than bone marrow origin of CD34+ cells were the dominant source of endothelial regeneration after denudation, while bone marrow-derived CD34+ cells differentiated into immune cells locally after vessel injury. (4) Using CD34+ cell depletion mouse models (Cd34-CreERT2;R26-DTA/tdTomato and Cd34-CreERT2;R26-DTR/tdTomato) we have found that specifically ablating different origins of CD34+ cells has different effects on neointimal formation, that ablating all sources of CD34+ cells alleviate neointima while ablating non-bone marrow CD34+ cells aggravate it. (5) Combining genetic lineage tracing and scRNA-seq, we reported the transcriptional profiling of 26,497 cells by scRNA-seq analysis and provided a cell atlas comparison between normal and lesioned femoral arteries in mouse. The lesioned arteries were described as a process of immune cell accumulation and SMC decreasing. By tracking the expression of lineage marker tdTomato in the scRNA-seq data, we characterized additional cell fate of CD34+ cell differentiating into myofibroblasts and pericytes beyond the EC and immune cell fate in the injured femoral artery. Using pseudotime trajectory analysis we constructed a differentiating direction of CD34+ cell towards EC. By gene enrichment pathway analysis and transcription factors prediction we identified TGF-β pathway involved. (6) Isolated adventitia CD34+ cells displayed endothelial differentiation, and TGF-β pathway were examined during the process, in which microRNA-21-Smad7-pSmad2/3 pathway was confirmed regulating endothelial gene expression and function during CD34+ cell to EC differentiation.

Conclusions and Translational Perspective: It has long been proposed, but quite controversial, that circulating CD34+ cells act as endothelial progenitors and contribute to vascular regeneration. Our studies using single-cell RNA sequencing and genetic cell lineage tracing strategy demonstrated that a heterogeneous population of CD34+ cells reside in the vessel wall. Importantly, non-bone marrow CD34+ cells acquired endothelial cell fate in the injured femoral artery, while bone marrow-derived CD34+ cells differentiated into immune cells locally. Depletion of non-bone marrow CD34+ cells exacerbate neointimal lesions of the injured vessel. Isolated vascular resident CD34+ cells displayed efficient endothelial differentiation potential. Our findings provided solid evidence that resident rather circulating CD34+ cells display a regenerative potential, indicating that CD34+ stem cell therapy for cardiovascular diseases could be strategically re-considered.
Date of Award1 May 2021
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorQingbo Xu (Supervisor), Yanhua Hu (Supervisor) & Alex Ivetic (Supervisor)

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