Circulating CD34⁺ Fibroblast Progenitors Engaged in Heart Fibrosis of Allograft.

Abstract

Fibrosis is one of the major causes of cardiac allograft malfunction and is mainly driven by fibroblasts. However, the role of recipient-derived cells in generating allograft fibroblasts and the underlying mechanisms remain to be explored. We analyzed human heart allograft samples and used murine transplant models (C57BL/6J, <i>Cd34</i> (cluster of differentiation 34)-CreER^T2; R26-tdTomato, mRFP (cell membrane labeled with red fluorescence protein) mice, Rosa26-iDTR, <i>Postn</i>-CreER^T2; R26-tdTomato, double-tdTomato, and immunodeficient mice with BALB/c donors). Human progenitor cells were cultivated from blood. Single-cell RNA sequencing, Western blotting, quantitative polymerase chain reaction, and immunohistochemistry, whole-mount staining with 3-dimensional reconstruction, and in vivo<i>/</i>in vitro experiments were applied to characterize allograft cellular composition and communication. Single-cell RNA sequencing was introduced to delineate the allograft cell atlas of patients and mice. Y chromosome analysis identified that recipient-derived cells contributed to allograft fibroblasts in both patients and murine models. Combining the genetic cell lineage tracing technique, we found that recipient-derived CD34⁺ cells could give rise to activated fibroblasts. Bone marrow transplantation and parabiosis models revealed that the recipient's circulating non-bone marrow <i>Cd34</i>⁺ cells could generate allograft fibroblasts. Human CD34⁺ cells could differentiate into fibroblasts both in vivo and in vitro. CD34⁺ fibroblast progenitors were recruited by CXCL12 (C-X-C motif chemokine ligand 12)-ACKR3 (atypical chemokine receptor 3) and MIF (macrophage migration inhibitory factor)-ACKR3 interactions and differentiated via the TGFβ (transforming growth factor beta)/GFPT2 (glutamine-fructose-6-phosphate transaminase 2)/SMAD2/4 (small mother against decapentaplegic 2/4) axis. Ablation of recipient <i>Cd34</i>⁺ cells reduced activated fibroblasts and alleviated allograft fibrosis. We identify circulating CD34⁺ cells as a novel source of fibroblast progenitors that contribute to cardiac allograft fibrosis, suggesting that targeting recipient CD34⁺ cells could be a novel therapeutic potential for treating cardiac fibrosis after heart transplantation.