Circulating CD34⁺ Fibroblast Progenitors Engaged in Heart Fibrosis of Allograft

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, Cd34 (cluster of differentiation 34)-CreER^T2; R26-tdTomato, mRFP (cell membrane labeled with red fluorescence protein) mice, Rosa26-iDTR, Postn-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/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 Cd34⁺ 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 Cd34⁺ 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.