Objective To elucidate the role of mesenchymal stem cell （MSC） in the development of lipochondrocyte （LC）

and to characterize the functional alterations of MSCs in patients with microtia. Methods Auricular cartilage from C57BL/6 mice at postnatal days P6， P13， P17， and P21 was subjected to single-cell RNA sequencing， with lipid droplet dynamics validated by BODIPY staining. Standard quality control， dimensionality reduction， clustering， and cell annotation were performed. Pseudotime trajectory analysis， RNA velocity inference， differential gene expression， pathway enrichment analyses， and intercellular communication modeling were applied， combined with patient-derived microtia data for comparison. Results The number and size of lipid droplets in auricular cartilage increased with development. MSCs progressively differentiated into mature lipochondrocytes （LCs） through progenitor states （LCP1/LCP2）. MSCs were classified into three subpopulations （Notch1 ⁺， Atf3 ⁺， and Mfap5 ⁺）， reflecting a dynamic transition from transcriptional activity to extracellular matrix （ECM） synthesis. At P13， ECM synthesis genes were markedly upregulated， accompanied by  activation of the PI3K-Akt pathway. MSCs served as the major source of Midkine （Mdk） and Periostin （Postn） signals， acting in a paracrine manner on lipochondrocytes and progenitors and in an autocrine manner to reinforce MSC function. In microtia， MSCs exhibited downregulation of ECM synthesis genes but upregulation of inflammatory mediators and TNF， IL-17， and NF- κB pathways， indicating functional shifts. Conclusion MSCs play a dual role in auricular lipocartilage development， serving both as direct progenitors of LCs and as regulators of the microenvironment through Mdk/Postn signaling to support ECM synthesis and tissue homeostasis. In microtia， MSCs display impaired matrix production and enhanced pro-inflammatory signaling.

Microtia, Auricular cartilage, Lipochondrocyte, Mesenchymal stem cells, Single-cell RNA sequencing,

Cell differentiation, Signal transduction