The Functional Sub-Population Of Hertwig’S Epithelial Root Sheath

This study aimed to explore subsets of Hertwig’s Epithelial Root Sheath (HERS) cells, isolate sub-populations, establish immortalized cell lines for functional analysis, and elucidate the mechanisms underlying epithelial-mesenchymal transition (EMT) and osteogenic differentiation in HERS cells. Single-cell RNA sequencing (scRNA-seq) identified HERS subsets, with gene clustering analyzed and key markers validated using multiplex immunohistochemistry (MIHC) and immunofluorescence staining. Blocking the BMP receptor Acvr1 with LDN-193189 assessed its role in EMT. Immortalized mesenchymal cells (rHM) were developed to evaluate osteogenic differentiation through RT-qPCR, Western blot, ALP staining, and Alizarin red staining. Cell sheet morphology were analyzed via scanning electron microscopy (SEM). A rat mandibular periodontal defect model was used to assess hard tissue repair, with micro-CT providing structural insights. Runx1’s role in osteogenesis was evaluated using siRNA knockdown. HERS cells express specific markers such as DSC3, CK5, and CK17, with six sub-populations identified. DSC3 was identified as a stable lineage-tracing marker in various states and conditions. Blocking Acvr1 promoted cell proliferation while suppressing mesenchymal markers. rHM exhibited the highest osteogenic potential, forming robust mineralized nodules and achieving superior periodontal tissue repair. Runx1 knockdown downregulated osteogenic differentiation markers, confirming its regulatory role. This study innovatively reveals DSC3 as a novel lineage-tracing marker and elucidates Runx1’s essential role in osteogenic regulation. These findings provide critical insights into HERS biology, paving the way for regenerative strategies in periodontal tissue repair and representing a significant advancement in the field.