Connexin 25 maintains self-renewal and functions of airway basal cells for airway regeneration

Abstract

Background The formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known. Methods Primary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca2+ in an individual cell to initiate an internal Ca2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air–liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential. Results Herein, we identify Connexin 25 (Cx25) dominates intercellular Ca2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis. Conclusion Cx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions.

1. Introduction

Stem-cell clones are believed to establish a microenvironment for self-renewal of each member inside during in vitro culture [1, 2]. The morphology of cell clone spheres ensures close contact of stem cells, probably for intercellular molecular signalings. Previous studies report the cell density, space, morphology and physical contact of clones are able to regulate the fate of stem cells [3–6]. The size and distance of cell clones greatly influence differentiation and self-renewal of human embryo stem cells [4, 7]. Therefore, the densely packed cells in clones might regulate the internal interactions of each cell to sustain their self-renewal capabilities.

Gap junctions, clusters of intercellular channels located on the cell membrane, facilitate the direct passage of ions and small molecules into the cytoplasm of neighboring cells, thereby playing a significant role in mediating intercellular communication [8–10]. It has been reported that gap junctions contribute to the differentiation in various stem cell types including adipose-derived stem cells and human-induced pluripotent stem cells [11, 12]. Specifically, calcium signals traverse gap junctions between cells without decrement [13], which serve as a guidance to many biological processes such as cell fate determination, proliferation and differentiation [14–17]. However, the role of gap junctions in molecular communications inside clones remains poorly understood.

Airway basal cells (BCs), a type of pluripotent stem cells, universally exist in airway epithelium to drive homeostasis of epithelium and hold good potential for tissue regeneration [18–20]. BCs are crucial for therapeutic intervention in lung diseases, encompassing both chronic lung conditions and acute epithelial injuries [21, 22], and thus widely used in medical researches and even in clinic which usually requires significant quantities of human BCs (hBCs) for transplantation [22–24]. For this purpose, hBCs need to be extracted from patients and cultured for tens of days to expand them to a large amount in vitro while maintaining their self-renewal ability. Different signaling pathways, such as the Notch and Rock pathways, as well as proteins like LC3B and GPR87, regulate the differentiation of hBCs [25–28].