BHLHE40 Maintains the Stemness of PαS Cells In Vitro by Targeting Zbp1 through the Wnt/β-Catenin Signaling Pathway

Menglong Hu; Yueming Tian; Xuenan Liu; Qian Guo; Dazhuang Lu; Xu Wang; Longwei Lv; Xiao Zhang; Yunsong Liu; Yongsheng Zhou; Ping Zhang

doi:10.3390/biomedicines11082190

BHLHE40 Maintains the Stemness of PαS Cells In Vitro by Targeting Zbp1 through the Wnt/β-Catenin Signaling Pathway

Biomedicines. 2023 Aug 3;11(8):2190. doi: 10.3390/biomedicines11082190.

Authors

Menglong Hu^{1

2}, Yueming Tian^{1

2}, Xuenan Liu^{1

2}, Qian Guo^{1

2}, Dazhuang Lu^{1

2}, Xu Wang^{1

2}, Longwei Lv^{1

2}, Xiao Zhang^{1

2}, Yunsong Liu^{1

2}, Yongsheng Zhou^{1

2}, Ping Zhang^{1

2}

Affiliations

¹ Department of Prosthodontics, Peking University School and Hospital of Stomatology, 22 Zhongguancun South Avenue, Haidian District, Beijing 100081, China.
² National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology, 22 Zhongguancun South Avenue, Haidian District, Beijing 100081, China.

Abstract

Primary bone mesenchymal stem cells (BMSCs) gradually lose stemness during in vitro expansion, which significantly affects the cell therapeutic effects. Here, we chose murine PαS (SCA-1⁺PDGFRα⁺CD45^-TER119^-) cells as representative of BMSCs and aimed to explore the premium culture conditions for PαS cells. Freshly isolated (fresh) PαS cells were obtained from the limbs of C57/6N mice by fluorescence-activated cell sorting (FACS). We investigated the differences in the stemness of PαS cells by proliferation, differentiation, and stemness markers in vitro and by ectopic osteogenesis and chondrogenesis ability in vivo, as well as the changes in the stemness of PαS cells during expansion in vitro. Gain- and loss-of-function experiments were applied to investigate the critical role and underlying mechanism of the basic helix-loop-helix family member E40 (BHLHE40) in maintaining the stemness of PαS cells. The stemness of fresh PαS cells representative in vivo was superior to that of passage 0 (P0) PαS cells in vitro. The stemness of PαS cells in vitro decreased gradually from P0 to passage 4 (P4). Moreover, BHLHE40 plays a critical role in regulating the stemness of PαS cells during in vitro expansion. Mechanically, BHLHE40 regulates the stemness of PαS cells by targeting Zbp1 through the Wnt/β-catenin signaling pathway. This work confirms that BHLHE40 is a critical factor for regulating the stemness of PαS cells during expansion in vitro and may provide significant indications in the exploration of premium culture conditions for PαS cells.

Keywords: BHLHE40; PαS cells; Wnt/β-catenin signaling pathway; Zbp1; culture in vitro; stemness.

Abstract

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