A modified system to promote stemness of mouse intestinal stem cells by activating Nrf2 and α2-adrenergic receptor signaling pathway

Abstract

Intestinal stem cells (ISCs) maintain epithelial homeostasis through continuous self-renewal and differentiation, but their regulatory mechanisms remain incompletely understood. Using a simplified culture system, we identify two novel pathways that synergistically enhance stem cell characteristics: antioxidant signaling through 2-phospho-L-ascorbic acid (pVc) and α2-adrenergic receptor (α2-AR) activation by dexmedetomidine (Dex). Mechanistic studies reveal that pVc promotes stem cell maintenance through Nrf2-mediated antioxidant responses, while α2-AR activation functions through suppression of cAMP signaling. In vivo administration of these compounds enhances intestinal epithelial renewal while maintaining proper stem cell positioning and identity. Notably, α2-AR activation promotes regeneration after radiation injury by enhancing proliferation of stem cells produced by Bmi1+ cells in the post-injury process, demonstrating therapeutic potential. These findings advance our understanding of ISC regulation and suggest new strategies for protecting intestinal integrity during injury or disease.

1. Introduction

The intestinal epithelium maintains homeostasis through continuous renewal driven by Lgr5-expressing intestinal stem cells (ISCs) located in crypt bases [1,2]. These stem cells maintain tissue architecture through balanced self-renewal and differentiation programs regulated by multiple niche signals [3,4]. Paneth cells provide essential Wnt, Notch, and EGF signals [5,6], while stromal cells contribute BMP and additional Wnt factors [7]. Current research shows that immune cells [8], metabolic factors [9,10], and enteric neurons [11] also influence ISC function, particularly during tissue regeneration and inflammatory responses [12,13].

Organoid culture systems have significantly advanced ISC biology by enabling controlled manipulation of niche components in vitro [2]. These experimental models incorporate defined factors that support ISC maintenance while allowing systematic investigation of regulatory mechanisms [14]. This approach has revealed new insights into stem cell regulation, such as IL-22’s role in regeneration [15], and enabled the development of Paneth cell-independent culture methods [16]. Small molecules like CHIR99021 and valproic acid have emerged as effective substitutes for key niche signals, simplifying experimental systems [16].

Despite advances in understanding ISC regulation, many signals show context-dependent and sometimes opposing effects. For example, both IL-22 and α2-adrenergic receptor (α2-AR) activation can promote [17,18] or inhibit [15,19] epithelial proliferation depending on experimental conditions. To address these complexities, we used minimal ISC cultures lacking support cells to identify direct regulators of stem cell function. This approach revealed that 2-phospho-L-ascorbic acid (pVc), a stable derivative of vitamin C, and α2-adrenergic signaling work together to promote ISC self-renewal. We show that pVc and α2-AR agonists enhance stem cell maintenance and intestinal regeneration both in vitro and in vivo, identifying new regulatory mechanisms with therapeutic potential.