Temporal induction of Lhx8 by optogenetic control system for efficient bone regeneration

Abstract

Background The spatiotemporal regulation of essential genes is crucial for controlling the growth and differentiation of cells in a precise manner during regeneration. Recently, optogenetics was considered as a potent technology for sophisticated regulation of target genes, which might be a promising tool for regenerative medicine. In this study, we used an optogenetic control system to precisely regulate the expression of Lhx8 to promote efficient bone regeneration. Methods Quantitative real-time PCR and western blotting were used to detect the expression of Lhx8 and osteogenic marker genes. Alkaline phosphatase staining and alizarin red staining were used to detect alkaline phosphatase activity and calcium nodules. A customized optogenetic expression system was constructed to regulate Lhx8, of which the expression was activated in blue light but not in dark. We also used a critical calvarial defect model for the analysis of bone regeneration in vivo. Moreover, micro-computed tomography (micro-CT), three-dimensional reconstruction, quantitative bone measurement, and histological and immunohistochemistry analysis were performed to investigate the formation of new bone in vivo. Results During the osteogenic differentiation of BMSCs, the expression levels of Lhx8 increased initially but then decreased thereafter. Lhx8 promoted the early proliferation of BMSCs but inhibited subsequent osteogenic differentiation. The optogenetic activation of Lhx8 in BMSCs in the early stages of differentiation by blue light stimulation led to a significant increase in cell proliferation, thus allowing a sufficient number of differentiating BMSCs to enter the later osteogenic differentiation stage. Analysis of the critical calvarial defect model revealed that the pulsed optogenetic activation of Lhx8 in transplanted BMSCs over a 5-day period led to a significant increase in the generation of bone in vivo. Conclusions Lhx8 plays a critical role in balancing proliferation and osteogenic differentiation in BMSCs. The optogenetic activation of Lhx8 expression at early stage of BMSCs differentiation led to better osteogenesis, which would be a promising strategy for precise bone regeneration.

Quantitative insights in tissue growth and morphogenesis with optogenetics

“…In terms of understanding and curing developmental and growth-related pathologies, great strides have been made in organoid engineering, tissue regeneration, etc. For example, Huang et al optogenetically activated the LIM Homeobox 8 (Lhx8) target gene to demonstrate the vital role of the gene in controlling complementary proliferation and osteogenic differentiation expressed in bone development [155][156][157]. As an optogenetic tool, FKF1 (flavinbinding, kelch repeat, f-box 1) receptor with a LOV domain temporally induced Lhx8 expression activatable with blue light.…”

Emerging advances in hydrogel-based therapeutic strategies for tissue regeneration

“…In recent years, MSC therapy has shown great potential to promote bone healing [ 109 ]. It has been shown that MSCs can directly differentiate into osteoblasts and osteocytes, secrete various bioactive substances and acquire similar functions to bone tissue [ 52 ]. For example, Sayanti Datta et al.…”

Precise modulation of embryonic development through optogenetics

“…Multiple PAP pairs undergo heterodimerization, such as blue‐light‐sensitive pairs CRY2/CIB (Kennedy et al, 2010), iLID (Guntas et al, 2015), TULIP (Strickland et al, 2012), Magnets (Kawano et al, 2015; Figure 1a). These optogenetic systems have been successfully used to control the activities of many proteins, including RhoGEFs and Rho GTPases activity for cytoskeleton reorganization (Deneke et al, 2019; Eritano et al, 2020; Krueger, Tardivo, Nguyen, & De Renzis, 2018), recombinase and Cas9 activity for genetic modification (Li et al, 2020; Nihongaki, Otabe, Ueda, & Sato, 2019), transcription factor activation, and gene expression (D. Huang et al, 2021; W. Wang et al, 2019), signaling effectors for signaling cascade activity modulation (Johnson, Djabrayan, Shvartsman, & Toettcher, 2020; Johnson et al, 2017; Johnson & Toettcher, 2019; Krishnamurthy et al, 2020; McFann, Dutta, Toettcher, & Shvartsman, 2021; Shao et al, 2017; Q. Wang et al, 2020), Rab11 activity for membrane trafficking regulation (Rathbun et al, 2020).…”