Probing Notch1-Dll4 signaling in regulating osteogenic differentiation of human mesenchymal stem cells using single cell nanobiosensor

Abstract

Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

Notch signaling and fluid shear stress in regulating osteogenic differentiation

“…The previous study has shown that Notch signaling is involved during hMSCs osteogenic differentiation, disruption of Notch signaling mediated ALP activity, and osteogenic differentiation efficiency. Our group also recently showed that Dll4 mRNA is a molecular biomarker of osteogenic differentiated hMSCs ( Zhao et al, 2022 ). Inhibition of Notch signaling reduces osteogenic differentiation with decreased ALP enzyme activity.…”

“…A fluorophore (6-FAM (fluorescein)) was labeled at the 5′ end of the LNA detecting probe for mRNA detection. Design, characterization, and optimization of LNA/DNA nanobiosensor have been reported previously ( Wang et al, 2015 ; Zheng et al, 2017 ; Wang et al, 2018a ; Zhao et al, 2022 ). Briefly, the LNA probe will bind to the quencher spontaneously to form a LNA - quencher complex.…”

“…This nanobiosensor has high thermal stability and specificity. Our previous studies showed the fluorescence intensity did not have a significant change as the incubation time increased up to 14 days ( Zhao et al, 2022 ). The specificity of this nanobiosensor was also previously characterized and compared ( Zhao et al, 2022 ).…”

“…This LNA/DNA nanobiosensor has been utilized to study spatiotemporal mRNA expression dynamics in collective cell migration ( Riahi et al, 2015 ), mice lung cancer ( Tao et al, 2014 ), wounded corneal tissue repair ( Wang et al, 2015 ), and liver tissue ( Riahi et al, 2014 ). Recently, our group utilized this nanobiosensor to monitor Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation ( Zhao et al, 2022 ). This nanobiosensor has high thermal stability and specificity.…”

“…The design process of the LNA probe for mRNA detection was reported previously. ( Wang et al, 2015 ; Wang et al, 2018b ; Wang et al, 2019 ; Zhao et al, 2022 ) Briefly, the target mRNA sequence was first acquired from GeneBank. A 20-base pair nucleotide sequence was selected and optimized using mFold server and NCBI Basic Local Alignment Search Tool (BLAST) database.…”