skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 5:00 PM ET until 11:00 PM ET on Friday, June 21 due to maintenance. We apologize for the inconvenience.


Title: 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.

 
more » « less
Award ID(s):
2143151
NSF-PAR ID:
10368100
Author(s) / Creator(s):
; ; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Scientific Reports
Volume:
12
Issue:
1
ISSN:
2045-2322
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Osteoporosis is a common bone and metabolic disease that is characterized by bone density loss and microstructural degeneration. Human bone marrow-derived mesenchymal stem cells (hMSCs) are multipotent progenitor cells with the potential to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes, which have been utilized extensively in the field of bone tissue engineering and cell-based therapy. Although fluid shear stress plays an important role in bone osteogenic differentiation, the cellular and molecular mechanisms underlying this effect remain poorly understood. Here, a locked nucleic acid (LNA)/DNA nanobiosensor was exploited to monitor mRNA gene expression of hMSCs that were exposed to physiologically relevant fluid shear stress to examine the regulatory role of Notch signaling during osteogenic differentiation. First, the effects of fluid shear stress on cell viability, proliferation, morphology, and osteogenic differentiation were investigated and compared. Our results showed shear stress modulates hMSCs morphology and osteogenic differentiation depending on the applied shear and duration. By incorporating this LNA/DNA nanobiosensor and alkaline phosphatase (ALP) staining, we further investigated the role of Notch signaling in regulating osteogenic differentiation. Pharmacological treatment is applied to disrupt Notch signaling to investigate the mechanisms that govern shear stress induced osteogenic differentiation. Our experimental results provide convincing evidence supporting that physiologically relevant shear stress regulates osteogenic differentiation through Notch signaling. Inhibition of Notch signaling mediates the effects of shear stress on osteogenic differentiation, with reduced ALP enzyme activity and decreased Dll4 mRNA expression. In conclusion, our results will add new information concerning osteogenic differentiation of hMSCs under shear stress and the regulatory role of Notch signaling. Further studies may elucidate the mechanisms underlying the mechanosensitive role of Notch signaling in stem cell differentiation. 
    more » « less
  2. Abstract

    Implantation of stem cells for tissue regeneration faces significant challenges such as immune rejection and teratoma formation. Cell‐free tissue regeneration thus has a potential to avoid these problems. Stem cell derived exosomes do not cause immune rejection or generate malignant tumors. Here, exosomes that can induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) are identified and used to decorate 3D‐printed titanium alloy scaffolds to achieve cell‐free bone regeneration. Specifically, the exosomes secreted by hMSCs osteogenically pre‐differentiated for different times are used to induce the osteogenesis of hMSCs. It is discovered that pre‐differentiation for 10 and 15 days leads to the production of osteogenic exosomes. The purified exosomes are then loaded into the scaffolds. It is found that the cell‐free exosome‐coated scaffolds regenerate bone tissue as efficiently as hMSC‐seeded exosome‐free scaffolds within 12 weeks. RNA‐sequencing suggests that the osteogenic exosomes induce the osteogenic differentiation by using their cargos, including upregulated osteogenic miRNAs (Hsa‐miR‐146a‐5p, Hsa‐miR‐503‐5p, Hsa‐miR‐483‐3p, and Hsa‐miR‐129‐5p) or downregulated anti‐osteogenic miRNAs (Hsa‐miR‐32‐5p, Hsa‐miR‐133a‐3p, and Hsa‐miR‐204‐5p), to activate the PI3K/Akt and MAPK signaling pathways. Consequently, identification of osteogenic exosomes secreted by pre‐differentiated stem cells and the use of them to replace stem cells represent a novel cell‐free bone regeneration strategy.

     
    more » « less
  3. Abstract

    Mesenchymal stem/stromal cells (MSCs) exhibit a rapid loss in osteogenic phenotype upon removal of osteoinductive cues, as commonly occurs during transplantation. Osteogenic differentiation can be more effectively but not fully maintained by aggregating MSCs into spheroids. Therefore, the development of effective strategies that prolong the efficacy of inductive growth factors would be advantageous for advancing cell‐based therapies. To address this challenge, osteoinductive bone morphogenetic protein‐2 (BMP‐2) is adsorbed to osteoconductive hydroxyapatite (HA) nanoparticles for incorporation into MSC spheroids. MSC induction is evaluated in osteogenic conditions and retention of the osteogenic phenotype in the absence of other osteogenic cues. HA is more uniformly incorporated into spheroids at lower concentrations, while BMP‐2 dosage is dependent upon initial morphogen concentration. MSC spheroids containing BMP‐2‐loaded HA nanoparticles exhibit greater alkaline phosphatase activity and more uniform spatial expression of osteocalcin compared to spheroids with uncoated HA nanoparticles. Spheroids cultured in media containing soluble BMP‐2 demonstrate differentiation only at the spheroid periphery. Furthermore, the osteogenic phenotype of MSC spheroids is better retained with BMP‐2‐laden HA upon the removal of soluble osteogenic cues. These findings represent a promising strategy for simultaneous delivery of osteoconductive and osteoinductive signals for enhancing MSC participation in bone formation.

     
    more » « less
  4. Abstract Mesenchymal stem cells (MSCs) are multipotent cells that can replicate and differentiate to different lineages of mesenchymal tissues, potentiating their use in regenerative medicine. Our previous work and other studies have indicated that mild heat shock enhances osteogenesis. However, the influence of pro-inflammatory cytokines on osteogenic differentiation during mildly elevated temperature conditions remains to be fully explored. In this study, human MSCs (hMSCs) were cultured with Tumor Necrosis Factor-alpha (TNF-a), an important mediator of the acute phase response, and Interleukin-6 (IL-6) which plays a role in damaging chronic inflammation, then heat shocked at 39ºC in varying frequencies - 1 hour per week (low), 1 hour every other day (mild), and 1 hour intervals three times per day every other day (high). DNA data showed that periodic mild heating inhibited suppression of cell growth caused by cytokines and induced maximal proliferation of hMSCs while high heating had the opposite effect. Quantitative osteogenesis assays show significantly higher levels of alkaline phosphatase activity and calcium precipitation in osteogenic cultures following mild heating compared to low heating or non-heated controls. These results demonstrate that periodic mild hyperthermia may be used to facilitate bone regeneration using hMSCs, and therefore may influence the design of heat-based therapies in vivo. 
    more » « less
  5. Abstract

    Current treatments for craniomaxillofacial (CMF) defects motivate the design of instructive biomaterials that can promote osteogenic healing of complex bone defects. We report methods to promote in vitro osteogenesis of human mesenchymal stem cells (hMSCs) within a model mineralized collagen scaffold via the incorporation of ascorbic acid (vitamin C), a key factor in collagen biosynthesis and bone mineralization. An addition of 5 w/v% ascorbic acid into the base mineralized collagen scaffold significantly changes key morphology characteristics including porosity, macrostructure, and microstructure. This modification promotes hMSC metabolic activity, ALP activity, and hMSC‐mediated deposition of calcium and phosphorous. Additionally, the incorporation of ascorbic acid influences osteogenic gene expression (BMP‐2,RUNX2,COL1A2) and delays the expression of genes associated with osteoclast activity and bone resorption (OPN,CTSK), though it reduces the secretion of OPG. Together, these findings highlight ascorbic acid as a relevant component for mineralized collagen scaffold design to promote osteogenic differentiation and new bone formation for improved CMF outcomes.

     
    more » « less