Widespread changes in transcriptome profile of human mesenchymal stem cells induced by two-dimensional nanosilicates.

Two-dimensional nanomaterials, an ultrathin class of materials such as graphene, nanoclays, transition metal dichalcogenides (TMDs), and transition metal oxides (TMOs), have emerged as a new generation of materials due to their unique properties relative to macroscale counterparts. However, little is known about the transcriptome dynamics following exposure to these nanomaterials. Here, we investigate the interactions of 2D nanosilicates, a layered clay, with human mesenchymal stem cells (hMSCs) at the whole-transcriptome level by high-throughput sequencing (RNA-seq). Analysis of cell-nanosilicate interactions by monitoring changes in transcriptome profile uncovered key biophysical and biochemical cellular pathways triggered by nanosilicates. A widespread alteration of genes was observed due to nanosilicate exposure as more than 4,000 genes were differentially expressed. The change in mRNA expression levels revealed clathrin-mediated endocytosis of nanosilicates. Nanosilicate attachment to the cell membrane and subsequent cellular internalization activated stress-responsive pathways such as mitogen-activated protein kinase (MAPK), which subsequently directed hMSC differentiation toward osteogenic and chondrogenic lineages. This study provides transcriptomic insight on the role of surface-mediated cellular signaling triggered by nanomaterials and enables development of nanomaterials-based therapeutics for regenerative medicine. This approach in understanding nanomaterial-cell interactions illustrates how change in transcriptomic profile can predict downstream effects following nanomaterial treatment.

Three-dimensional in vitro modeling of malignant bone disease recapitulates experimentally accessible mechanisms of osteoinhibition.

Malignant bone disease (MBD) occurs when tumors establish in bone, causing catastrophic tissue damage as a result of accelerated bone destruction and inhibition of repair. The resultant so-called osteolytic lesions (OL) take the form of tumor-filled cavities in bone that cause pain, fractures, and associated morbidity. Furthermore, the OL microenvironment can support survival of tumor cells and resistance to chemotherapy. Therefore, a deeper understanding of OL formation and MBD progression is imperative for the development of future therapeutic strategies. Herein, we describe a novel in vitro platform to study bone-tumor interactions based on three-dimensional co-culture of osteogenically enhanced human mesenchymal stem cells (OEhMSCs) in a rotating wall vessel bioreactor (RWV) while attached to micro-carrier beads coated with extracellular matrix (ECM) composed of factors found in anabolic bone tissue. Osteoinhibition was recapitulated in this model by co-culturing the OEhMSCs with a bone-tumor cell line (MOSJ-Dkk1) that secretes the canonical Wnt (cWnt) inhibitor Dkk-1, a tumor-borne osteoinhibitory factor widely associated with several forms of MBD, or intact tumor fragments from Dkk-1 positive patient-derived xenografts (PDX). Using the model, we observed that depending on the conditions of growth, tumor cells can biochemically inhibit osteogenesis by disrupting cWnt activity in OEhMSCs, while simultaneously co-engrafting with OEhMSCs, displacing them from the niche, perturbing their activity, and promoting cell death. In the absence of detectable co-engraftment with OEhMSCs, Dkk-1 positive PDX fragments had the capacity to enhance OEhMSC proliferation while inhibiting their osteogenic differentiation. The model described has the capacity to provide new and quantifiable insights into the multiple pathological mechanisms of MBD that are not readily measured using monolayer culture or animal models.