The Role of Prep1 in the Regulation of Mesenchymal Stromal Cells

Abstract

Molecular mechanisms governing cell fate decision events in bone marrow mesenchymal stromal cells (MSC) are still poorly understood. Herein, we investigated the homeobox gene Prep1 as a candidate regulatory molecule, by adopting Prep1 hypomorphic mice as a model to investigate the effects of Prep1 downregulation, using in vitro and in vivo assays, including the innovative single cell RNA sequencing technology. Taken together, our findings indicate that low levels of Prep1 are associated to enhanced adipogenesis and a concomitant reduced osteogenesis in the bone marrow, suggesting Prep1 as a potential regulator of the adipo-osteogenic differentiation of mesenchymal stromal cells. Furthermore, our data suggest that in vivo decreased Prep1 gene dosage favors a pro-adipogenic phenotype and induces a "browning" effect in all fat tissues.

Keywords: adipogenesis; homeobox gene; in vivo imaging; mesenchymal stromal cells (MSC); murine models; osteogenesis; single cell RNA sequencing.

Figure 1

Hematoxylin Eosin staining shows differences in adipose depots between wt and Prep1^i/i mice. HE staining has been performed on (A) bone marrow sections of wt (left panel) and Prep1^i/i mice (right panel), 10× (upper panel), 20× (middle panel) and 40× (lower panel) magnifications are shown. Black raws indicate the presence of adipocytes. HE on (B) different adipose tissues, i.e. Brown Adipose Tissue (BAT) (upper panel), subcutaneous White Adipose Tissue (sWAT) (middle panel) and visceral White Adipose Tissue (vWAT) (lower panel), 40× magnification is shown.

Figure 2

Volumetric rendering of the whole body micro-CT reconstructions indicates on average a smaller, lighter, shorter and less adipose tissue in hypomorphic mice. A wt mouse (left) and a Prep1^i/i mouse (right) are represented. In the right panel of each group, the highlighted region represents the segmented adipose tissue.

Figure 3

Hypomorph subjects show a higher density of the interscapular BAT than wt. Transaxial slices of the micro-CT scans. Figure shows density of interscapular BAT of the wt mouse (left) and the Prep1^i/i mouse (center). The histogram on the right panel shows the distribution of gray level intensities inside the highlighted BAT Region of Interest (ROI).

Figure 4

Single Cell RNA Sequencing reveals different BM cell composition between wt and hypomorphic samples. (A) t-SNE plot of single Cell RNA sequencing, performed on wt (upper panel) and Prep1^i/i (lower panel) fresh Bone Marrow Stromal Cells, shows eight distinct transcriptional clusters, through Loupe Cell Browser visualization. Clusters specific for each sample are highlighted by dotted squares. (B) The heatmap shows unique molecular signatures displayed by each cluster.

Figure 5

Cluster annotation highlights adipogenic and osteogenic subpopulations. (A) Annotation, using specific gene lists (GO White Fat Cell Differentiation, LEPR+ Adipo, GO Brown Fat Cell Differentiation, Progenitors and Osteo), shows the presence of specific cell types in each cluster. Dotted squares highlights clusters (most uniquely expressed per each genotype). (B) Pie charts depict percentages of cells present in each cluster. (C) Histograms show the percentage of cells per cluster which are positive to the indicated annotation. Blue bars indicate wt cells, light blue bars refer to hypomorphic cells. GO, Gene Ontology; WFCD, White Fat Cell Differentiation; LEPR, Leptin Receptor; BFCD, Brown Fat Cell Differentiation.

Figure 6

Alizarin Red O’Staining highlights altered osteogenic ability in culture Prep1^i/i cells. Staining is used to evaluate in culture osteogenic differentiation efficiency. Analysis has been performed on undifferentiated wt (upper left) and Prep1^i/i (lower left) cells as negative controls and on cells at terminal osteogenic differentiation, wt (upper right) and Prep1^i/i (lower right). Red staining labels the extracellular matrix secreted by mature osteobalsts.