Protocol to analyze and validate transcriptomic changes in PDGFRß-KO mesenchymal stem cell osteogenic potential in the mouse embryo.

Mesenchymal stem/stromal cells (MSCs) can differentiate into osteoblasts under appropriate conditions. PDGFRß signaling controls MSC osteogenic potential both transcriptomically and in culture. Here, we present a "computer to the bench" protocol to analyze changes in MSC osteogenic potential at transcriptomic and cellular level in the absence of PDGFRß. We detail the preparation of cells from mouse embryos, the analysis of transcriptomic changes from single-cell RNA-sequencing data, the procedure for MSC derivation and culture, and an osteogenic assay for functional validation. For complete details on the use and execution of this protocol, please refer to Sá da Bandeira et al. (2022).1.

Aldehyde Dehydrogenase, a Marker of Normal and Malignant Stem Cells, Typifies Mesenchymal Progenitors in Perivascular Niches.

Innate mesenchymal stem cells exhibiting multilineage differentiation and tissue (re)generative-or pathogenic-properties reside in perivascular niches. Subsets of these progenitors are committed to either osteo-, adipo-, or fibrogenesis, suggesting the existence of a developmental organization in blood vessel walls. We evaluated herein the activity of aldehyde dehydrogenase, a family of enzymes catalyzing the oxidation of aldehydes into carboxylic acids and a reported biomarker of normal and malignant stem cells, within human adipose tissue perivascular areas. A progression of ALDHLow to ALDHHigh CD34+ cells was identified in the tunica adventitia. Mesenchymal stem cell potential was confined to ALDHHigh cells, as assessed by proliferation and multilineage differentiation in vitro of cells sorted by flow cytometry with a fluorescent ALDH substrate. RNA sequencing confirmed and validated that ALDHHigh cells have a progenitor cell phenotype and provided evidence that the main isoform in this fraction is ALDH1A1, which was confirmed by immunohistochemistry. This demonstrates that ALDH activity, which marks hematopoietic progenitors and stem cells in diverse malignant tumors, also typifies native, blood vessel resident mesenchymal stem cells.

PDGFRß+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny.

Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRß signaling controls hematopoietic stem/progenitor cell (HSPC) generation and is required in the HSC specification niche. Little is known about murine HSPC specification in vivo and whether PDGF-B/PDGFRß is involved. Here, we show that PDGFRß is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRß+ cells play a dual role in murine hematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRß+ embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findings provide crucial information for the controlled production of HSPCs in vitro.