Single cell transcriptomics analysis of bone marrow mesenchymal lineage cells

Bone marrow mesenchymal lineage cells are a heterogeneous cell population involved in bone homeostasis and diseases such as osteoporosis. While it is long postulated that they originate from mesenchymal stem cells (MSCs), the true identity of MSCs and their in vivo bifurcated differentiation routes into osteoblasts and adipocytes remain poorly understood. Here, by employing large scale single cell transcriptome analysis, we computationally defined MSCs and delineated their bi-lineage differentiation paths in young, adult and aging mice. One identified subpopulation is a unique cell type that expresses adipocyte markers but contains no lipid droplets. As non-proliferative precursors for adipocytes, they exist abundantly as pericytes and stromal cells that form a ubiquitous 3D network inside the marrow cavity. Functionally they play critical roles in maintaining marrow vasculature and suppressing bone formation. Therefore, we name them marrow adipogenic lineage precursors (MALPs) and conclude that they are a new component of marrow adipose tissue. Overall design: 4 batches of single cell libraries for sequencing: endosteal Td+ bone marrow cells from 1-month-old (n=2), 1.5-month-old (n=3), 3-month-old (n=3) and 16-month-old (n=3) male Col2/Td mice. 20,000 cells were loaded in aim of acquiring one single library of 10,000 cell for each age group by Chromium controller (V3 chemistry version, 10X Genomics Inc, San Francisco, USA), barcoded and purified as described by the manufacturer, and sequenced using a 2x150 pair-end configuration on an Illumina HiSeq platform at a sequencing depth of ~400 million reads.