Bulk RNA-seq detects cMPCs HO signature in B/T mouse blood samples - dataset #2

Heterotopic ossification (HO), the aberrant bone in soft tissues, is one of the most debilitating complications associated with severe burn and traumatic injuries as well as joint replacement surgeries due to its insidious development. Currently, no technologies exist to either support early HO detection or to guide early prophylactic strategies. Furthermore, no technologies exist to assess treatment efficacy. In this study, we used microfluidic iChip, designed to isolate circulating rare non-hematopoietic cells from whole blood, to isolate and analyze circulating mesenchymal progenitor cells (cMPCs) released in a clinically relevant mouse model of traumatic HO. RNA sequencing of cMPCs revealed the unique expression of HO-associated MPC genes observed as soon as 6 hours post HO-inducing injury, 41 days earlier than gold standard radiographic diagnostic strategies. Using multiple lineage tracing systems, we determined that the cMPCs derived from the periosteum rather than the bone marrow. We then formulated a cMPC HO score and evaluated the diagnostic ability of the liquid biopsy approach for the early detection of HO. By using Youden's J Statistic for optimizing sensitivity and specificity, our score yielded a sensitivity of 82% and specificity of 100% for detecting HO in mice. Overall design: iChip enriched cells were lysed with RLT buffer (Qiagen Micro kit) and stored at -80C. Before proceeding to library preparation, RNA was purified with Qiagen micro kit. Total RNA libraries were prepared using Seqwell Rapid kit using manufacturer instructions.