title: Single cell gene expression profiling of cortical osteoblast lineage cells.
abstract: In tissues with complex architectures such as bone, it is often difficult to purify and characterize specific cell types via molecular profiling. Single cell gene expression profiling is an emerging technology useful for characterizing transcriptional profiles of individual cells isolated from heterogeneous populations. In this study we describe a novel procedure for the isolation and characterization of gene expression profiles of single osteoblast lineage cells derived from cortical bone. Mixed populations of different cell types were isolated from adult long bones of C57BL/6J mice by enzymatic digestion, and subsequently subjected to FACS to purify and characterize osteoblast lineage cells via a selection strategy using antibodies against CD31, CD45, and alkaline phosphatase (AP), specific for mature osteoblasts. The purified individual osteoblast lineage cells were then profiled at the single cell level via nanofluidic PCR. This method permits robust gene expression profiling on single osteoblast lineage cells derived from mature bone, potentially from anatomically distinct sites. In conjunction with this technique, we have also shown that it is possible to carry out single cell profiling on cells purified from fixed and frozen bone samples without compromising the gene expression signal. The latter finding means the technique can be extended to biopsies of bone from diseased individuals. Our approach for single cell expression profiling provides a new dimension to the transcriptional profile of the primary osteoblast lineage population in vivo, and has the capacity to greatly expand our understanding of how these cells may function in vivo under normal and diseased states.
authors.affiliation: Buck Institute for Research on Aging, 8001 Redwood Blvd. Novato, CA 94945, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Flynn
authors.forename: Steven C
authors.initials: SC
authors.lastname: Spusta
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Rosen
authors.forename: Simon
authors.initials: S
authors.lastname: Melov
mesh_terms: D000818:Animals; D019070:Cell Lineage; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods