Dataset Information

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.

SUBMITTER: Flynn JM

PROVIDER: S-EPMC3589579 | biostudies-other | 2013 Mar

REPOSITORIES: biostudies-other

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Single cell gene expression profiling of cortical osteoblast lineage cells.

Flynn James M JM Spusta Steven C SC Rosen Clifford J CJ Melov Simon S

Bone 20121210 1

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. Mixe ...[more]

PMID: 23238121

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Project description:The osteoblast-lineage consists of cells at various stages of maturation that are essential for skeletal development, growth, and maintenance. Over the past decade, many of the signaling cascades that regulate this lineage have been elucidated; however, little is known of the networks that coordinate, modulate, and transmit these signals. Here, we identify a gene network specific to the osteoblast-lineage through the reconstruction of a bone co-expression network using microarray profiles collected on 96 Hybrid Mouse Diversity Panel (HMDP) inbred strains. Of the 21 modules that comprised the bone network, module 9 (M9) contained genes that were highly correlated with prototypical osteoblast maker genes and were more highly expressed in osteoblasts relative to other bone cells. In addition, the M9 contained many of the key genes that define the osteoblast-lineage, which together suggested that it was specific to this lineage. To use the M9 to identify novel osteoblast genes and highlight its biological relevance, we knocked-down the expression of its two most connected "hub" genes, Maged1 and Pard6g. Their perturbation altered both osteoblast proliferation and differentiation. Furthermore, we demonstrated the mice deficient in Maged1 had decreased bone mineral density (BMD). It was also discovered that a local expression quantitative trait locus (eQTL) regulating the Wnt signaling antagonist Sfrp1 was a key driver of the M9. We also show that the M9 is associated with BMD in the HMDP and is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In conclusion, we have identified a physiologically relevant gene network and used it to discover novel genes and regulatory mechanisms involved in the function of osteoblast-lineage cells. Our results highlight the power of harnessing natural genetic variation to generate co-expression networks that can be used to gain insight into the function of specific cell-types.

Dataset Information

Single cell gene expression profiling of cortical osteoblast lineage cells.

Publications

Single cell gene expression profiling of cortical osteoblast lineage cells.

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