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.
[Show abstract][Hide abstract] ABSTRACT: Aim:
To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate.
Materials & methods:
MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction.
Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45(-)CD271(+)CD73(+)CD90(+)CD105(+) MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate.
Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings.
Regenerative Medicine 03/2014; 9(5). DOI:10.2217/rme.14.17 · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circular RNAs (circRNAs) are a new class of non-polyadenylated non-coding RNAs that may play important roles in many biological processes. Here we develop a single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq) method to sequence both polyadenylated and non-polyadenylated RNAs from individual cells. This method exhibits robust sensitivity, precision and accuracy. We discover 2891 circRNAs and 913 novel linear transcripts in mouse preimplantation embryos and further analyze the abundance of circRNAs along development, the function of enriched genes, and sequence features of circRNAs. Our work is key to deciphering regulation mechanisms of circRNAs during mammalian early embryonic development.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0706-1) contains supplementary material, which is available to authorized users.
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