The Unmixing Problem: A Guide to Applying Single-Cell RNA Sequencing to Bone

Scollan

Journal of Bone and Mineral Research

et al. 2020

Self Cite

Single-cell RNA sequencing (scRNA-Seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-Seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-Seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-Seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-Seq on freshly recovered long bone endocortical cells from mice that received either vehicle or sclerostin-neutralizing antibody for 1 week. We were unable to detect significant changes in bone anabolism-associated transcripts in immature and mature osteoblasts recovered from mice treated with sclerostin-neutralizing antibody; this might be a consequence of being underpowered to detect modest changes in gene expression, because only 7% of the sequenced endocortical cells were osteoblasts and a limited portion of their transcriptomes were sampled. We conclude that scRNA-Seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single-cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

Section: Discussionmentioning

confidence: 99%

“…( 15,16 ) In order to study freshly isolated osteoblasts or their precursors, skeletal tissues need to be subjected to sequential enzymatic digestions for an hour or longer. ( 14,15,41 )…”

Section: Discussionmentioning

confidence: 99%

Single-Cell RNA Sequencing of Calvarial and Long-Bone Endocortical Cells

Scollan

Journal of Bone and Mineral Research

et al. 2020

Self Cite

“…Chondrocytes are regulated by complex and multifactorial molecular signaling pathways, which govern their ability to self-renew and differentiate into diverse cell types, such as columnar chondrocytes in the growth plate as well as osteoblasts and bone marrow stromal cells in the primary spongiosa and marrow cavity (Figure 1). Given the scientific era where we observe explosive technological advances on a daily basis, the application of single cell RNA transcriptomics and other omic-based technologies will enable researchers to further elucidate the fundamental molecular properties of individual chondrocytes in a spatiotemporal manner during various stages of skeletal development [83,84]. By doing so, we become one step closer to bridging the gap between the clinic and the bench, using novel regenerative therapies for patients suffering from congenital skeletal defects or those who have suffered through traumatic injuries to the skeleton.…”

Section: Discussionmentioning

confidence: 99%

Growth Plate Chondrocytes: Skeletal Development, Growth and Beyond

Hallett

Ono

2019

IJMS

Self Cite

117

Growth plate chondrocytes play central roles in the proper development and growth of endochondral bones. Particularly, a population of chondrocytes in the resting zone expressing parathyroid hormone-related protein (PTHrP) is now recognized as skeletal stem cells, defined by their ability to undergo self-renewal and clonally give rise to columnar chondrocytes in the postnatal growth plate. These chondrocytes also possess the ability to differentiate into a multitude of cell types including osteoblasts and bone marrow stromal cells during skeletal development. Using single-cell transcriptomic approaches and in vivo lineage tracing technology, it is now possible to further elucidate their molecular properties and cellular fate changes. By discovering the fundamental molecular characteristics of these cells, it may be possible to harness their functional characteristics for skeletal growth and regeneration. Here, we discuss our current understanding of the molecular signatures defining growth plate chondrocytes.

“…For this reason, in vitro studies of osteoblast differentiation and mineralization typically expand cells in culture and then chemically induce matrix mineralization[15, 16]. In order to study freshly isolated osteoblasts or their precursors, skeletal tissues need to be subjected to sequential enzymatic digestions for an hour or longer [14, 15, 40]. We employed scRNA-seq to assess similarities and differences between osteoblasts that were freshly obtained from calvaria versus long bone endocortices, between fresh calvarial cells and those that had been expanded in culture, and between long bone endocortical cells from PBS- and SclAbIII-treated animals.…”

Section: Discussionmentioning

confidence: 99%

Single cell RNA sequencing of calvarial and long bone endocortical cells

Scollan

Vesprey

et al. 2019

Preprint

Single cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.