Single‐Cell RNA Sequencing of Calvarial and Long‐Bone Endocortical Cells
ABSTRACT 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 vit...
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Published in | Journal of bone and mineral research Vol. 35; no. 10; pp. 1981 - 1991 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
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Hoboken, USA
John Wiley & Sons, Inc
01.10.2020
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Abstract | ABSTRACT
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. © 2020 American Society for Bone and Mineral Research. |
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AbstractList | 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. We were unable to detect significant changes in bone anabolism-associated transcripts in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this might be 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. 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. © 2020 American Society for Bone and Mineral Research. ABSTRACT 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. © 2020 American Society for Bone and Mineral Research. |
Author | Vesprey, Alexander Goz Ayturk, Didem Ayturk, Ugur M Jacobsen, Christina M Scollan, Joseph P Suh, Eun Sung Divieti Pajevic, Paola Warman, Matthew L |
AuthorAffiliation | 1. Musculoskeletal Integrity Program, Hospital for Special Surgery, New York, NY 8. Department of Genetics, Harvard Medical School, Boston, MA 2. Department of Orthopaedic Surgery, Weill Cornell Medical College, New York, NY 3. Department of Orthopaedic Surgery, Boston Children’s Hospital, Boston, MA 4. Department of Orthopaedic Surgery, Cleveland Clinic Foundation, Cleveland, OH 5. Divisions of Endocrinology and Genetics and Genomics, Boston Children’s Hospital, Boston, MA 6. Department of Pediatrics, Harvard Medical School, Boston MA 7. Department of Translational Dental Medicine, Boston University Goldman School of Dental Medicine, Boston, MA |
AuthorAffiliation_xml | – name: 2. Department of Orthopaedic Surgery, Weill Cornell Medical College, New York, NY – name: 7. Department of Translational Dental Medicine, Boston University Goldman School of Dental Medicine, Boston, MA – name: 5. Divisions of Endocrinology and Genetics and Genomics, Boston Children’s Hospital, Boston, MA – name: 6. Department of Pediatrics, Harvard Medical School, Boston MA – name: 8. Department of Genetics, Harvard Medical School, Boston, MA – name: 3. Department of Orthopaedic Surgery, Boston Children’s Hospital, Boston, MA – name: 4. Department of Orthopaedic Surgery, Cleveland Clinic Foundation, Cleveland, OH – name: 1. Musculoskeletal Integrity Program, Hospital for Special Surgery, New York, NY |
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Keywords | CELLS OF BONE STROMAL/STEM CELLS ANIMAL MODELS STATISTICAL METHODS OSTEOBLASTS |
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Single‐cell RNA sequencing (scRNA‐Seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether... Single-cell RNA sequencing (scRNA-Seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-Seq... Single‐cell RNA sequencing (scRNA‐Seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA‐Seq... 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... |
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SubjectTerms | ANIMAL MODELS Animals CELLS OF BONE Gene expression Gene Expression Profiling Kinases Long bone Macrophages Mice Neonates OSTEOBLASTS Osteocytes Ribonucleic acid RNA Sequence Analysis, RNA Single-Cell Analysis Skeleton SOST protein STATISTICAL METHODS STROMAL/STEM CELLS Transcriptome |
Title | Single‐Cell RNA Sequencing of Calvarial and Long‐Bone Endocortical Cells |
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