Protocol to analyze and validate transcriptomic changes in PDGFRβ-KO mesenchymal stem cell osteogenic potential in the mouse embryo

Alastair Morris Kilpatrick; Madalena Marques; Mario Gomez Salazar; Dorota Stefancova; Diana Sa da Bandeira; Mihaela Crisan

doi:10.1016/j.xpro.2022.102016

Protocol to analyze and validate transcriptomic changes in PDGFRβ-KO mesenchymal stem cell osteogenic potential in the mouse embryo

Alastair Morris Kilpatrick, Madalena Marques, Mario Gomez Salazar, Dorota Stefancova, Diana Sa da Bandeira, Mihaela Crisan^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract / Description of output

Mesenchymal stem/stromal cells (MSCs) can differentiate into osteoblasts under appropriate conditions. PDGFRβ signaling controls MSC osteogenic potential both transcriptomically and in culture. Here, we present a “computer to the bench” protocol to analyze changes in MSC osteogenic potential at transcriptomic and cellular level in the absence of PDGFRβ. We detail the preparation of cells from mouse embryos, the analysis of transcriptomic changes from single-cell RNA-sequencing data, the procedure for MSC derivation and culture, and an osteogenic assay for functional validation. For complete details on the use and execution of this protocol, please refer to Sá da Bandeira et al. (2022).

Original language	English
Article number	102016
Journal	STAR Protocols
Volume	4
Issue number	1
Early online date	12 Jan 2023
DOIs	https://doi.org/10.1016/j.xpro.2022.102016
Publication status	Published - 17 Mar 2023

Keywords / Materials (for Non-textual outputs)

Bioinformatics
Cell Biology
Cell Differentiation
Developmental Biology
Gene Expression
Molecular Biology
RNAseq
Single Cell
Stem Cells

Access to Document

10.1016/j.xpro.2022.102016Licence: Creative Commons: Attribution (CC-BY)

R3 Crisan CellPress_STARProtocols FinalAccepted author manuscript, 2.94 MBLicence: Creative Commons: Attribution (CC-BY)
1-s2.0-S2666166722008966-mainFinal published version, 3.23 MBLicence: Creative Commons: Attribution (CC-BY)

1 Finished
1 Active

Chancellors fellowship – Takanori Kitamura (G37048)
Kitamura, T.
31/07/12 → …
Project: University Awarded Project Funding
REA3 pump priming
Baker, A.
UK-based charities
1/04/19 → 31/03/24
Project: Research

Cite this

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title = "Protocol to analyze and validate transcriptomic changes in PDGFRβ-KO mesenchymal stem cell osteogenic potential in the mouse embryo",

abstract = "Mesenchymal stem/stromal cells (MSCs) can differentiate into osteoblasts under appropriate conditions. PDGFRβ signaling controls MSC osteogenic potential both transcriptomically and in culture. Here, we present a “computer to the bench” protocol to analyze changes in MSC osteogenic potential at transcriptomic and cellular level in the absence of PDGFRβ. We detail the preparation of cells from mouse embryos, the analysis of transcriptomic changes from single-cell RNA-sequencing data, the procedure for MSC derivation and culture, and an osteogenic assay for functional validation. For complete details on the use and execution of this protocol, please refer to S{\'a} da Bandeira et al. (2022).",

keywords = "Bioinformatics, Cell Biology, Cell Differentiation, Developmental Biology, Gene Expression, Molecular Biology, RNAseq, Single Cell, Stem Cells",

author = "Kilpatrick, {Alastair Morris} and Madalena Marques and {Gomez Salazar}, Mario and Dorota Stefancova and {Sa da Bandeira}, Diana and Mihaela Crisan",

note = "Funding Information: This work was performed at the University of Edinburgh and has made use of the resources provided by the Edinburgh Compute and Data Facility (ECDF). For the purpose of open access, the author has applied a CC-BY public copyright license to any Author-Accepted Manuscript version arising from this submission. We are grateful to the staff members of the University of Edinburgh imaging and animal units for their help and Edinburgh Genomics for library sequencing. We acknowledge the University of Edinburgh Chancellor{\textquoteright}s Fellowship ( G37048 ), the Association Francaise contre la Myopathie (AFM) Trampoline Award ( 22134/6744359 I190311-2308 ), the British Heart Foundation (BHF) Research Excellence Award (REA3) pump-priming grant ( RE/18/5/34216 ), and the University of Edinburgh Wellcome Trust Institutional Strategic Support Fund (ISSF3, 199MCR/J22739 ) to M.C. We thank Christer Betsholtz for PDGFRβ+/− mice. Publisher Copyright: {\textcopyright} 2022 The Author(s)",

year = "2023",

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doi = "10.1016/j.xpro.2022.102016",

language = "English",

volume = "4",

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T1 - Protocol to analyze and validate transcriptomic changes in PDGFRβ-KO mesenchymal stem cell osteogenic potential in the mouse embryo

AU - Kilpatrick, Alastair Morris

AU - Marques, Madalena

AU - Gomez Salazar, Mario

AU - Stefancova, Dorota

AU - Sa da Bandeira, Diana

AU - Crisan, Mihaela

N1 - Funding Information: This work was performed at the University of Edinburgh and has made use of the resources provided by the Edinburgh Compute and Data Facility (ECDF). For the purpose of open access, the author has applied a CC-BY public copyright license to any Author-Accepted Manuscript version arising from this submission. We are grateful to the staff members of the University of Edinburgh imaging and animal units for their help and Edinburgh Genomics for library sequencing. We acknowledge the University of Edinburgh Chancellor’s Fellowship ( G37048 ), the Association Francaise contre la Myopathie (AFM) Trampoline Award ( 22134/6744359 I190311-2308 ), the British Heart Foundation (BHF) Research Excellence Award (REA3) pump-priming grant ( RE/18/5/34216 ), and the University of Edinburgh Wellcome Trust Institutional Strategic Support Fund (ISSF3, 199MCR/J22739 ) to M.C. We thank Christer Betsholtz for PDGFRβ+/− mice. Publisher Copyright: © 2022 The Author(s)

PY - 2023/3/17

Y1 - 2023/3/17

N2 - Mesenchymal stem/stromal cells (MSCs) can differentiate into osteoblasts under appropriate conditions. PDGFRβ signaling controls MSC osteogenic potential both transcriptomically and in culture. Here, we present a “computer to the bench” protocol to analyze changes in MSC osteogenic potential at transcriptomic and cellular level in the absence of PDGFRβ. We detail the preparation of cells from mouse embryos, the analysis of transcriptomic changes from single-cell RNA-sequencing data, the procedure for MSC derivation and culture, and an osteogenic assay for functional validation. For complete details on the use and execution of this protocol, please refer to Sá da Bandeira et al. (2022).

AB - Mesenchymal stem/stromal cells (MSCs) can differentiate into osteoblasts under appropriate conditions. PDGFRβ signaling controls MSC osteogenic potential both transcriptomically and in culture. Here, we present a “computer to the bench” protocol to analyze changes in MSC osteogenic potential at transcriptomic and cellular level in the absence of PDGFRβ. We detail the preparation of cells from mouse embryos, the analysis of transcriptomic changes from single-cell RNA-sequencing data, the procedure for MSC derivation and culture, and an osteogenic assay for functional validation. For complete details on the use and execution of this protocol, please refer to Sá da Bandeira et al. (2022).

KW - Bioinformatics

KW - Cell Biology

KW - Cell Differentiation

KW - Developmental Biology

KW - Gene Expression

KW - Molecular Biology

KW - RNAseq

KW - Single Cell

KW - Stem Cells

U2 - 10.1016/j.xpro.2022.102016

DO - 10.1016/j.xpro.2022.102016

M3 - Article

SN - 2666-1667

VL - 4

JO - STAR Protocols

JF - STAR Protocols

IS - 1

M1 - 102016

ER -

Protocol to analyze and validate transcriptomic changes in PDGFRβ-KO mesenchymal stem cell osteogenic potential in the mouse embryo

Abstract / Description of output

Keywords / Materials (for Non-textual outputs)

Access to Document

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Projects

Chancellors fellowship – Takanori Kitamura (G37048)

REA3 pump priming

Cite this