Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

Daisuke Sakai; Jordy Schol; Frances C Bach; Adel Tekari; Nobuho Sagawa; Yoshihiko Nakamura; Samantha C W Chan; Tomoko Nakai; Laura B Creemers; Daniela A Frauchiger; Rahel D May; Sibylle Grad; Masahiko Watanabe; Marianna A Tryfonidou; Benjamin Gantenbein

doi:10.1002/jsp2.1018

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

Daisuke Sakai
, Jordy Schol
, Frances C Bach
, Adel Tekari
, Nobuho Sagawa
, Yoshihiko Nakamura
, Samantha C W Chan
, Tomoko Nakai
, Laura B Creemers
, Daniela A Frauchiger
, Rahel D May
, Sibylle Grad
, Masahiko Watanabe
, Marianna A Tryfonidou
, Benjamin Gantenbein

Center for Regenerative Medicine Tokai University School of Medicine Isehara Japan.
Laboratory of Molecular and Cellular Screening Processes Centre of Biotechnology of Sfax, University of Sfax Sfax Tunisia.
Department for Orthopaedic Surgery Tokai University School of Medicine Isehara Japan.
Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, Medical Faculty University of Bern Bern Switzerland.
University Medical Center Utrecht
Department of Musculoskeletal Regeneration, AO Research Institute Davos Switzerland.
AO Spine Research Network, AO Spine International Davos Switzerland.

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

Abstract

Background: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin-1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.

Purpose: Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs.

Methods: We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2 across species is presented.

Results: Our protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.

Conclusions: Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.

Original language	English
Article number	e1018
Journal	Spine
Volume	1
Issue number	2
DOIs	https://doi.org/10.1002/jsp2.1018
Publication status	Published - Jun 2018

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

biologic therapies
culture systems
stem cell
tissue-specific progenitor cells

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Sakai_et_al-2018-JOR_Spine (1)Final published version, 3.26 MB

Cite this

Sakai, D., Schol, J., Bach, F. C., Tekari, A., Sagawa, N., Nakamura, Y., Chan, S. C. W., Nakai, T., Creemers, L. B., Frauchiger, D. A., May, R. D., Grad, S., Watanabe, M., Tryfonidou, M. A., & Gantenbein, B. (2018). Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species. Spine, 1(2), Article e1018. https://doi.org/10.1002/jsp2.1018

@article{65d2ccc8ae4241259829bcbf651f392d,

title = "Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species",

abstract = "Background: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin-1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.Purpose: Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs.Methods: We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2 across species is presented.Results: Our protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.Conclusions: Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.",

keywords = "biologic therapies, culture systems, stem cell, tissue-specific progenitor cells",

author = "Daisuke Sakai and Jordy Schol and Bach, \{Frances C\} and Adel Tekari and Nobuho Sagawa and Yoshihiko Nakamura and Chan, \{Samantha C W\} and Tomoko Nakai and Creemers, \{Laura B\} and Frauchiger, \{Daniela A\} and May, \{Rahel D\} and Sibylle Grad and Masahiko Watanabe and Tryfonidou, \{Marianna A\} and Benjamin Gantenbein",

year = "2018",

month = jun,

doi = "10.1002/jsp2.1018",

language = "English",

volume = "1",

journal = "Spine",

issn = "0362-2436",

publisher = "Wolters Kluwer Health",

number = "2",

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TY - JOUR

T1 - Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

AU - Sakai, Daisuke

AU - Schol, Jordy

AU - Bach, Frances C

AU - Tekari, Adel

AU - Sagawa, Nobuho

AU - Nakamura, Yoshihiko

AU - Chan, Samantha C W

AU - Nakai, Tomoko

AU - Creemers, Laura B

AU - Frauchiger, Daniela A

AU - May, Rahel D

AU - Grad, Sibylle

AU - Watanabe, Masahiko

AU - Tryfonidou, Marianna A

AU - Gantenbein, Benjamin

PY - 2018/6

Y1 - 2018/6

N2 - Background: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin-1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.Purpose: Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs.Methods: We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2 across species is presented.Results: Our protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.Conclusions: Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.

AB - Background: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin-1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.Purpose: Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs.Methods: We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2 across species is presented.Results: Our protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.Conclusions: Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.

KW - biologic therapies

KW - culture systems

KW - stem cell

KW - tissue-specific progenitor cells

U2 - 10.1002/jsp2.1018

DO - 10.1002/jsp2.1018

M3 - Article

C2 - 31463445

SN - 0362-2436

VL - 1

JO - Spine

JF - Spine

IS - 2

M1 - e1018

ER -

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species

Abstract

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Keywords

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