Suppression of dedifferentiation and hypertrophy in canine chondrocytes through lentiviral vector expression of Sox9 and induced pluripotency stem cell factors

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Objectives: Prolonged in vitro culture of primary articular chondrocytes results in dedifferentiation to a fibroblast-like cell with reduced expression of the Sox9 transcription factor and the extracellular matrix protein collagen II. The ability to genetically-modify chondrocytes to allow both proliferation and maintenance of an articular phenotype may provide increased numbers of appropriate cells for regeneration of large cartilage defects. Results: Canine chondrocytes were expanded in monolayer culture and transduced with a lentiviral vector expressing Sox9 or in combination with a multicistronic lentiviral vector expressing the four induced pluripotency stem (iPS) cell factors, Oct4, Klf4, Sox2 and c-Myc (OSKM). 3D pellet cultures of transduced cells in the presence of TGFβ-3 revealed increased pellet size and higher levels of total glycosaminoglycan in both Sox9 and Sox9+ OSKM co-transduced chondrocytes compared to untransduced and green fluorescent protein expressing controls. Immunohistochemical detection of Sox9 and collagen II was evident in transduced cells (Sox9, OSKM, or Sox9+ OSKM) with very low levels in untransduced chondrocytes, demonstrating a dedifferentiated state (P < 0.01). The marker for chondrocyte hypertrophy, collagen X was highly expressed in Sox9 transduced chondrocytes but lower in OSKM or Sox9+ OSKM cells (P < 0.05). Conclusion: A combination of Sox9 and OSKM gene delivery to canine chondrocytes allows continuous proliferation in monolayer culture with a higher expression of col2a1 without an increase in the hypertrophy marker collagen X in 3D pellet cultures.
Original languageEnglish
Pages (from-to)1495-1504
Number of pages10
JournalBiotechnology Letters
Volume37
Issue number7
DOIs
Publication statusPublished - Mar 2015

Fingerprint

Stem Cell Factor
Chondrocytes
Stem cells
Collagen
Hypertrophy
Canidae
Monolayers
Proteins
Transcription factors
Extracellular Matrix Proteins
Cartilage
Fibroblasts
Green Fluorescent Proteins
Glycosaminoglycans
Cell culture
Transcription Factors
Genes
Joints
Defects
Regeneration

Cite this

@article{a49b0adac252448ab2e387d5adb06df1,
title = "Suppression of dedifferentiation and hypertrophy in canine chondrocytes through lentiviral vector expression of Sox9 and induced pluripotency stem cell factors",
abstract = "Objectives: Prolonged in vitro culture of primary articular chondrocytes results in dedifferentiation to a fibroblast-like cell with reduced expression of the Sox9 transcription factor and the extracellular matrix protein collagen II. The ability to genetically-modify chondrocytes to allow both proliferation and maintenance of an articular phenotype may provide increased numbers of appropriate cells for regeneration of large cartilage defects. Results: Canine chondrocytes were expanded in monolayer culture and transduced with a lentiviral vector expressing Sox9 or in combination with a multicistronic lentiviral vector expressing the four induced pluripotency stem (iPS) cell factors, Oct4, Klf4, Sox2 and c-Myc (OSKM). 3D pellet cultures of transduced cells in the presence of TGFβ-3 revealed increased pellet size and higher levels of total glycosaminoglycan in both Sox9 and Sox9+ OSKM co-transduced chondrocytes compared to untransduced and green fluorescent protein expressing controls. Immunohistochemical detection of Sox9 and collagen II was evident in transduced cells (Sox9, OSKM, or Sox9+ OSKM) with very low levels in untransduced chondrocytes, demonstrating a dedifferentiated state (P < 0.01). The marker for chondrocyte hypertrophy, collagen X was highly expressed in Sox9 transduced chondrocytes but lower in OSKM or Sox9+ OSKM cells (P < 0.05). Conclusion: A combination of Sox9 and OSKM gene delivery to canine chondrocytes allows continuous proliferation in monolayer culture with a higher expression of col2a1 without an increase in the hypertrophy marker collagen X in 3D pellet cultures.",
keywords = "Cartilage, Cell-culture, Cells, Chondrogenesis, Collagen, Cytology, Gene-expression, Gene-transfer, Hypertrophy, Induced-pluripotent-stem-cells, Lentiviral-vector, Monolayers, Pathology, Pelletizing, Proteins, Reprogra, Stem-cells, Transcription, Vectors",
author = "Saliya Gurusinghe and Peter Young and Jacob Michelsen and Padraig Strappe",
note = "Includes bibliographical references.",
year = "2015",
month = "3",
doi = "10.1007/s10529-015-1805-5",
language = "English",
volume = "37",
pages = "1495--1504",
journal = "Biotechnology Techniques",
issn = "0141-5492",
publisher = "Springer Netherlands",
number = "7",

}

TY - JOUR

T1 - Suppression of dedifferentiation and hypertrophy in canine chondrocytes through lentiviral vector expression of Sox9 and induced pluripotency stem cell factors

AU - Gurusinghe, Saliya

AU - Young, Peter

AU - Michelsen, Jacob

AU - Strappe, Padraig

N1 - Includes bibliographical references.

PY - 2015/3

Y1 - 2015/3

N2 - Objectives: Prolonged in vitro culture of primary articular chondrocytes results in dedifferentiation to a fibroblast-like cell with reduced expression of the Sox9 transcription factor and the extracellular matrix protein collagen II. The ability to genetically-modify chondrocytes to allow both proliferation and maintenance of an articular phenotype may provide increased numbers of appropriate cells for regeneration of large cartilage defects. Results: Canine chondrocytes were expanded in monolayer culture and transduced with a lentiviral vector expressing Sox9 or in combination with a multicistronic lentiviral vector expressing the four induced pluripotency stem (iPS) cell factors, Oct4, Klf4, Sox2 and c-Myc (OSKM). 3D pellet cultures of transduced cells in the presence of TGFβ-3 revealed increased pellet size and higher levels of total glycosaminoglycan in both Sox9 and Sox9+ OSKM co-transduced chondrocytes compared to untransduced and green fluorescent protein expressing controls. Immunohistochemical detection of Sox9 and collagen II was evident in transduced cells (Sox9, OSKM, or Sox9+ OSKM) with very low levels in untransduced chondrocytes, demonstrating a dedifferentiated state (P < 0.01). The marker for chondrocyte hypertrophy, collagen X was highly expressed in Sox9 transduced chondrocytes but lower in OSKM or Sox9+ OSKM cells (P < 0.05). Conclusion: A combination of Sox9 and OSKM gene delivery to canine chondrocytes allows continuous proliferation in monolayer culture with a higher expression of col2a1 without an increase in the hypertrophy marker collagen X in 3D pellet cultures.

AB - Objectives: Prolonged in vitro culture of primary articular chondrocytes results in dedifferentiation to a fibroblast-like cell with reduced expression of the Sox9 transcription factor and the extracellular matrix protein collagen II. The ability to genetically-modify chondrocytes to allow both proliferation and maintenance of an articular phenotype may provide increased numbers of appropriate cells for regeneration of large cartilage defects. Results: Canine chondrocytes were expanded in monolayer culture and transduced with a lentiviral vector expressing Sox9 or in combination with a multicistronic lentiviral vector expressing the four induced pluripotency stem (iPS) cell factors, Oct4, Klf4, Sox2 and c-Myc (OSKM). 3D pellet cultures of transduced cells in the presence of TGFβ-3 revealed increased pellet size and higher levels of total glycosaminoglycan in both Sox9 and Sox9+ OSKM co-transduced chondrocytes compared to untransduced and green fluorescent protein expressing controls. Immunohistochemical detection of Sox9 and collagen II was evident in transduced cells (Sox9, OSKM, or Sox9+ OSKM) with very low levels in untransduced chondrocytes, demonstrating a dedifferentiated state (P < 0.01). The marker for chondrocyte hypertrophy, collagen X was highly expressed in Sox9 transduced chondrocytes but lower in OSKM or Sox9+ OSKM cells (P < 0.05). Conclusion: A combination of Sox9 and OSKM gene delivery to canine chondrocytes allows continuous proliferation in monolayer culture with a higher expression of col2a1 without an increase in the hypertrophy marker collagen X in 3D pellet cultures.

KW - Cartilage

KW - Cell-culture

KW - Cells

KW - Chondrogenesis

KW - Collagen

KW - Cytology

KW - Gene-expression

KW - Gene-transfer

KW - Hypertrophy

KW - Induced-pluripotent-stem-cells

KW - Lentiviral-vector

KW - Monolayers

KW - Pathology

KW - Pelletizing

KW - Proteins

KW - Reprogra

KW - Stem-cells

KW - Transcription

KW - Vectors

U2 - 10.1007/s10529-015-1805-5

DO - 10.1007/s10529-015-1805-5

M3 - Article

VL - 37

SP - 1495

EP - 1504

JO - Biotechnology Techniques

JF - Biotechnology Techniques

SN - 0141-5492

IS - 7

ER -