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Both Chondroinduction and Proliferation Account for Growth of Cartilage Nodules in Mouse Limb Bud Cultures

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Abstract

High density micromass culture of limb bud mesenchymal stem cells isolated from mouse embryos represents a well-established model to study chondro- and osteogenesis. In spite of wide usage of the limb bud model, the mechanisms underlying cartilage nodule growth remain unclear. To determine whether cartilage nodules grow solely by induction of surrounding cells or proliferation of cells within the nodules, we performed BrdU/Collagen II (Col II) double-labelling and 3D reconstruction of growing cartilage nodules. We demonstrated that Col II-positive replicating chondrocytes are present throughout the nodules with the majority of replicating cells localized on the top (cell-medium interface) and periphery/sides of nodules. Kinetic analysis of cellular proliferation within the nodules demonstrated the time-dependent reduction in number of Col II-positive replicating cells. The sequential expression of Col I, Col II, Col X, parathyroid hormone related peptide receptor 1 (Pthr1), bone sialoprotein (Bsp) and osteocalcin (Ocn) mRNAs was similar to that characterizing chondrocyte differentiation and maturation in vivo. We conclude that the limb bud model recapitulates events seen during endochondral bone formation: cellular aggregation, proliferation, differentiation and maturation to hypertrophy. We also conclude that not only induction of peri-nodular mesenchymal cells but also proliferation of chondrocytes within cartilage nodules contribute to cartilage nodule growth.

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Abbreviations

ALP:

Alkaline phosphatase

BrdU:

Bromodeoxyuridine

BSP:

Bone sialoprotein

Col:

Collagen

FITC:

Fluorescein isothiocyanate

OCN:

Osteocalcin

PBS:

Phosphate buffered saline

PI:

Propidium iodide

PSA:

Puck’s saline without glucose

PSG:

Puck’s saline with glucose

PTHR1:

Parathyroid hormone related peptide receptor 1

SEM:

Standard error of the mean

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Acknowledgements

M.O. and J.E.A. are members of the Ontario Stem Cell initiative, J.E.A. is a member of the Stem Cell Network of Centres of Excellence, and M.O. is a member of the Heart & Stroke/Richard Lewar Centre of Excellence.

Conflict of Interest

None

Author information

Authors and Affiliations

  1. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

    Andrei V. Malko, Maria Villagomez & Michal Opas

  2. Department of Molecular Genetics, University of Toronto, Toronto, Canada

    Jane E. Aubin

  3. Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building, room 6326, Toronto, ON, M5S 1A8, Canada

    Michal Opas

Authors
  1. Andrei V. Malko
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  2. Maria Villagomez
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  3. Jane E. Aubin
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Corresponding author

Correspondence to Michal Opas.

Additional information

Contract grant sponsor: CIHR; Contract grant number: MOP-106461.

Contract grant sponsor: CIHR; Contract grant number: MOP-102549.

Contract grant sponsor: CIHR; Contract grant number: MOP-83704.

Authors’ contributions

Andrei V. Malko: Collection and assembly of data Data analysis and interpretation, Manuscript writing

Maria Villagomez: Collection and assembly of data Data analysis and interpretation, Manuscript writing

Jane E. Aubin: Conception and design Data analysis and interpretation, Manuscript writing and Final approval

M. Opas: Conception and design Data analysis and interpretation, Manuscript writing and Final approval

Electronic supplementary material

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Supplemental Figure 1

Morphological characterization of mouse limb bud cultures by phase-contrast microscopy of live cultures on days 2, 4, 6 and 8. On day 2, cells were already confluent and condensations, which will give rise to cartilage nodules, have started to form. On day 4, well defined oval-shaped cartilage nodules were present and characterized by rounded cells and refractile matrix. On days 6 and 8, nodules were progressively bigger in size. Note gradual change in shape of cells within the nodules. C - condensation; N - nodule. (TIFF 2633 kb)

Supplemental Table 1

Primers are listed in 5′ to 3′ orientation. The expected product size as well as the annealing temperature and number of PCR cycles used for each set of primers are indicated. (DOC 35 kb)

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Malko, A.V., Villagomez, M., Aubin, J.E. et al. Both Chondroinduction and Proliferation Account for Growth of Cartilage Nodules in Mouse Limb Bud Cultures. Stem Cell Rev and Rep 9, 121–131 (2013). https://doi.org/10.1007/s12015-013-9434-7

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  • DOI: https://doi.org/10.1007/s12015-013-9434-7

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