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Morphological examination during in vitro cartilage formation by human mesenchymal stem cells

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Abstract

The formation of the skeleton through endochondral ossification is one of the most complex processes in development. One approach to resolving this complexity is to examine simplified systems. In vitro cartilage formation by mesenchymal stem cells (MSCs) is observed when the cells are cultured as a micromass. Several studies have confirmed the molecular events, showing the usefulness of these cells as a differentiation model. We have elucidated the process of cartilage formation in MSCs from the morphological point of view by light and transmission electron microscopy and immunohistochemical examination. The morphology of the MSCs changed from spherical to spindle-shaped, and the cells aggregated and formed junctional complexes during Day 1. At Day 7, three layers were observed. The superficial zone consisted of several layers of elongated cells with junctional complexes. The middle zone was composed of apoptotic bodies, and the deep zone was occupied by chondrocyte-like cells excreting extracellular matrices. At Day 14, the middle zone had disappeared, and the chondrocyte-like cells in the deep zone were detected within cartilage lacuna. They were covered by cartilage matrices containing collagen types I, II, and X and chondroitin sulfate. By Day 21, the outer layer consisting of spindle-shaped cells had disappeared in places. As the pellet grew, the outer layer seemed to be unable to stretch to maintain a constant covering around the pellet. Our findings have thus revealed that MSCs change their morphology depending upon their microenvironment during differentiation. In vitro cartilage formation by MSCs makes it possible to clarify the detailed morphological events that occur during chondrogenesis.

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Acknowledgements

The authors thank Yusuke Sakaguchi and Izumi Nakagawa for help with the cell cultures, Miyoko Ojima for help with the immunostaining, Masako Kawano and Kumiko Takeda for technical assistance, and Alexandra Peister for proofreading.

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Authors and Affiliations

  1. Instrumental Analysis Research Center, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan

    Shizuko Ichinose

  2. Department of Medicine, Sanraku Hospital, Chiyoda-ku, Tokyo, 101-0062, Japan

    Motoki Tagami

  3. Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Tokyo, Japan

    Takeshi Muneta

  4. Section of Orthopedic Surgery, Division of Bio-Matrix, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan

    Takeshi Muneta & Ichiro Sekiya

Authors
  1. Shizuko Ichinose
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  2. Motoki Tagami
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  3. Takeshi Muneta
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  4. Ichiro Sekiya
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Corresponding author

Correspondence to Shizuko Ichinose.

Additional information

S. Ichinose and I. Sekiya contributed equally to this study

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Ichinose, S., Tagami, M., Muneta, T. et al. Morphological examination during in vitro cartilage formation by human mesenchymal stem cells. Cell Tissue Res 322, 217–226 (2005). https://doi.org/10.1007/s00441-005-1140-6

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  • DOI: https://doi.org/10.1007/s00441-005-1140-6

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