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Improved Protocol for Chondrogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells -Effect of PTHrP and FGF-2 on TGFβ1/BMP2-Induced Chondrocytes Hypertrophy

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Abstract

Growth factors have a pivotal role in chondrogenic differentiation of stem cells. The differential effects of known growth factors involved in the maintenance and homeostasis of cartilage tissue have been previously studied in vitro. However, there are few reported researches about the interactional effects of growth factors on chondrogenic differentiation of stem cells. The aim of this study is to examine the combined effects of four key growth factors on chondrogenic differentiation of mesenchymal stem cells (MSCs). Isolated and expanded rabbit bone marrow-derived MSCs underwent chondrogenic differentiation in a micromass cell culture system that used a combination of the following growth factors: transforming growth factor beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP2), parathyroid hormone related protein (PTHrP), and fibroblast growth factor 2 (FGF2) according to a defined program. The chondrogenic differentiation program was analyzed by histochemistry methods, quantitative RT-PCR (qRT-PCR), and measurement of matrix deposition of sulfated glycosaminoglycan (sGAG) and collagen content at days 16, 23, and 30. The results showed that the short-term combination of TGF-β1 and BMP-2 increased sGAG and collagen content, Alkaline phosphates (ALP) activity, and type X collagen (COL X) expression. Application of either PTHrP or FGF2 simultaneously decreased TGF-β1/BMP-2 induced hypertrophy and chondrogenic markers (at least for FGF2). However, successive application of PTHrP and FGF2 dramatically maintained the synergistic effects of TGF-β1/BMP-2 on the chondrogenic differentiation potential of MSCs and decreased unwanted hypertrophic markers. This new method can be used effectively in chondrogenic differentiation programs.

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Acknowledgments

The authors would like to thanks Dr. Mohammadreza Gharaati for his kindly technical advisement. This study was supported by grants from Semnan University of medical science (grant No. H-4155-2014) and Royan institute (grant No. 91000211). All the authors declare that there is no conflict of interest regarding the publication of this article.

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Authors and Affiliations

  1. Medical Biotechnology Department, Semnan University of Medical Sciences, Semnan, Iran

    Davood Nasrabadi & Aliakbar Shabani

  2. Department of Stem Cell and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box: 19395-4644, Tehran, Iran

    Davood Nasrabadi & Mohamadreza Baghaban Eslaminejad

  3. Student Research Committee, Semnan University of Medical Sciences, Semnan, Iran

    Davood Nasrabadi

  4. Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

    Siamak Rezaeiani

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  1. Davood Nasrabadi
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Correspondence to Mohamadreza Baghaban Eslaminejad or Aliakbar Shabani.

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Nasrabadi, D., Rezaeiani, S., Eslaminejad, M.B. et al. Improved Protocol for Chondrogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells -Effect of PTHrP and FGF-2 on TGFβ1/BMP2-Induced Chondrocytes Hypertrophy. Stem Cell Rev and Rep 14, 755–766 (2018). https://doi.org/10.1007/s12015-018-9816-y

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