Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system

Authors

  • Vedrana Mužić Radović Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Hospital for Medical Rehabilitation of the Heart and Lung Diseases and Rheumatism-Thalassotherapia Opatija, Opatija, Croatia https://orcid.org/0000-0003-2087-1674
  • Paula Bunoza Department of Medical Biology, School of Medicine, Zagreb, Croatia https://orcid.org/0000-0002-4183-8083
  • Tihana Marić Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Medical Biology, School of Medicine, Zagreb, Croatia https://orcid.org/0000-0002-6238-9811
  • Marta Himelreich-Perić Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia https://orcid.org/0000-0002-2143-6968
  • Floriana Bulić-Jakuš Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Medical Biology, School of Medicine, Zagreb, Croatia https://orcid.org/0000-0002-5538-4692
  • Marta Takahashi Department of Communicology, Catholic University of Croatia, Croatia; Department of Histology and Embryology, School of Medicine, Zagreb, Croatia https://orcid.org/0000-0002-5645-5421
  • Gordana Jurić-Lekić Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Histology and Embryology, School of Medicine, Zagreb, Croatia https://orcid.org/0000-0001-5876-647X
  • Nino Sinčić Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Medical Biology, School of Medicine, Zagreb, Croatia
  • Davor Ježek Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Histology and Embryology, School of Medicine, Zagreb, Croatia https://orcid.org/0000-0002-1528-5462
  • Ana Katušić-Bojanac Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Medical Biology, School of Medicine, Zagreb, Croatia https://orcid.org/0000-0002-9078-4966

DOI:

https://doi.org/10.17305/bjbms.2021.6584

Keywords:

Chondrogenesis, global DNA methylation, limb bud, development, rat, organ-culture, ex vivo, in vitro, serum-free, embryo

Abstract

Although DNA methylation epigenetically regulates development, data on global DNA methylation during development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum-supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at 13th and 14th gestation days (GD) were cultivated at the air-liquid interface in Eagle's Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (SINE rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by Safranin O. Upon isolation, weak Safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures towards day 14, except in the SF-cultivated GD13 hind-LBs, where Safranin O staining was almost absent on day 3. That was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day three. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th day was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day three and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive towards differentiation in vitro, which is of importance for regenerative medicine strategies.

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Author Biographies

Vedrana Mužić Radović, Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Hospital for Medical Rehabilitation of the Heart and Lung Diseases and Rheumatism-Thalassotherapia Opatija, Opatija, Croatia

Hospital for Medical Rehabilitation of the Heart and Lung Diseases and Rheumatism-Thalassotherapia Opatija, Opatija, Croatia; MD Specialist in Physiotherapy

Paula Bunoza, Department of Medical Biology, School of Medicine, Zagreb, Croatia

Student

Tihana Marić, Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Medical Biology, School of Medicine, Zagreb, Croatia

Department of Medical Biology, School of Medicine, Zagreb, Croatia, PhD student

Marta Himelreich-Perić, Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia

Department of Medical Biology, School of Medicine, Zagreb, Croatia, PhD student

Floriana Bulić-Jakuš, Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Medical Biology, School of Medicine, Zagreb, Croatia

Department of Medical Biology, School of Medicine, Zagreb, Croatia, MD, PhD Tenured Professor

Marta Takahashi, Department of Communicology, Catholic University of Croatia, Croatia; Department of Histology and Embryology, School of Medicine, Zagreb, Croatia

Department of Histology and Embryology, School of Medicine, Zagreb, Croatia, M.Ed.

Gordana Jurić-Lekić, Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Histology and Embryology, School of Medicine, Zagreb, Croatia

Department of Histology and Embryology, School of Medicine, Zagreb, Croatia, MD, PhD, Tenured Professor

Nino Sinčić, Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Medical Biology, School of Medicine, Zagreb, Croatia

Department of Medical Biology, School of Medicine, Zagreb, Croatia, MD, PhD, Associate Professor

Davor Ježek, Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Histology and Embryology, School of Medicine, Zagreb, Croatia

Department of Histology and Embryology, School of Medicine, Zagreb, Croatia, MD, PhD, Tenured Professor

Ana Katušić-Bojanac, Center of Excellence in Reproductive and Regenerative Medicine, School of Medicine, Zagreb, Croatia; Department of Medical Biology, School of Medicine, Zagreb, Croatia

Department of Medical Biology, School of Medicine, Zagreb, Croatia, PhD, Associate Professor

Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system

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Published

2022-02-21

How to Cite

1.
Mužić Radović V, Bunoza P, Marić T, Himelreich-Perić M, Bulić-Jakuš F, Takahashi M, Jurić-Lekić G, Sinčić N, Ježek D, Katušić-Bojanac A. Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system. Bosn J of Basic Med Sci [Internet]. 2022Feb.21 [cited 2022Jul.5];. Available from: https://bjbms.org/ojs/index.php/bjbms/article/view/6584

Issue

Section

Molecular Biology