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Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions

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Abstract

Summary

Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs.

Introduction

Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation.

Methods

cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, β-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies.

Results

In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition.

Conclusions

The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts.

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Acknowledgements

This study was supported by a grant from the German Federal Ministry of Education and Research (no. 0315121A) to NzN. We are grateful to Dr. Gesine Fleischmann for help with setting up marmoset ESC culture and Dr. Ludovic Vallier for assistance with feeder-free culture.

Conflicts of interest

None.

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Authors and Affiliations

  1. Group Applied Stem Cell Technology, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany

    S. Trettner, A. Findeisen, S. Taube & N. I. zur Nieden

  2. Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany

    P. A. Horn

  3. Central Institute for Experimental Animals, 1430 Nogawa, Miyamae-ku, Kawasaki, Kanagawa, 216-0001, Japan

    E. Sasaki

  4. Department of Cell Biology and Neuroscience and Stem Cell Center, College of Natural and Agricultural Sciences, University of California Riverside, 1113 Biological Sciences Building, Riverside, CA, 92521, USA

    N. I. zur Nieden

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  1. S. Trettner
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Correspondence to N. I. zur Nieden.

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Trettner, S., Findeisen, A., Taube, S. et al. Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions. Osteoporos Int 25, 1255–1266 (2014). https://doi.org/10.1007/s00198-013-2566-4

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  • DOI: https://doi.org/10.1007/s00198-013-2566-4

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