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Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells

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Abstract

Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.

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Acknowledgment

We wish to thank Dr Ricardo Pimenta Bertolla, PhD for his assistance in statistical analysis.

Conflict of interest

None declared.

Financial support

FAPESP—Fundação de Amparo à Pesquisa do estado de São Paulo—grant number 06/57479-2.

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Authors and Affiliations

  1. Department of Urology, Paulista School of Medicine, São Paulo Federal University, São Paulo, SP, Brazil

    Letícia Siqueira de Sá Barretto, Camila Lessio, Ahy Natally Sawaki e Nakamura, Edson Guimarães Lo Turco, Camila Gonzaga da Silva, João Paulo Zambon & Fernando Gonçalves de Almeida

  2. Dalton Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas, Campinas, SP, Brazil

    Fábio César Gozzo & Eduardo Jorge Pilau

  3. Rua Barata Ribeiro, 414, Bela Vista, São Paulo, SP, 01308-000, Brazil

    Fernando Gonçalves de Almeida

Authors
  1. Letícia Siqueira de Sá Barretto
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  2. Camila Lessio
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  3. Ahy Natally Sawaki e Nakamura
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  4. Edson Guimarães Lo Turco
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  5. Camila Gonzaga da Silva
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  6. João Paulo Zambon
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  8. Eduardo Jorge Pilau
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  9. Fernando Gonçalves de Almeida
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Correspondence to Fernando Gonçalves de Almeida.

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Editor: T. Okamoto

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Sá Barretto, L.S., Lessio, C., Nakamura, A.N.S. et al. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells. In Vitro Cell.Dev.Biol.-Animal 50, 831–839 (2014). https://doi.org/10.1007/s11626-014-9782-x

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  • DOI: https://doi.org/10.1007/s11626-014-9782-x

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