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Home / Equine Research Bank / Animals (Basel) / Optimizing the Adipogenic Induction Protocol Using Rosiglita...
Animals : an open access journal from MDPI2023; 13(20); 3224; doi: 10.3390/ani13203224

Optimizing the Adipogenic Induction Protocol Using Rosiglitazone Improves the Physiological Parameters and Differentiation Capacity of Adipose Tissue-Derived Mesenchymal Stem Cells for Horses, Sheep, Dogs, Murines, and Humans.

Abstract: The investigation of adipose tissue-derived mesenchymal stem cells (ASCs) has received considerable interest in regenerative medicine. A nontoxic adipogenic induction protocol valid for cells of different mammalian species has not been described. This study aims to establish an adipogenic differentiation protocol suitable for horses, sheep, dogs, murines, and human cells. An optimized rosiglitazone protocol, consisting of 5% fetal calf serum in Dulbecco's Modified Eagle's Medium, 10 μg/mL insulin, 0.55 μg/mL transferrin, 6.8 ng sodium selenite, 1 μM dexamethasone, and 1-5 μM of rosiglitazone, is compared to the 3-isobutyl-1-methylxantine (IBMX) protocol, where rosiglitazone was replaced with 0.5 mM IBMX and 0.2 mM indomethacin. Cell viability, cytotoxicity, a morphometric analysis of the lipid, and the expression of adipogenic markers for 14 days were assessed. The data revealed that using 5 µM of rosiglitazone promotes the adipogenic differentiation capacity in horse, sheep, and dog cells compared to IBMX induction. Meanwhile, marked reductions in the cell viability and cell number with the IBMX protocol were detected, and rosiglitazone increased the cell number and lipid droplet size, prevented apoptosis, and upregulated and expression in the cells of most of the species. Our data revealed that the rosiglitazone protocol improves the adipogenesis of ASCs, together with having less toxicity, and should be considered for cell reproducibility and clinical applications targeting obesity.
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Publication Date: 2023-10-15 PubMed ID: 37893949PubMed Central: PMC10603751DOI: 10.3390/ani13203224Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study developed and tested an improved method using rosiglitazone to induce fat cell differentiation in mesenchymal stem cells derived from adipose tissue across multiple mammalian species.
  • The new protocol showed better cell viability, less toxicity, and enhanced fat differentiation compared to the traditional IBMX-based method.

Background and Purpose

  • Adipose tissue-derived mesenchymal stem cells (ASCs) are important in regenerative medicine due to their ability to differentiate into various cell types, including adipocytes (fat cells).
  • Inducing adipogenic differentiation reliably and safely is crucial for studying fat biology and developing therapies, especially those targeting metabolic diseases such as obesity.
  • Existing protocols commonly use IBMX and indomethacin for induction, but these can be toxic and vary in effectiveness across species.
  • The purpose of this study was to create a nontoxic, standardized adipogenic induction protocol using rosiglitazone, applicable to ASCs from horses, sheep, dogs, murines (mice/rats), and humans.

Methods

  • Two protocols were compared:
    • Rosiglitazone Protocol: Utilized Dulbecco’s Modified Eagle’s Medium with 5% fetal calf serum, supplemented with 10 μg/mL insulin, 0.55 μg/mL transferrin, 6.8 ng sodium selenite, 1 μM dexamethasone, and varying rosiglitazone concentrations (1-5 μM).
    • IBMX Protocol: The rosiglitazone was replaced with 0.5 mM IBMX and 0.2 mM indomethacin.
  • ASCs from the five species were cultured under these different conditions for 14 days.
  • Evaluations included:
    • Cell viability and cytotoxicity assays to assess cell health.
    • Morphometric analysis of lipid accumulation, measuring lipid droplet size and distribution.
    • Quantification of key adipogenic gene expression markers to assess differentiation at a molecular level.

Key Findings

  • Rosiglitazone at 5 μM was most effective in promoting adipogenic differentiation in cells from horses, sheep, and dogs, as demonstrated by:
    • Greater lipid droplet accumulation and larger lipid droplet size.
    • Upregulation of adipogenic gene markers, indicating active fat cell formation.
  • Compared to the IBMX protocol, rosiglitazone-treated cells showed:
    • Higher cell viability and increased cell number.
    • Reduced signs of apoptosis (programmed cell death) and cytotoxicity.
  • The IBMX and indomethacin protocol caused:
    • Significant reduction in cell viability and total cell number.
    • Potential toxicity across multiple species, limiting its clinical and research utility.
  • Rosiglitazone’s ability to improve differentiation and maintain cell health was relatively consistent across species tested, suggesting a broadly applicable protocol.

Implications and Conclusions

  • Replacing IBMX/indomethacin with rosiglitazone in adipogenic induction protocols offers significant advantages by enhancing differentiation capacity while reducing toxicity.
  • This optimized protocol can lead to more reproducible research outcomes involving ASCs across diverse mammalian species.
  • Clinically, using rosiglitazone for adipogenic induction could improve the development of cell-based therapies targeting obesity and related metabolic disorders.
  • Overall, the study provides a valuable standardized approach to adipogenic differentiation to advance both regenerative medicine research and therapeutic applications.

Cite This Article

APA
Heimann M, Elashry MI, Klymiuk MC, Eldaey A, Wenisch S, Arnhold S. (2023). Optimizing the Adipogenic Induction Protocol Using Rosiglitazone Improves the Physiological Parameters and Differentiation Capacity of Adipose Tissue-Derived Mesenchymal Stem Cells for Horses, Sheep, Dogs, Murines, and Humans. Animals (Basel), 13(20), 3224. https://doi.org/10.3390/ani13203224

Publication

Animals : an open access journal from MDPI
ISSN: 2076-2615
NlmUniqueID: 101635614
Country: Switzerland
Language: English
Volume: 13
Issue: 20
PII: 3224

Researcher Affiliations

Heimann, Manuela
  • Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
Elashry, Mohamed I
  • Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
Klymiuk, Michele C
  • Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
Eldaey, Asmaa
  • Clinic of Small Animals, c/o Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
Wenisch, Sabine
  • Clinic of Small Animals, c/o Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.
Arnhold, Stefan
  • Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany.

Grant Funding

  • AR333/11-1 / Deutsche Forschungsgemeinschaft

Conflict of Interest Statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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