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Home / Equine Research Bank / Reproduction / Isolation, characterization and differentiation of mesenchym...
Reproduction (Cambridge, England)2012; 143(4); 455-468; doi: 10.1530/REP-10-0408

Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton’s jelly in the horse.

Abstract: Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2  h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 days) (P<0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.
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Publication Date: 2012-01-24 PubMed ID: 22274885DOI: 10.1530/REP-10-0408Google Scholar: Lookup
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  • Journal Article
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  • Non-U.S. Gov't

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.

This study explores the extraction, identification, and characterization of mesenchymal stem cells from horse amniotic fluid, umbilical cord blood, and a substance in the umbilical cord called Wharton’s Jelly. The researchers found these stem cells to be readily accessible, capable of rapid growth, and potentially beneficial for understanding stem cell biology and treatment applications in horses.

Sample Collection and Preparation

  • The researchers collected samples from 13 mares at the time of labor. These samples included amniotic fluid (AF), cord blood (CB), and Wharton’s Jelly (WJ).
  • The extraction of AF and CB cells was done by centrifugation, a process that uses centrifugal force to separate cells from liquids.
  • WJ was prepared by incubating it in an enzymatic solution for 2 hours to aid in the extraction of cells.

Culturing and Characterization of Cells

  • All the cell lines obtained were cultured in a medium that included DMEM/TCM199 and fetal bovine serum to support their growth.
  • Fibroblast-like cells, which resemble the type of cells found in connective tissue, were observed in the majority of AF, CB and WJ samples.
  • There were statistically significant differences found between the doubling times (the time needed for a quantity to double, often used to measure cell growth) of the cells from different sources.
  • Cells from Wharton’s Jelly expanded the fastest, followed by the cells from amniotic fluid and cord blood.

Positive Markers for Stem Cell Identification

  • Several tests were performed to confirm the capability of these cell lines to differentiate into multiple types of cells – a key trait of mesenchymal stem cells (MSCs).
  • Staining with von Kossa and Alizarin Red S, which detect the presence of calcium deposits indicative of bone formation, showed positive results, suggesting that the cells could differentiate into bone cells (osteogenesis).
  • Alcian Blue staining, which highlights the presence of glycosaminoglycans, a major component of the matrix of cartilage, indicated that the cells could leading to cartilage cells (chondrogenesis).
  • Oil Red O staining was used to detect lipid droplets, suggesting that these cells could differentiate into fat cells (adipogenesis).
  • The cell lines were also positive for CD90, CD44, CD105 – markers indicative of mesenchymal stem cells and negative for CD34, CD14, CD45 – markers related to other cell types such as hematopoietic stem cells and immune cells.

Conclusion and Further Research

  • The researchers concluded that equine MSCs from AF, UCB, and WJ can be easily obtained and proliferate rapidly, making them a potential model for studying stem cell biology and for use in reparative therapy in horses.
  • However, more research is needed to evaluate the expression of markers related to cell immunogenicity to assess the potential use of these cells as an allogeneic (from a donor) cell source.

Cite This Article

APA
Iacono E, Brunori L, Pirrone A, Pagliaro PP, Ricci F, Tazzari PL, Merlo B. (2012). Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton’s jelly in the horse. Reproduction, 143(4), 455-468. https://doi.org/10.1530/REP-10-0408

Publication

Reproduction (Cambridge, England)
ISSN: 1741-7899
NlmUniqueID: 100966036
Country: England
Language: English
Volume: 143
Issue: 4
Pages: 455-468

Researcher Affiliations

Iacono, Eleonora
  • Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy. eleonora.iacono2@unibo.it
Brunori, Lara
    Pirrone, Alessandro
      Pagliaro, Pasquale Paolo
        Ricci, Francesca
          Tazzari, Pier Luigi
            Merlo, Barbara

              MeSH Terms

              • Amniotic Fluid / cytology
              • Animals
              • Cell Culture Techniques
              • Cell Differentiation
              • Cells, Cultured
              • Female
              • Fetal Blood / cytology
              • Flow Cytometry
              • Horses
              • Immunophenotyping
              • Mesenchymal Stem Cells / cytology
              • Pregnancy
              • Wharton Jelly / cytology

              Citations

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