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. 2021 Feb 7;8(2):24.
doi: 10.3390/vetsci8020024.

Isolation and Characterization of Feline Wharton's Jelly-Derived Mesenchymal Stem Cells

Min-Soo Seo  1 , Kyung-Ku Kang  1 , Se-Kyung Oh  1 , Soo-Eun Sung  1 , Kil-Soo Kim  1   2 , Young-Sam Kwon  3 , Sungho Yun  3
Affiliations

Isolation and Characterization of Feline Wharton's Jelly-Derived Mesenchymal Stem Cells

Min-Soo Seo et al. Vet Sci. .

Abstract

Wharton's jelly is a well-known mesenchymal stem cell source in many species, including humans. However, there have been no reports confirming the presence of mesenchymal stem cells in Wharton's jelly in cats. The purpose of this study was to isolate mesenchymal stem cells (MSCs) from the Wharton's jelly of cats and to characterize stem cells. In this study, feline Wharton's jelly-derived mesenchymal stem cells (fWJ-MSCs) were isolated and successfully cultured. fWJ-MSCs were maintained and the proliferative potential was measured by cumulative population doubling level (CPDL) test, scratch test, and colony forming unit (CFU) test. Stem cell marker, karyotyping and immunophenotyping analysis by flow cytometry showed that fWJ-MSCs possessed characteristic mesenchymal stem cell markers. To confirm the differentiation potential, we performed osteogenic, adipogenic and chondrogenic induction under each differentiation condition. fWJ-MSCs has the ability to differentiate into multiple lineages, including osteogenic, adipogenic and chondrogenic differentiation. This study shows that Wharton's jelly of cat can be a good source of mesenchymal stem cells. In addition, fWJ-MSCs may be useful for stem cell-based therapeutic applications in feline medicine.

Keywords: Wharton’s jelly; characterization; feline; mesenchymal stem cell.

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Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of the article.

Figures

Figure 1
Figure 1
Primary culture of fWJ-MSCs and identification of cumulative population doubling level (CPDL). (A) Extracted feline umbilical cord tissue. (B) Image of isolated fWJ-MSCs. Cells exhibited a spindle shape, similar to other mesenchymal stem cells. Scale bar: 100 μm. (C) Cumulative growth curve of fWJ-MSCs. CPDL was measured from passage 5 to 16. (D) Scratch test. Figure shows representative images of fWJ-MSCs migration post-scratch assay obtained after 24 h. Scale bar: 10 μm. (E) Colony forming unit (CFU) test. At 2 weeks of culture, the CFUs were stained with toluidine blue to visualize the colonies generated. (F) RT-PCR. OCT4, SOX2, KLF4, MYC expression determined stemness.
Figure 2
Figure 2
Karyotyping and flow cytometry analysis. (A) Karyotype of fWJ-MSCs at passage 5 showing a euploid number of chromosomes. (B) FACS analysis was performed at passage 5. Values show the intensity of the indicated antigen.
Figure 3
Figure 3
Osteogenic differentiation. (A–J) Osteogenic differentiation of fWJ-MSCs. (A,F) Bright field images of cells in osteogenic condition and control condition. (B–E,G–J) Alizarin Red S and Von Kossa staining after 3 weeks of osteogenic induction and control condition. Osteogenic differentiated cells (A–E) were grown in osteogenic induction medium. Negative control cells (F–J) were grown in low glucose DMEM with 10% FBS. Differentiated cells stained strongly with Alizarin Red S (B,C) and Von Kossa (D,E). Scale bar: 25 μm. (K) For quantification, Alizarin Red S-stained cells were solubilized with 100 mM cetylpyridinium chloride, and the absorbance was measured by spectrophotometer at 570 nm for 0.5 s. Compared with the negative control, differentiated cells showed 67-fold greater absorbance values. (L,M) qRT-PCR for detection of mRNA expression level of osteogenic-specific markers: MSX2, COL1A1, and GAPDH were used as references for evaluating the quality of mRNA. (Control, undifferentiated fWJ-MSC). Means ± standard deviations are plotted (*** p < 0.001), ** p < 0.01).
Figure 4
Figure 4
Adipogenic differentiation. (A–F) Adipogenic differentiation of fWJ-MSCs. Oil Red O staining was conducted after 3 weeks of adipogenic induction. (A–C) Adipogenic differentiated cells were grown in adipogenic induction media. Fatty droplets were strongly stained with Oil Red O (arrow: fatty droplet). (D–F) Negative controls showed no staining with Oil Red O. Scale bar: 10 μm. (G) For quantification, stained cells were solubilized with 100% isopropanol, and the absorbance was measured spectrophotometrically at 500 nm for 0.5 s. Compared with the negative control, differentiated cells showed 3-fold greater absorbance values. (H–J) qRT-PCR for detection of mRNA expression level of adipogenic-specific markers: LPL, LEPTIN and FABP4. GAPDH was used as the reference for evaluating the quality of mRNA. (Control, undifferentiated fWJ-MSC). Means ± standard deviations are plotted (*** p < 0.001), ** p < 0.01).
Figure 5
Figure 5
Chondrogenic differentiation. (A–F) Chondrogenic differentiation of fWJ-MSCs. Toluidine blue scheme after 3 weeks of chondrogenic induction. Scale bar: 50 μm. (G) qRT-PCR for detection of mRNA expression level of chondrogenic-specific markers: COL2A1. GAPDH was used as a reference for evaluating the quality of mRNA. (H) The shape of chondrogenic pellet. (I) Toluidine blue staining for chondrogenic pellet. Scale bar: 100 µm. (J) qRT-PCR for detection of mRNA expression level of osteogenic-specific markers: COL2A1. GAPDH was used as a reference for evaluating the quality of mRNA. (Control, undifferentiated fWJ-MSC). Means ± standard deviations are plotted (*** p < 0.001), * p < 0.05).
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References

    1. Diaz-Prado S., Muinos-Lopez E., Hermida-Gomez T., Rendal-Vazquez M.E., Fuentes-Boquete I., de Toro F.J., Blanco F.J. Isolation and characterization of mesenchymal stem cells from human amniotic membrane. Tissue Eng. Part C Methods. 2011;17:49–59. doi: 10.1089/ten.tec.2010.0136. - DOI - PubMed
    1. La Rocca G., Anzalone R., Corrao S., Magno F., Loria T., Lo Iacono M., Di Stefano A., Giannuzzi P., Marasà L., Cappello F., et al. Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: Differentiation potential and detection of new markers. Histochem. Cell Biol. 2009;131:267–282. doi: 10.1007/s00418-008-0519-3. - DOI - PubMed
    1. Ullah I., Subbarao R.B., Rho G.J. Human mesenchymal stem cells—Current trends and future prospective. Biosci. Rep. 2015;35:e00191. doi: 10.1042/BSR20150025. - DOI - PMC - PubMed
    1. Huang H.I., Chen S.K., Ling Q.D., Chien C.C., Liu H.T., Chan S.H. Multilineage differentiation potential of fibroblast-like stromal cells derived from human skin. Tissue Eng. Part A. 2010;16:1491–1501. doi: 10.1089/ten.tea.2009.0431. - DOI - PubMed
    1. Kang S.G., Shinojima N., Hossain A., Gumin J., Yong R.L., Colman H., Marini F., Andreeff M., Lang F.F. Isolation and perivascular localization of mesenchymal stem cells from mouse brain. Neurosurgery. 2010;67:711–720. doi: 10.1227/01.NEU.0000377859.06219.78. - DOI - PMC - PubMed

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