A Robust and Highly Efficient Approach for Isolation of Mesenchymal Stem Cells From Wharton's Jelly for Tissue Repair

Abstract

Mesenchymal stem cells derived from umbilical cord Wharton's Jelly (WJ-MSCs) are emerging as promising therapeutics for a variety of diseases due to their ability of regeneration and immunomodulation, and their non-tumorigenic and non-immunogenic properties. Although multiple protocols have been developed for WJ-MSC isolation, insufficient cell numbers, heterogeneous cell population, and variations in procedures between different laboratories impede further clinical applications. Here, we compared six widely used WJ-MSC isolation methods regarding cell morphology, yield, purity, proliferation rate, and differentiation potential. Based on these analyses, we identified that the inefficiency of the extracellular matrix digestion results in low cell yield. Thus, we developed a new method called "Mince-Soak-Digest (MSD)" to isolate MSCs from WJ by incorporating a soaking step to facilitate the digestion of the extracellular matrix and release of the cells. Our newly developed method generates significantly higher cell yield (4- to 10-fold higher) than six widely used methods that we tested with high purity and consistency. Importantly, by transplantation of WJ-MSCs to the rat uterus, we repair the endometrial injury and restore the fertility of the rats. In conclusion, our results provide a robust and highly efficient approach for the isolation of WJ-MSCs to restore injured tissue. The higher efficiency of MSD assures the abundance of WJ-MSCs for clinical applications. Furthermore, the reliability of MSD contributes to the standardization of WJ-MSC isolation, which eliminates the discrepancies due to isolation procedures, thus facilitating the evaluation of the efficacy of WJ-MSCs across various human clinical applications.

Keywords: Wharton’s jelly; intrauterine adhesions; mesenchymal stem cells; tissue repair; transplantation.

Figure 1.

Comparison of the six widely used isolation methods. (A) The morphology of cells isolated from M1 to M6; scale bars represent 100 µm; (B) counts of cell number using M1 to M6; (C) growth curve of isolated mesenchymal stem cells from M1 and M6.

Figure 2.

Immunophenotype of isolated cells from M1 to M6. Isolated cells at passage 3 were used for flow cytometry analyses. Each histogram is a representative result of three biological repeats. Values represent the mean percentage of all assessed cells positively stained by the respective antibodies in the flow cytometry analyses.

Figure 3.

Development of the Mince-Soak-Digest (MSD) method for isolation of WJ-MSCs. (A) Hematoxylin and eosin staining of WJ tissue after explant isolation methods. Arrows indicate cells that are trapped in the tissue. Scale bar represents 1,000 µm; (B) mRNA expression of collagen genes in WJ. VIM (Vimentin) gene was used as a positive control. (C) Masson’s trichrome staining of collagen fibers in fresh and soaked WJ tissues. Scale bars on the left original tissue images represent 1,000 µm and on the right magnified images represent 100 µm. (D) BCA protein assays were used to quantify the total protein concentration of media used to soak WJ tissues. (E) Schematic diagram of the MSD method. WJ-MSCs: mesenchymal stem cells derived from umbilical cord Wharton’s Jelly; BCA: bicinchoninic acid.

Figure 4.

Comparison of the MSD method with the six widely used methods. (A) Counts of cell numbers of MSD and M1–M6. (B) Growth curve of cell number of M1, M6, and MSD. (C) Flow cytometry analysis on MSD-isolated cells. (D) Immunofluorescence staining of MSD-isolated cells after directed differentiation. Scale bars represent 100 µm. MSD: Mince-Soak-Digest.

Figure 5.

Endometrial regeneration by Wharton’s jelly-mesenchymal stem cells (Mince-Soak-Digest). (A) Hematoxylin and eosin staining of rat uteri under different treatments. Scale bars on the left original tissue images represent 1,000 µm and on the right magnified images represent 100 µm. Statistical analysis of endometrial thickness (B) and the number of endometrial glands (C) under different treatments.

Figure 6.

Fertility restoration by Wharton’s jelly-mesenchymal stem cells (Mince-Soak-Digest). (A) Embryo implantation images of rat uteri under different treatment. Black arrows indicate treatment received for each side of uteri horn. Statistical analysis of pregnancy rate (B) and the number of received well-developed embryos (C).