A Simple High Yield Technique for Isolation of Wharton's Jelly-derived Mesenchymal Stem Cell

Bahare Niknam; Arezou Azizsoltani; Neda Heidari; Samaneh Tokhanbigli; Helia Alavifard; Mahsa Haji Valili; Davar Amani; Hamid Asadzadeh Aghdaei; Seyed Mahmoud Hashemi; Kaveh Baghaei

doi:10.18502/ajmb.v16i2.14860

A Simple High Yield Technique for Isolation of Wharton's Jelly-derived Mesenchymal Stem Cell

Avicenna J Med Biotechnol. 2024 Apr-Jun;16(2):95-103. doi: 10.18502/ajmb.v16i2.14860.

Affiliations

¹ Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
² Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
³ Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
⁴ Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Abstract

Background: The isolation of Mesenchymal Stem Cells (MSCs) from various tissues is possible, with the umbilical cord emerging as a competitive alternative to bone marrow. In order to fulfill the demands of cell therapy, it is essential to generate stem cells on a clinical scale while minimizing time, cost, and contamination. Here is a simple and effective protocol for isolating MSC from Wharton's Jelly (WJ-MSC) using the explant method with various supplements.

Methods: Utilizing the explant method, small fragments of Wharton's jelly from the human umbilical cord were cultured in a flask. The multipotency of the isolated cells, were confirmed by their differentiation ability to osteocyte and adipocyte. Additionally, the immunophenotyping of WJ-MSCs showed positive expression of CD73, CD90, and CD105, while remaining negative for hematopoietic markers CD34 and CD45, meeting the criteria for WJ-MSC identification. Following that, to evaluate cells' proliferative capacity, various supplements, including basic Fibroblast Growth Factor (bFGF), Non-Essential amino acids (NEA), and L-Glutamine (L-Gln) were added to either alpha-Minimal Essential Medium (α-MEM) or Dulbecco's Modified Eagle's Medium-F12 (DMEM-F12), as the basic culture media.

Results: WJ-MSCs isolated by the explant method were removed from the tissue after seven days and transferred to the culture medium. These cells differentiated into adipocyte and osteocyte lineages, expressing CD73, CD90, and CD105 positively and CD34 and CD45 negatively. The results revealed that addition of bFGF to α-MEM or DMEMF12 media significantly increased the proliferation of MSCs when compared to the control group. However, there were no significant differences observed when NEA or LGln were added.

Conclusion: Although bFGF considerably enhances cell proliferation, our study demonstrates that MSCs can grow and expand when properly prepared Wharton's jelly tissues of the human umbilical cord.

Keywords: Fibroblast growth factor 2; Mesenchymal stem cells; Umbilical cord.