Assessment of Long-Term in vitro Multiplied Human Wharton’s Jelly-Derived Mesenchymal Stem Cells prior to Their Use in Clinical Administration

Abstract

The quantity of mesenchymal stem/stromal cells (MSCs) required for a particular therapy demands their subsequent expansion through ex vivo culture. During in vitro multiplication, they undergo replicative senescence which may alter their genetic stability. Therefore, this study was aimed to analyze cellular, molecular, and chromosomal alterations in Wharton’s jelly-derived MSCs (WJ-MSCs) during their in vitro sequential passages, where WJ-MSCs were sequentially passaged up to P14 and cells were evaluated at an interval of P2, P6, P10, and P14. They were examined for their morphology, tumorigenicity, surface markers, stemness markers, DNA damage, chromosomal aberration, and telomere length. We have processed five full-term delivered human umbilical cord samples to obtain WJ-MSCs. Morphological appearance observed at initial stages was small fine spindle-shaped WJ-MSCs which were transformed to flat, long, and broader cells in later passages. The cell proliferation rate was gradually decreased after the 10th passage. WJ-MSCs have expressed stemness markers OCT-4 and NANOG, while they showed high expression of positive surface markers CD90 and CD105 and lower expression of CD34 and CD45. They were non-tumorigenic with slow cellular aging during subsequent passages. There was no chromosomal abnormality up to the 14th passage, while increase in comet score and decrease in telomere length were observed in later passages. Hence, our study suggests that early and middle passaged (less than P10) WJ-MSCs are good candidates for clinical administration for treatment.

Wharton’s jelly mesenchymal stem cells: a concise review of their secretome and prospective clinical applications

“…However, the expression of senescence-related gene, p21, and oncogene, c-Myc, was significantly upregulated at late passages (>20 cell passages). Furthermore, at low (<10) cell passages, WJ-MSCs adopt small fine-spindle shape which then transforms into flat, long, and broader cell morphology at later passages associated with low proliferation rate ( Panwar et al, 2021 ). The late passage cells are non-tumorigenic, show slow cellular aging and do not exhibit chromosomal abnormalities.…”

“…The late passage cells are non-tumorigenic, show slow cellular aging and do not exhibit chromosomal abnormalities. However, further passages demonstrate shorter telomere length ( Panwar et al, 2021 ). Due to their embryonic nature, WJ-MSCs have low senescence rate relative to adult MSCs.…”

Aging of mesenchymal stem cell: machinery, markers, and strategies of fighting

“…Meanwhile, in vitro expansion of Wharton's jelly MSCs led to chromosomal changes, which may affect their clinical usage. Thus, quality-control measures should be applied before transplantation [57]. On the other hand, adipose-tissue-derived MSCs expressed low percentages of aneuploidy cells in early passages 0-4, whereas prolonged culture expansion for 5-16 passages was characterized by significantly high aneuploidy percentages without malignant transformations [58].…”