Senescence State in Mesenchymal Stem Cells at Low Passages: Implications in Clinical Use

Abstract

Mesenchymal stem cells (MSCs) are multipotent cells found in various tissues and are easily cultivated. For use in clinical protocols, MSCs must be expanded to obtain an adequate number of cells, but a senescence state may be instituted after some passages, reducing their replicative potential. In this study, we report a case where MSC derived from an elderly donor acquired a senescence state after three passages. The bone marrow was aspirated from a female patient submitted to a cell therapy for the incontinency urinary protocol; MSCs were cultivated with DMEM low glucose, supplemented with 10% autologous serum (AS) plus 1% L-glutamine and 1% antibiotic/antimycotic. Senescence analysis was performed by β-galactosidase staining after 24 and 48 h. Controls were established using BM-MSC from healthy donors and used for senescence and gene expression assays. Gene expression was performed using RT-PCR for pluripotency genes, such as SOX2, POU5F1, NANOG, and KLF4. MSC telomere length was measured by the Southern blotting technique, and MSCs were also analyzed for their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. The patient’s MSC expansion using AS displayed an early senescence state. In order to understand the role of AS in senescence, MSCs were then submitted to two different culture conditions: 1) with AS or 2) with FBS supplementation. Senescence state was assessed after 24 h, and no statistical differences were observed between the two conditions. However, patients’ cells cultured with AS displayed a higher number of senescence cells than FBS medium after 48 h (p = 0.0018). Gene expression was performed in both conditions; increased expression of KLF4 was observed in the patient’s cells in comparison to healthy controls (p = 0.0016); reduced gene expression was observed for NANOG (p = 0.0016) and SOX2 (p = 0.0014) genes. Telomere length of the patient’s cells was shorter than that of a healthy donor and that of a patient of similar age. Osteocyte differentiation seemed to be more diffuse than that of the healthy donor and that of the patient of similar age. MSCs could enter a senescence state during expansion in early passages and can impact MSC quality for clinical applications, reducing their efficacy when administered.

Suicide gene‐enabled cell therapy: A novel approach to scalable human pluripotent stem cell quality control

“…Therefore, to provide clinically relevant numbers of MSCs for both trials and application this hurdle will need to be overcome. [27,28] In addition to issues surrounding expansion of MSCs, the heterogeneity of these cell populations can be significant and impacted by; donor, tissue source, age, health condition, culture condition and other factors. [9] Therefore, many pre-clinical and clinical studies have suggested that allogenic therapies may be more practical as an off the self-solution, which would also decrease the cost per therapy.…”

“…Therefore, to provide clinically relevant numbers of MSCs for both trials and application this hurdle will need to be overcome. [ 27,28 ]…”

Improved senescent cell segmentation on bright‐field microscopy images exploiting representation level contrastive learning

“…MSCs gradually lose their potential therapeutic value with aging and cellular senescence. Cellular senescence is a complex state in which irreversible cell cycle arrest will limit their ability to proliferate, as well as their ability to regenerate and homing capacity 3,4 . In addition, the senescence of the MSCs changes their secretions profile.…”

“…Cellular senescence is a complex state in which irreversible cell cycle arrest will limit their ability to proliferate, as well as their ability to regenerate and homing capacity. 3,4 In addition, the senescence of the MSCs changes their secretions profile. Senescenceassociated secretory phenotype (SASP) is a multicomponent phenotype released in large amounts by senescent MSCs that comprises pro-inflammatory chemicals, immune-modulators, and growth factors.…”

Vibrational phenomics decoding of the stem cell stepwise aging process at single-cell resolution

“…6 Due to the relevant cell numbers required for clinical therapies, MSCs typically need to be expanded in vitro for long passages. 7 Similar to cell division that occurs in vivo , this expansion of MSCs results in many senescent phenotypes. 8 Many studies based on single cell data have reported a change in transcriptional heterogeneity with age.…”