Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy

Abstract

Ex vivo expansion and manipulation of human mesenchymal stem cells are important approaches to immunoregulatory and regenerative cell therapies. Although these cells show great potential for use, issues relating to their overall nature emerge as problems in the field. The need for extensive cell quantity amplification in vitro to obtain sufficient cell numbers for use, poses a risk of accumulating genetic and epigenetic abnormalities that could lead to sporadic malignant cell transformation. In this study, we have examined human mesenchymal stem cells derived from bone marrow, over extended culture time, using cytogenetic analyses, mixed lymphocyte reactions, proteomics and gene expression assays to determine whether the cultures would retain their potential for use in subsequent passages. Results indicate that in vitro cultures of these cells demonstrated chromosome variability after passage 4, but their immunomodulatory functions and differentiation capacity were maintained. At the molecular level, changes were observed from passage 5 on, indicating initiation of differentiation. Together, these results lead to the hypothesis that human mesenchymal stem cells cultures can be used successfully in cell therapy up to passage 4. However, use of cells from higher passages would have to be analysed case by case.

Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation

“…Based on traditional karyotype analysis, some studies found that abnormal karyotype appeared in the culture of ADMSCs and BMMSCs. 10,11,37 However, whether MSCs with genomic anomalies gained the ability of forming tumor in vivo was unclear, even though chromosomal aberrations were found in cultured ADMSCs and BMMSCs. High-throughput methods were also used to investigate the genomic mutation of cultured ADMSCs and BMMSCs, 12,14 whereas, these studies failed to find significant genomic changes and stated that MSCs expanded in vitro confirmed an overall stability of genomic profile.…”

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

“…Following isolation of the BM-MNCs using the Sepax method, a total of 10.82x10 9 BM-MNCs were collected from 8 donors. The absolute number of BM-MNCs per 1 mL of bone marrow after isolation was 4.1x10 6 ±7.8x10 5 .…”

“…The cells from each donor were re-suspended in cryomedium and frozen individually in bags. The total number of frozen BM-MNCs was 9.86x10 9 .…”

“…8 In contrast, several other reports demonstrated that the immunosuppressive effect of MSCs remains unchanged for up to 7 or 8 passages in culture. 9,10 Another important issue regarding the clinical application of MSCs is their culture under serum-free conditions. The majority of clinical studies have used MSCs that were expanded in media supplemented with fetal bovine serum (FBS).…”

Asymmetric Aneuploidy in Mesenchymal Stromal Cells Detected by In Situ Karyotyping and Fluorescence In Situ Hybridization: Suggestions for Reference Values for Stem Cells

“…In a study using nine MSCs derived from bone marrow, a conventional cytogenetic analysis revealed random aneuploidies. In particular, one MSC presented with clonal trisomy 16 in p6 and p8 [31].…”