Hurdles to overcome for mesenchymal stem cell translation from bench to bedside

Table 2 Methods to detect genetic stability of in vitro expanded mesenchymal stem cells

Assay	What it detects	Strengths	Limitations	Suggested use/frequency	Ref.
Conventional karyotype (G-banding)	Large chromosomal abnormalities	Low cost; detects numerical and structural chromosomal abnormalities	Low resolution (approximately 5-10 Mb), not sufficient to predict full genetic stability, laborious	At master cell bank and before clinical release	[96-98]
FISH	Low mosaicism, minor structural abnormalities	Rapid, sensitive, easy data interpretation	The chromosomal aberration being searched for must be known beforehand	Not a screening technique, complementary to karyotyping for further proof of genotypic stability	[96-98]
M-FISH	Overall view of all chromosomes in a single assay	High resolution (approximately 1.5 Mb)	Highly expensive, difficult to analyze and interpret data	Not to be used to replace karyotyping	[96,99]
a-CGH	Copy number variations	Higher resolution than karyotype (≤ 50 kb), medium cost, no need for metaphases, requires only the genomic DNA	Cannot detect balanced translocations, inversion and intragenic rearrangement; need of experienced cytogenetic specialist	At defined passage thresholds and for new donor lines or when recurrent abnormalities are found	[96-98]
WGS	Single-nucleotide changes and copy number alterations	Highest resolution; comprehensive	Costly; complex analysis; uncertain clinical significance for many variants	As confirmatory test for master bank or if abnormalities suspected	[100]
Telomere length (qPCR)	Telomere attrition	Simple surrogate for replicative senescence	Not definitive for malignancy risk	Periodic, after a certain number of passages	[101]
DNA methylation profiling	Epigenetic drift; aging signatures	Useful for senescence, potency	Interpretation still evolving	For research and characterization; consider for potency correlation	[102-104]

FISH: Fluorescence in situ hybridization; M-FISH: Multiplex/multicolor fluorescence in situ hybridization; a-CGH: Array comparative genomic hybridization; WGS: Whole-genome sequencing; qPCR: Quantitative polymerase chain reaction.