Soluble immunosuppressive checkpointome of mesenchymal stromal cells: Immunoregulatory mechanisms and a conceptual framework for potency assessment

Table 4 Determinants shaping the composition of the mesenchymal stromal cell soluble checkpointome and the corresponding standardisation requirement

Determinant	Reported influence	Standardisation requirement
Tissue source	Bone marrow, adipose and birth-associated MSCs differ in immunomodulatory behaviour; direct comparative data remain limited[32]	Report source explicitly; do not pool analyte reference ranges across sources
Licensing regimen	IFN-γ is the canonical licensing signal[8]; IFN-γ and TNF-α act synergistically on PD-L1[9,21]; interleukin-1 beta further enhances ligand expression and in vivo benefit[22]; licensed cells acquire a distinct potency marker profile[36]	Fix cytokine identity, concentration and exposure duration; treat licensing as part of the assay, not as pre-treatment
Culture and manufacturing	Medium composition, supplement choice and passage number alter MSC function; standardisation is a recognised prerequisite for immunotherapeutic use[37]	Define passage window and medium; qualify reagents, since biological variability of assay reagents degrades reproducibility[38]
Cryopreservation and cell state	Freezing alters functional properties; apoptotic MSCs nevertheless retain efficacy[29,30]	Report the interval between thaw and sampling; viability alone is an inadequate surrogate for potency
MSC subpopulation heterogeneity	Distinct MSC subpopulations may differ in transcriptional state, secretory activity and immunomodulatory function	Characterize cellular composition using subpopulation-resolved/single-cell approaches and determine its relationship with checkpoint output

MSC: Mesenchymal stromal cell; IFN-γ: Interferon gamma; TNF-α: Tumor necrosis factor alpha; PD-L1: Programmed death ligand-1.