Table 4.

Proposed experiments design for evaluation the potential therapeutic mechanism of UC-MSCs in the treatment of COPD

Proposed experiments	Criteria	Cell type/method	Expected outcomes
UC-MSC characterisation	MSC marker analysis	UC-MSCs/flow cytometry	Meet ISCT guideline
	Differentiation potential	UC-MSCs/In vitro differentiation using commercial kits.	Adipogenic, chondrogenic, and osteogenic differentiation
	Karyotype	UC-MSCs/G-banding method	Normal post-expansion
	Growth factor, cytokines secretion	UC-MSCs/ProcartaPlex Immunoassays	Detection of cytokines and growth factors involves in anti-inflammatory and tissue regeneration process.
Metabolic evaluation	Mitochondrial activities	UC-MSCs/agilent seahorse XF cell mito stress test	Measurement of mitochondrial activities of UC-MSCs pre-administration
	Glycolysis	UC-MSCs/agilent seahorse XF glycolysis stress test	Measurement of glycolysis process of UC-MSC pre-administration
Immunoregulatory assessment	Lymphocyte Proliferation Assay	UC-MSCs+peripheral mononuclear cells from healthy donors	UC-MSCs inhibit the proliferation rate of lymphocytes in the present of PHA.
		UC-MSCs+peripheral mononuclear cells from patients with COPD	UC-MSCs inhibit the proliferation rate of lymphocytes in the present of PHA in a similar manner to healthy donor counterpart
Growth factors and cytokines analysis	UC-MSC secretion profiles under xeno-free and serum-free culture conditions	UC-MSCs/procartaPlex immunoassays	Detection of cytokines and growth factors involves in anti-inflammatory and tissue regeneration process.
	Cytokine profiles from patients with COPD plasma	Patients with COPD plasma/procartaPlex immunoassays	Evaluation of cytokines involves in inflammatory response obtain from patients with COPD plasma before and after UC-MSC administration.
	Interaction between UC-MSCs and patients with COPD’ lymphocytes	Media obtained from co-culture of UC-MSCs and peripheral mononuclear cells from patients with COPD/ProcartaPlex Immunoassays	Detection of cytokines involves in the anti-inflammatory functions of UC-MSCs.

COPD, chronic obstructive pulmonary disease; GOLD, Global Initiative for Chronic Obstructive Lung Disease; ISCT, International Society for Cellular Therapy.