Table 7.

Assessment of differentiation potential.

Author	Results	Passage number	Method of assessment
Bacigalupo et al. (47)	ND	NR	Incubation with chondrogenic medium for 14 days; Immunohistochemical and alcian blue staining Incubation with osteogenic medium for 14 days; Alkaline phosphatase activity staining and calcium deposition Incubation with adipogenic medium for 21 days; Oil-Red O staining
Bueno et al. (37)	• No difference in differentiation potential	3-5	Incubation with osteogenic medium for 14 days; Alizarin red staining and RT-qPCR (osteocalcin, alkaline phosphatase, osterix) Incubation with adipogenic medium for 14 days; Oil-red O staining and RT-qPCR (PPAR, CEBPα)
Chao et al. (44)	• AA-MSC lower osteogenic potential  - less intense von Kossa stain and lower    ALP activity  - lower Cbfa1 expression • AA-MSC lower adipogenic potential  - lower Oil-Red O activity  - lower LPL expression	3	Incubation with osteogenic medium for 21 days; Alkaline phosphatase activity and von Kossa staining, quantification of alkaline phosphatase activity by spectrophotometry and RT-qPCR (Cbfa1) Incubation with adipogenic medium for 14 days; Oil-red O staining, quantification by spectrophotometry and RT-qPCR (LPL)
Chaturvedi et al. (31)	• AA-MSC lower osteogenic potential  - less alizarin red staining • AA-MSC higher adipogenic potential  - higher density and larger size of lipid    droplets/vesicles	3	Incubation with osteogenic medium for 21 days; Alizarin red staining Incubation with adipogenic medium for 18 days; Oil-red O staining
Cheng et al. (35)	• AA-MSC lower osteogenic potential  - less alizarin red staining  - ALPL expression decreased to 68%    in AA-patients • AA-MSC higher adipogenic potential  - higher density and larger size of lipid    droplets/vesicles  - Higher PPARγ expression	3	Incubation with osteogenic medium for 21 days; Alizarin red staining, quantification by spectrophotometry and RT-qPCR (BMP2, BMP4, BMP6, BMP7, ALPL) Incubation with adipogenic medium for 21 days; Oil-red O staining, quantification by spectrophotometry and RT-qPCR (PPARγ)
El-Mahgoub et al. (38)	• AA-MSC lower osteogenic potential  - less mineralization and von-Kossa    staining • AA-MSC lower adipogenic potential  - less lipid-containing cells and smaller size    of lipid droplets	3	Incubation with osteogenic medium for 21 days; von Kossa staining Incubation with adipogenic medium for 14 days; Oil-red O staining
Huo et al. (29)	• AA-MSC lower chondrogenic potential  - less alcian blue staining  - lower ACAN and SOX9 expression • AA-MSC lower osteogenic potential  - less alizarin red staining  - lower RUNX2 and BGLAP expression • AA-MSC higher adipogenic potential  - more lipid droplets  - higher ADIPOQ and PPARγ expression	3	Incubation with chondrogenic medium for 14 days; Alcian blue staining and RT-qPCR (ACAN, SOX9) Incubation with osteogenic medium for 14 days; Alizarin red S staining and RT-qPCR (RUNX2, BGLAP) Incubation with adipogenic medium for 14 days; Oil-Red O staining and RT-qPCR (PPARγ, ADIPOQ)
Jiang et al. (39)	ND	NR	Incubation with osteogenic medium; von Kossa staining and colony forming unit-osteoblast ability (CFU-O) Incubation with adipogenic medium; Oil red O staining and colony forming unit-adipocyte ability (CFU-Ad)
Li, H. et al. (26)	• AA-MSC lower osteogenic potential  - less alizarin red staining  - higher DNMT1 expression, lower MEG3 expression, lower BMP4 expression	3-6	Incubation with osteogenic medium for 21 days; Alizarin red staining and quantification with spectrophotometry, RT-qPCR/western blot analysis (DNMT1, MEG3, BMP4) and methylation of MEG3 promoter
Li, H. et al. (27)	• AA-MSC higher adipogenic potential  - more lipid droplets  - higher miR-146b-5p expression, lower   SIAH2 expression, higher PPARγ expression	3	Incubation with adipogenic medium for; Oil red O staining, RT-qPCR/western blot analysis (miR-146b-5p, SIAH2, PPARγ)
Li, J et al. (42)	• AA-MSC lower osteogenic potential  - less von Kossa, alizarin red and alkaline phosphatase activity staining • AA-MSC higher adipogenic potential  - more Oil red O staining	4	Incubation with osteogenic medium; von Kossa, alizarin red and alkaline phosphatase activity staining Incubation with adipogenic medium; Oil red O staining
Li, J et al. (43)	• AA-MSC lower osteogenic potential  - less von Kossa, alizarin red and    alkaline phosphatase    activity staining • AA-MSC higher adipogenic potential  - more Oil red O staining	4	Incubation with osteogenic medium; von Kossa, alizarin red and alkaline phosphatase activity staining Incubation with adipogenic medium; Oil red O staining
Li, S. et al. (28)	• AA-MSC lower osteogenic potential  - less alkaline phosphatase activity    staining • AA-MSC higher adipogenic potential  - more Oil red O staining	3	Incubation with osteogenic medium for 21 days; Alkaline phosphatase activity staining Incubation with adipogenic medium for 14 days; Oil red O staining
Michelozzi et al. (33)	• No difference in differentiation potential	3	Incubation with chondrogenic medium for 21 days; Histological analysis, RT-qPCR (COL2A1, COL10A1, SOX9, ACAN) Incubation with osteogenic medium for 21 days; Alizarin red S staining, RT-qPCR (SPP1, SPARC, ALPL, COL1A2) Incubation with adipogenic medium for 21 days; Oil red O staining, RT-qPCR (FABP4, LPL, PPARγ)
Sharma et al. (25)	• No difference in differentiation potential	3	Incubation with chondrogenic medium for 14 days; Alcian blue staining Incubation with osteogenic medium for 28 days; Alizarin Red S staining Incubation with adipogenic medium for 21 days; Oil red O staining
Shipounova et al. (45)	• AA-MSC lower osteogenic potential  - Among patient MSCs that failed  osteogenic differentiation, 80% had    SAA • AA-MSC lower adipogenic potential  - 1 patient without differentiation, 2    patients changed morphology of  MSCs    without lipid droplet formation, 3    patients with lipid droplet formation    without changed morphology	NR	Incubation with osteogenic medium; Alizarin red staining Incubation with adipogenic medium; Oil red O staining
Tripathy et al. (40)	• No difference in osteogenic potential • AA-MSC higher adipogenic potential  - higher density and larger size of lipid    droplets/vesicles  - higher expression of adiponectin    and FABP4	3	Incubation with osteogenic medium for 21 days; Alizarin Red S staining, RT-qPCR/western blot analysis (osteopontin) Incubation with adipogenic medium for 18 days; Oil red O staining, RT-qPCR/western blot analysis (adiponectin, FABP4)
Wei et al. (34)	• AA-MSC higher adipogenic potential  - higher density and larger size of lipid    droplets/vesicles at every time point    of differentiation  - higher FABP4, mTOR, PPARγ, S6K1    and p-S6K1 expression at every time    point of differentiation  - higher p-mTOR expression	3	Incubation with adipogenic medium for 7, 14 and 21 days; Oil red O staining, western blot analysis/RT-qPCR/immunofluorescence analysis (PPAR-γ, mTOR, p-mTOR, S6K1, p-S6K1, FABP4)
Xu et al. (46)	• No difference in osteogenic potential • AA-MSC higher adipogenic potential  - no difference in Oil red O staining  - lower GATA-2 expression  - higher PPARγ expression	3	Incubation with osteogenic medium for 21 days; Silver nitrate staining Incubation with adipogenic medium for 21 days; Oil red O staining, RT-qPCR/western blot analysis (GATA-2, PPARγ)
Zhao et al. (41)	• AA-MSC lower osteogenic potential  - fewer and smaller mineralized nodes  - lower Runx2 expression (P < 0.05) • AA-MSC higher adipogenic potential  - more and larger lipid droplets  - higher LPL, PPARγ and miR-204 expression	3	Incubation with osteogenic medium for 21 days, Alizarin red staining and quantification with spectrophotometry, RT-qPCR (Runx2) Incubation with adipogenic medium for 21 days, Oil red O staining and quantification with spectrophotometry, RT-qPCR/western blot analysis (LPL, PPARγ, miR-204)

ND, not determinable; NR, not reported.