Fig 1.
Spheroids formation from primary hMSCs.
(A) Schematic representation of experimental plan. (B) 30,000 primary hMSCs per well were seeded into U-bottomed 96-well in medium containing 0.25%, 0.5% or 1% of methylcellulose (MethocultTM H4100 or SF H4236). Microscopy analysis was performed after 24 h (scale bars = 500 μm).
Fig 2.
Follow up of the spheroids from various MSCs.
(A) Microscopy analysis of primary hMSC-, HS-27a-, HS-5- and MS-5-spheroids over 21 days in culture (scale bars = 100 μm). (B) Perimeter was measured with an arbitrary unit; each experiment is the mean of at least 10 spheroids from n = 3 experiments. Data are shown as mean ± SD; * compared to day 1; * p ≤ 0.01. (C) Number of living cells per spheroid over 21 days in culture (primary hMCSs and MS-5 n = 3; HS-27a and HS-5 n = 4). Data are shown as mean ± SD; *, **, *** compared to day 0; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Fig 3.
Scanning electron microscopy (SEM) observation of MSC-spheroids.
(A) Spheroids from primary hMSCs are round with a smooth surface and show progressive shrinking. HS-27a- and HS-5-spheroids are more irregular and granular. (B) Higher magnification show that cells are cohesive at the surface of primary hMSC-spheroids and more chaotic with distinguishable cells of different shapes for human cell lines. ECM deposition (arrow heads) is visible on all spheroids. Scale bars = 100 μm (A) and 20 μm (B).
Fig 4.
Determination of proliferation and apoptosis of MSC-spheroids.
(A, B and D) Cell cycle analysis of spheroids over 21 days in culture. (A) Representative gating strategy from primary hMSCs at day 0, (B) sub-G1 apoptosis quantification (primary hMSCs n = 6; HS-27a and HS-5 n = 3) and (D) cell cycle quantification (primary hMSCs n = 6; HS-27a and HS-5 n = 5; * for G0; ǂ for G1; # for S/G2/M) (data are mean ± SD; *, ǂ, # compared to day 0; *, ǂ p ≤ 0.05; **, ǂǂ, ## p ≤ 0.01). (C and E) Immunohistochemistry of (C) caspase-3 and (E) Ki-67 at days 1, 3 and 7 for primary hMSC-, HS-27a- and HS-5-spheroids (scale bars = 50 μm (C) and 100 μm (E)). Arrow heads indicate Ki-67-positive cells.
Fig 5.
Hypoxia detection of primary hMSC- and HS-27a-spheroids over 7 days in culture.
(A) Immunohistochemistry of CA-IX. (B) Immunohistochemistry and mRNA of HIF-1α. (C) Immunohistochemistry and mRNA expression of VEGF-A. (primary hMSCs n = 5; HS-27a n = 3; * p ≤ 0.05; ** p ≤ 0.01; scale bars = 100 μm).
Fig 6.
Oxidative stress detection in primary hMSC- and HS-27a-spheroids.
(A) Immunohistochemistry of HO-1 (scale bars = 100 μm). (B) Expression of antioxidant genes (n = 3; data are mean; * compared to 2D control (CTL); * p ≤ 0.05; ** p ≤ 0.01).
Fig 7.
Dedifferentiation detection of hMSC- and HS-27a-spheroids over 7 days in culture.
(A and B) Gene expression of OCT4, NANOG and SOX2 for (A) primary hMSC- and (B) HS-27a-spheroids (primary hMSCs n = 5; HS-27a n = 3; * p ≤ 0.05; ** p ≤ 0.01).