Alizarin Red Mineralization Assay.

<p>Only small, diffusely distributed mineral deposits are detected in phase contrast images of stained cultures that are more prominent in hMSCs in osteogenic differentiation than in propagation media. Quantitative analysis by absorption at 405 nm of eluted alizarin red stain reveals significantly more staining of NO cells than HO (denoted by asterisk). N = 6.</p

RT-PCR.

<p>Transcript levels of COL 1a1, COL 2a1, BSP and LPL were assessed for each imaging time point relative to GAPDH. Statistically significant differences in mRNA transcript levels are denoted by a single asterisk (in comparison to the propagation medium at the same oxygen tension) or by a double asterisk (significance in comparison to cells of the same medium condition, at 20% oxygen tension). N = 4.</p

Lysosomal localization of lipofuscin.

<p>Lipofuscin autofluorescence (green channel) is co-localized with (A) Lysotracker red (red channel) and not (B) Mitotracker Orange (red channel). Co-localization is indicated by the yellow color in panel A. Note that not all lysosomes contain lipofuscin. Bar  = 30 µm.</p

SHG evaluation of fibrous collagen.

<p>The gradual deposition of fibrous collagens is apparent in SHG images acquired on days 4, 8,12 and 16 from hMSCs in osteogenic medium at (A) 5% oxygen and (B) 20% oxygen. Quantitative evaluation of pixel density in SHG images reveals that collagen deposition evolves earlier for HO conditions than for NO cells. N = 4. (Bar = 50 µm).</p

Shorter, earlier depolarization times are sufficient to suppress AD differentiation.

<p>Cells were exposed to 80 mM K⁺ (AD-K) or 10 nM ouabain (AD-ouab) during Days 1–2 (A) or Days 1–4 (B), then washed and continued in culture in AD medium. Gene expression was evaluated on Day 7. Two days of exposure to 80 mM K⁺ or four days of exposure to 10 nM ouabain was sufficient to effect a change in AD marker expression. Data points are mean relative expression±standard deviation (N = 6). Marked samples are statistically different, * relative to PPARG expression of untreated AD samples (p<0.002), # relative to LPL expression of untreated AD samples (p<0.002). Undiff, hMSCs cultured in control medium; AD, hMSCs cultured in AD medium; AD-80K, hMSCs cultured in AD medium supplemented with 80 mM K⁺; AD-ouab, hMSCs cultured in AD medium supplemented with 10 nM ouabain.</p

Components of hMSC TPEF emission spectra.

<p>(A) hMSC TPEF emission spectra collected at either 755 nm or 860 nm excitation with corresponding fits by the ALS script. (B) linear unmixing methods reveal the contributing cellular fluorophores to be NAD(P)H, Lipofuscin and FP. When components of intrinsic cellular fluorescence are used to produce the fits displayed in panel A the NAD(P)H component does not contribute to cellular spectra excited at 860 nm.</p

Oil Red - O Staining for lipid droplets.

<p>hMSCs maintained in adipogenic differentiation medium were stained with Oil Red - O to highlight the lipid contents of cells (top images, bar  = 100 µm). Higher magnification transmission images of HA and NA (bottom images, bar = 50 µm) before staining reveal larger, more numerous lipid droplets in hMSCs cultured in adipogenic differentiation medium at 20% oxygen. Quantification of eluted Oil Red - O by absorption of 490 nm reveals an increase in total Oil Red - O staining amongst HA or NA cells, but the differences are not statistically significant. N = 6.</p

Shorter, earlier depolarization times are sufficient to suppress OS differentiation.

<p>Cells were exposed to 80 mM K⁺ or 10 nM ouabain during Days 1–2 (A) or Days 1–4 (B), then washed and continued in culture in OS medium. Gene expression was evaluated on Day 7. Two or four days of exposure to depolarization treatment was sufficient to effect a change in OS marker expression. Data points are mean relative expression±standard deviation (N = 6). Marked samples are statistically different * relative to ALP expression of untreated OS samples (p<0.009), # relative to BSP expression of untreated OS samples (p<0.04). Undiff, hMSCs cultured in control medium; OS, hMSCs cultured in OS medium; OS-80K, hMSCs cultured in OS medium supplemented with 80 mM K⁺; OS-ouab, hMSCs cultured in OS medium supplemented with 10 nM ouabain.</p

Hyperpolarization upregulates OS gene expression.

<p>(A) K-_ATP-channel openers pinacidil and diazoxide hyperpolarized hMSCs undergoing OS differentiation. Cells were impaled individually and the V_mem recorded until a stable baseline was reached (pre-treatment), then 10 µM pinacidil or diazoxide was added and the V_mem recorded until a new equilibrium was reached (post-treatment). Data points are mean potentials±standard deviation (N = 5 cells). Marked samples are statistically different * relative to respective pre-treatment samples (p<0.04). (B, C) Exposure to K-_ATP-channel openers pinacidil (B) and diazoxide (C) resulted in slight upregulation of OS markers compared to untreated cells. When treated with 1 and 10 µM pinacidil (OS-1pin and OS-10pin, respectively), cells showed upregulated BSP expression compared to untreated OS cells (p<0.04). When treated with 10 and 100 µM diazoxide, cells upregulated ALP and BSP expression compared to untreated OS cells. Data points are mean relative expression±standard deviation (N = 6). Marked samples are statistically different * relative to ALP expression of untreated OS samples (p<0.05), # relative to BSP expression of untreated OS samples (p<0.05). Undiff, hMSCs cultured in control medium; OS, hMSCs cultured in OS medium; OS-80K, hMSCs cultured in OS medium supplemented with 80 mM K⁺; OS-ouab, hMSCs cultured in OS medium supplemented with 10 nM ouabain.</p

Membrane potential of AD and OS cells recovers after washout of early depolarization treatments.

<p>hMSCs in AD or OS differentiation media were depolarized with 80 mM K⁺ (AD-K, OS-K) or 10 nM ouabain (AD-ouab, OS-ouab) during Days 1–4. Control cells were cultured in normal AD or OS media (AD or OS). Depolarization treatment was washed out after Day 4 and replaced with normal AD or OS media. Intracellular recordings were performed after washout on Days 5 or 6. Data points are mean potentials±standard deviation (N = 7–10 cells). Neither treated AD cells nor treated OS cells were statistically different from their respective untreated controls (p<0.05).</p