Membrane Potential Controls Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells

Background: Control of stem cell behavior is a crucial aspect of developmental biology and regenerative medicine. While the functional role of electrophysiology in stem cell biology is poorly understood, it has become clear that endogenous ion flows represent a powerful set of signals by means of which cell proliferation, differentiation, and migration can be controlled in regeneration and embryonic morphogenesis. Methodology/Principal Findings: We examined the membrane potential (Vmem) changes exhibited by human mesenchymal stem cells (hMSCs) undergoing adipogenic (AD) and osteogenic (OS) differentiation, and uncovered a characteristic hyperpolarization of differentiated cells versus undifferentiated cells. Reversal of the progressive polarization via pharmacological modulation of transmembrane potential revealed that depolarization of hMSCs prevents differentiation. In contrast, treatment with hyperpolarizing reagents upregulated osteogenic markers. Conclusions/Significance: Taken together, these data suggest that the endogenous hyperpolarization is a functiona

Efficient isolation, biophysical characterisation and molecular composition of extracellular vesicles secreted by primary and immortalised cells of reproductive origin

Effective communication between the maternal reproductive tract, gametes and the pre-implantation embryo is essential for the successful establishment of pregnancy. Recent studies have recognised extracellular vesicles (EVs) as potent vehicles for intercellular communication, potentially via their transport of microRNAs (miRNAs). The aim of the current investigation was to determine the size, concentration and electrical surface properties (zeta potential) of EVs secreted by; (1) primary cultures of porcine oviductal epithelial cells (POECs) from the isthmus and ampullary regions of the female reproductive tract; (2) Ishikawa and RL95-2 human endometrial epithelial cell line cultures; and (3) the non-reproductive epithelial cell line HEK293T. In addition, this study investigated whether EVs secreted by POECs contained miRNAs. All cell types were cultured in EV-depleted medium for 24 or 48 h. EVs were successfully isolated from conditioned culture media using size exclusion chromatography. Nanoparticle tracking analysis (NTA) was performed to evaluate EV size, concentration and zeta potential. QRT-PCR was performed to quantify the expression of candidate miRNAs (miR-103, let-7a, miR-19a, miR-203, miR-126, miR-19b, RNU44, miR-92, miR-196a, miR-326 and miR-23a). NTA confirmed the presence of EVs with diameters of 50–150 nm in all cell types. EV size distribution was significantly different between cell types after 24 and 48 h of cell culture and the concentration of EVs secreted by POECs and Ishikawa cells was also time dependent. The distribution of EVs with specific electrokinetic potential measurements varied between cell types, indicating that EVs of differing cellular origin have varied membrane components. In addition, EVs secreted by POECs exhibited significantly different time dependant changes in zeta potential. QRT-PCR confirmed the presence of miR-103, let-7a, miR-19a, miR-203, miR-126, and miR-19b in EVs secreted by POECs (CT ≥ 29). Bioinformatics analysis suggests that these miRNAs are involved in cell proliferation, innate immune responses, apoptosis and cellular migration. In conclusion, reproductive epithelial cells secrete distinct populations of EVs containing miRNAs, which potentially act in intercellular communication in order to modulate the periconception events leading to successful establishment of pregnancy

Role of membrane potential in the regulation of cell proliferation and differentiation. Stem Cell Rev 5

Abstract Biophysical signaling, an integral regulator of long-term cell behavior in both excitable and non-excitable cell types, offers enormous potential for modulation of important cell functions. Of particular interest to current regenerative medicine efforts, we review several examples that support the functional role of transmembrane potential (V mem ) in the regulation of proliferation and differentiation. Interestingly, distinct V mem controls are found in many cancer cell and precursor cell systems, which are known for their proliferative and differentiation capacities, respectively. Collectively, the data demonstrate that bioelectric properties can serve as markers for cell characterization and can control cell mitotic activity, cell cycle progression, and differentiation. The ability to control cell functions by modulating bioelectric properties such as V mem would be an invaluable tool for directing stem cell behavior toward therapeutic goals. Biophysical properties of stem cells have only recently begun to be studied and are thus in need of further characterization. Understanding the molecular and mechanistic basis of biophysical regulation will point the way toward novel ways to rationally direct cell functions, allowing us to capitalize upon the potential of biophysical signaling for regenerative medicine and tissue engineering

Depolarization suppresses AD gene expression.

<p>PPARG and LPL expression were suppressed on Days 2, 7, 14, and 22 by addition of 80 mM K⁺ (AD-80K) during AD differentiation. Similarly, PPARG and LPL expression were suppressed on Days 7, 14, and 22 by addition of 10 nM ouabain (AD-ouab) during AD differentiation. Data points are mean relative expression±standard deviation (N = 6). Marked samples are statistically different, * relative to PPARG expression of untreated AD samples (p<0.05), † relative to LPL expression of untreated AD samples (p<0.003), ‡ relative to LPL expression of AD-80K samples (p<0.0005). (For clarity, statistical significances are reported among samples taken within the same day.) 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

Measurement of resting and depolarized membrane potentials during OS and AD differentiation.

<p>(A) Intracellular recordings of resting and depolarized membrane potentials (V_mem) in hMSCs during OS and AD differentiation. Cells were impaled individually and the V_mem recorded until a stable baseline was reached (pre-treatment), then 10 nM ouabain (OS-ouab, AD-ouab samples) or 80 mM K⁺ (OS-K, AD-K) was added and the V_mem recorded until a new equilibrium was reached (post-treatment). Data points are mean potentials±standard deviation (N = 6–7 cells). Marked samples are statistically different, * relative to respective pre-treatment samples (p<0.03), # relative to AD-ouab post-treatment sample (p<0.04). (For clarity, statistical significances marked by # are reported among post-treatment samples only.) (B) Intensities of DiSBAC₂(3)-loaded cells at resting and depolarized potentials during OS and AD differentiation. Pre-treatment values are the fluorescence intensities of OS and AD cells at rest, while post-treatment values are the fluorescence intensities after depolarization with 10 nM ouabain (OS-ouab, AD-ouab) or 80 mM K⁺ (OS-K, AD-K). Data points are mean pixel intensity±standard deviation (N = 15–20 cell fields). Marked samples are statistically different, * relative to respective pre-treatment samples (p≪0.0001), # relative to OS-K post-treatment sample (p≪0.0001), † relative to AD-K post-treatment sample (p≪0.0001). (For clarity, statistical significances marked by # and † are reported among post-treatment samples only).</p

V_mem hyperpolarization exhibited by OS- and AD-differentiated cells.

<p>(A) Cell culture timeline for V_mem studies. Cells were seeded in control medium, then switched to OS or AD differentiation medium (OS or AD) at various time points over the course of 4 weeks. After 4 weeks, cells that had differentiated for a total of 0, 1, 2, 3, or 4 weeks (samples 0wk-diff, 1wk-diff, 2wk-diff, 3wk-diff, and 4wk-diff, respectively) were imaged on the same day. (B) Fluorescence measurements from cells cultured according to the timeline in OS or AD media. Cells were stained with the voltage-sensitive dye DiSBAC, which exhibits higher intensity with membrane depolarization. Data points are mean pixel intensity±standard deviation (N = 5–15 cell fields). Marked samples are statistically different, * relative to 0wk-diff OS sample (p<0.0005), § relative to 4wk-diff OS sample (p<0.0005), # relative to 0wk-diff AD sample (p<0.0005), † relative to 3wk-diff AD sample (p<0.0002), ‡ relative to 4wk-diff AD sample (p<0.005).</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

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