Membrane potential controls adipogenic and osteogenic differentiation of mesenchymal stem cells

Our data extend the previous analyses of somatic vs. embryonic/cancer cell membrane voltage [1], [43], [49] to stem cells. Crucially, our functional results show that the relationship between depolarization and an undifferentiated state is not merely a correlation but rather that transmembrane potential is a functional determinant of differentiation state in hMSCs. Moreover, in complement to recent studies implicating specific ion channels in cell behavior, our data implicate Vmem as the salient parameter, not specific ion gradients or ion-independent functions of specific proteins. This is important for biomedical applications because it suggests that rational changes in cell behavior can be induced by techniques that do not depend on specific channel or pump genes being expressed natively in a cell population. A number of mechanisms have been proposed to explain how Vmem levels are transduced into transcriptional cascades by second messenger systems (reviewed in [1]), and we are currently focused on testing Ca2+ influx, voltage sensor-containing phosphatase (VSP)-PTEN transduction, and other mechanisms in the voltage control of hMSC differentiation. It clear, however, that control of bioelectrical properties of stem cells is not only a fascinating fundamental aspect of cell regulation, but also potentially a useful tool in tissue engineering efforts. Future work in regenerative medicine may be able to capitalize on Vmem as a powerful and tractable control point for rational modulation of adult stem cell function.

hMSCs were obtained from bone marrow aspirates from Cambrex Bio Science Walkersville, Inc., from a 25 yr old healthy male and prepared as we have previously reported [57]. Whole bone marrow aspirates were plated at a density of 10 µL of aspirate per centimeter squared in 185 cm2 tissue culture flasks in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL), 0.1 mM non-essential amino acids, and basic fibroblast growth factor (bFGF, 1 ng/mL) (Invitrogen, Carlsbad, CA). Cells were maintained in a humidified incubator at 37°C with 5% CO2. hMSCs were separated from hematopoietic stem cells on the basis of their adherence to tissue culture plastic; hematopoietic stem cells in suspension were removed after approximately 5 days of culture. hMSCs were expanded until confluent, with medium changes every 3–4 days. Cells were trypsinized with 0.25% trypsin-1mM EDTA (Invitrogen), and frozen in liquid nitrogen in FBS with 10% DMSO as first passage cells (P1). P1-P3 cells were used for all experiments.

hMSCs were thawed and plated on glass bottom dishes or on tissue culture polystyrene at a density of 5000 cells/cm2 for osteogenic (OS) differentiation or 10000 cells/cm2 for adipogenic (AD) differentiation. For the first 24 hr post-thaw, cells were cultured in control medium, consisting of DMEM supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL), and 0.1 mM non-essential amino acids. Differentiation inducers were then added to the medium in order to study temporal-dependent changes in membrane potential during OS and AD differentiation. Control medium was supplemented with 10 mM β-glycerophosphate, 0.05 mM L-ascorbic acid-2-phosphate, and 100 nM dexamethasone for OS differentiation; or 0.5 mM 3-isobutyl-1-methyl-xanthine, 1 µM dexamethasone, 5 µg/mL insulin, and 50 µM indomethacin for AD differentiation (Sigma-Aldrich, St. Louis, MO). Undifferentiated hMSCs were maintained in control medium.

To assess the effects of disruption of membrane potential (Vmem), several methods were employed to change Vmem. (1) Na+/K+-ATPase-inhibitor ouabain (10 nM, Sigma-Aldrich) was added to the medium from a fresh 10 mM stock solution in distilled water. (2) The concentration of extracellular K+ was increased by adding potassium gluconate (Sigma-Aldrich) to the medium to final concentrations of 10–80 mM. (3) The ATP-sensitive potassium channel (KATP ) openers pinacidil and diazoxide (Sigma-Aldrich) were added to the medium to final concentrations of 1, 10, or 100 µM from 10 mM stock solutions in ethanol.

After 0, 1, 2, 3, or 4 weeks of AD or OS differentiation (culture schedule presented in Fig. 1A), cells were dyed with a fluorescent dye that is sensitive to membrane potential. Bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3) or DiSBAC, Invitrogen) is an anionic voltage-sensitive dye whose uptake into cells is voltage-dependent: higher uptake is seen in more depolarized cells. A fresh solution of 10 mM DiSBAC in DMSO was prepared and diluted to 0.5 µM in Hank's Buffered Salt Solution (HBSS, Invitrogen). Cells grown in glass-bottom dishes (poly-d-lysine coated, No. 1.5, MatTek Corp., Ashland, MA) were incubated in DiSBAC for 30 minutes at 37°C, then imaged while submerged in dye at room temperature. Images were acquired on a Leica TCS SP2 laser scanning confocal microscope with an inverted DM IRE2 stand (Wetzlar, Germany) and a Leica PL APO 63× (NA 1.2) water-immersion objective. DiSBAC was excited with a 543 nm HeNe laser; images were collected at 570±5 nm by a non-descanned PMT controlled by Leica Confocal Software. A double dichroic filter was used to eliminate 543 nm excitation light. Confocal images for all samples in an experimental set were taken on the same day to minimize instrumental and other variations. Since fluorescence intensity was quantified for each image, the gain and offset settings of the microscope were kept constant over the duration of each experiment. To visualize membrane potential depolarization, cells at resting potential were imaged as above, then exposed to depolarization agents and allowed to equilibrate for 5 min, then imaged once more. MATLAB software (The MathWorks, Inc.) was used to assist in the drawing of regions of interest (ROI) around cells and in calculating pixel intensities within the ROIs. ROIs were drawn on thresholded images by using the function bwboundaries to trace cells and their nuclei.

Fluorescence intensities of cells encircled by ROIs were calculated by averaging corresponding pixel intensities in the original image, excluding pixels within encircled nuclei, after background correction using a blank (no cell) region of the image.

Membrane potentials of hMSCs were recorded during OS and AD differentiation before and after depolarization treatments. Electrodes were pulled from borosilicate glass capillary tubing (1.0 mm OD, Warner Instrument Corp., Hamden, CT) using a Flaming/Brown Micropipette Puller (Model P-87, Sutter Instrument Co., Novato, CA), filled with a filtered 2 M KCl electrode solution, and loaded onto a Burleigh PCS PS60 micromanipulator (EXFO Life Sciences, Mississauga, Ontario). Electrode resistances were in the range of 40–60 MΩ. Signals were passed through a preamplifier (8100-1 Single Electrode System, Dagan Corp., Minneapolis, MN) and an amplifier (Instrumentation Amplifier Model 210, Brownlee Precision Co., San Jose, CA). Data was collected using a DI-720 Series #3 Data Acquisition System (Dataq Instruments, Inc., Akron, OH) controlled by WinDaq Waveform Recording Software, and was analyzed using WinDaq Waveform Browser (Dataq Instruments, Inc.). Individual cells were impaled, and membrane potential was recorded relative to the bath solution (HBSS, Invitrogen). After obtaining a stable signal, depolarizing or hyperpolarizing agents were added to the bath solution, and the membrane potential was recorded for an additional 5 min, or until the signal stabilized.

RNA was isolated from hMSCs using Trizol reagent (Invitrogen) following the single step acid-phenol guanidinium method, and purified using the Qiagen RNEasy kit (Qiagen, Valencia, CA). To synthesize cDNA, reverse transcription was performed on the purified RNA using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Real time RT-PCR was performed on differentiated cells with and without depolarization treatment to track the expression of markers characteristic of the differentiated cell types. Osteoblast differentiation markers include alkaline phosphatase (ALP) and bone sialoprotein (BSP). Adipogenic differentiation markers include perixosome proliferator-activated receptor γ (PPARG) and lipoprotein lipase (LPL). Primers and probes for the bone-related and adipose-related genes above were obtained from TaqMan® Gene Expression Assay kits (Applied Biosystems). Transcript expression levels were quantified with an ABI Prism 7000 Real Time PCR System (Applied Biosystems) or a Stratagene Mx3000P QPCR System (Stratagene, La Jolla, CA). Expression levels were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and reported relative to positive control reactions (untreated OS or AD) [58]–[60]. We have previously reported PCR reaction conditions and primers [61]–[63].

Total calcium content was determined by a colorimetric assay using the Calcium (CPC) Liquicolor Test (Stanbio Laboratory, Boerne, TX). Calcium was dissociated with trichloroacetic acid and reacted with o-cresolphthalein complexone, forming a purple color which was measured spectrophotometrically at 575 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA).

Enzyme activity was assessed using a biochemical assay from Sigma-Aldrich that detects the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol by ALP. Cultured cells were lysed with 0.2% v/v Triton X-100 in 5 mM MgCl2, and incubated with pNPP substrate in 2-amino-2-methyl-1-propanol buffer. The hydrolysis reaction was stopped by 0.2 M NaOH, and the colored end product was detected spectrophotometrically at 405 nm.

Cells undergoing AD differentiation were stained with Oil Red O to visualize lipid droplet accumulation. Cells were fixed overnight in 4% neutral buffered formalin, then washed with 60% isopropanol and air-dried. A fresh 60% Oil Red O working solution was prepared from a stock solution (0.7 g Oil Red O in 200 mL isopropanol) and filtered through a 45 µM syringe filter. Cells were stained with the working solution for 45 min, washed five times with distilled water, and imaged at room temperature with an inverted microscope (Axiovert S100, Carl Zeiss, Inc.) equipped with Zeiss A-Plan 10× (NA 0.25) and LD A-Plan 32× (0.40) objectives. Images captured by a Sony Exwave HAD CCD camera were acquired using ImageJ software (NIH). Photoshop software (Adobe Systems Inc.) was used to adjust levels and color balance. To measure the amount of Oil Red O in the stained samples, Oil Red O was eluted from the cells by adding 100% isopropanol and incubating for 1 hr, and then transferred to a 96-well plate and measured spectrophotometrically at 500 nm.

Cell viability was assessed using the alamarBlue assay (Invitrogen). Cells were incubated at 37°C in a solution of 3% alamarBlue in control medium, and the resulting fluorometric change was detected with an excitation wavelength of 560 nm and emission wavelength of 590 nm using a microplate spectrofluorometer (SpectraMax Gemini EM, Molecular Devices, Sunnyvale, CA).

Statistically significant differences between groups were determined by performing a two-tailed Student's t-test. Differences were considered significant if p<0.05, unless otherwise noted.