Loading Human Mesenchymal Stem Cells with ...

CELL PRESERVATION TECHNOLOGY Volume 2, Number 1, 2004 © Mary Ann Liebert, Inc.

Loading Human Mesenchymal Stem Cells with Trehalose by Fluid-Phase Endocytosis ANN E. OLIVER,1,2 KAMRAN JAMIL,1,3 JOHN H. CROWE,1,2 and FERN TABLIN1,3

ABSTRACT Human mesenchymal stem cells (MSCs) have shown great promise in the area of tissue engineering. Regardless of their regenerative potential, however, they will not be useful on a large scale unless an improved and more stable form of cellular storage is developed. An ideal storage condition would be dehydrated cells, as this would allow room temperature storage and would not require refrigeration or freezing equipment. As a first step toward developing a method for storing MSCs in a desiccated state, we have characterized the ability of these cells to take up solutes from the extracellular milieu, as the introduction of protective solutes into the cytosol is a critical step in the dehydration process. Lucifer yellow (LYCH), a well-known probe in the study of fluid-phase endocytosis, indicated the uptake process was inhibited below 20°C. Fourier transfer infrared spectroscopy studies suggested that this inhibition is associated with the membrane physical state. In addition, fluorescence microscopy revealed endosomes stained with LYCH accumulated in the MSCs, and suggested that the dye entered the cytosol over time. Trehalose, a protective disaccharide, was accumulated by the MSCs as well. Uptake was proportional to the length of incubation and showed a nonsaturating dependence on extracellular concentration, characteristic of fluid-phase endocytosis. Endocytosis inhibitors were used to investigate further the mechanism of uptake. Colchicine and nocodazole, both of which depolymerize microtubules, blocked trehalose uptake. Dimethyl sulfoxide, which affects microtubules differently, by forming shorter and more abundant microtubules, also inhibited trehalose uptake. However, cytochalasin B, which depolymerizes actin filaments, and thus blocks both macropinocytosis and caveolae-dependent pinocytosis, did not cause a decrease in trehalose uptake. Amiloride, which blocks sodium channels and inhibits clathrin-independent pinocytosis, also did not inhibit trehalose uptake. Taken together, these results suggest that human MSCs are capable of loading trehalose from the extracellular space by a clathrin-dependent fluid-phase endocytotic mechanism that is microtubule-dependent but actinindependent.

followed by culture expansion,6 and can be stored in liquid nitrogen with 10% dimethyl sulfoxide (DMSO). The washing and growth following a freeze–thaw cycle are slow processes; therefore transport, storage, and manipulation of the final cell product are severely limited. An alternative method, in which the cells were dehydrated for storage and rehydrated with a sterile solution prior to transplant, would provide an enormous benefit to the use of MSCs in clinical practice.

INTRODUCTION

H

(MSCs), found in adult bone marrow, are capable of differentiating along several distinct pathways. Tissue regeneration studies with these cells have shown exciting potential in the production of bone, muscle, cartilage, adipose, and possibly neural tissue.1–5 MSCs are usually harvested from bone marrow aspirates by centrifugation on a Percoll cushion, UMAN M ESENCH YMAL STEM CELLS

1 Center

for Biostabilization and 2Section of Molecular and Cellular Biology, University of California, Davis, Cali-

fornia. 3 Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California.

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Lyophilization represents an ideal method for dehydration, as large samples can be dried simultaneously, producing homogeneous products. Human blood platelets have been successfully freeze-dried, and functional cells responding to physiological agonists were obtained.7 Furthermore, the freeze-dried platelets are stable during dry storage under nitrogen, giving more than 85% recovery after 2 years.8 Similar storage for MSCs would be a significant advancement in the area of tissue engineering research. However, nucleated cells have more types of internal structures than platelets, all of which must be protected. Also, since the cells must be capable of cell division following rehydration, obtaining viable cells after lyophilization represents a complex hurdle. Lyophilization requires a protectant to inhibit the damage caused by the removal of water. Recent studies have used trehalose to protect mammalian cells during drying.7,9,10 Trehalose, a protective disaccharide, is found in extremely high concentrations in many organisms capable of surviving dehydration. For instance, in cysts of the brine shrimp Artemia, trehalose represents approximately 15% of the dry weight of the embryos.11 Other examples include bacteria, yeast, tardigrades, and nematodes (reviewed by Crowe et al.12,13). Trehalose protects the membranes, proteins, and nucleic acids (reviewed by Crowe and Crowe14 and Crowe et al.15 ) in such organisms because of its capacity to replace water molecules 16–18 and form a high-melting carbohydrate glass.13,19–21 In any successful protocol for lyophilizing human nucleated cells, introducing trehalose into the cytosol is likely to be an important step, though other protective methods are also likely to be required.22 As a first step toward preserving MSCs by lyophilization, we have investigated the loading of these cells with extracellular solutes. The fluorescent dye Lucifer yellow (LYCH) is anionic and thus does not cross the cell membrane by passive diffusion.23,24 LYCH is a well-studied probe for fluid-phase endocytosis (FPE), which has been shown to occur in many different cell types, such as yeast,25,26 rat hepatocytes, 27 epithelial cells,28 and plant cell protoplasts.29 FPE is characterized by time- and temperature-dependent uptake.

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Further, as there is no receptor to mediate the process, FPE shows a nonsaturating dependence on the extracellular concentration and cannot result in an intracellular concentration that exceeds that in the extracellular environment. LYCH and trehalose uptake studies indicated that FPE is indeed occurring in the MSCs and that it correlates well with the membrane phase behavior of these cells. Finally, various endocytosis inhibitors were used to characterize further the mechanism of uptake. Colchicine, nocodazole, and DMSO, which affect microtubules, cytochalasin B, which depolymerizes actin filaments, and amiloride, which blocks clathrin-independent pinocytosis, were all investigated for their effect on trehalose uptake. The results of these studies suggest the uptake mechanism is microtubule-dependent, but actin-independent, and is most likely a collateral result of constitutive clathrin-mediated endocytosis.

MATERIALS AND METHODS Materials Tissue culture reagents were from Invitrogen (Carlsbad, CA), unless otherwise stated. Tissue culture disposables were from Nalge Nunc International (Rochester, NY). LYCH was from Sigma-Aldrich (St. Louis, MO), and trehalose was from Ferro Pfanstiehl Laboratories, Inc. (Waukegan, IL). Colchicine and N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride (amiloride) were obtained from Sigma-Aldrich. Methyl[5-(2thienylcarbonyl)-1H-benzimidazol-2-yl]carbamate (nocodazole) and cytochalasin B were obtained from Calbiochem (La Jolla, CA). DMSO was from Edward Life Sciences (West Midvale, VT). Cell culture Human MSCs previously isolated from bone marrow and expanded in vitro to passage number 1 were a gift from Osiris Therapeutics (Baltimore, MD) and were shipped to the University of California, Davis in liquid nitrogen. The cells were grown in Dulbecco’s modified Eagle medium-low glucose, with 10% fetal bovine

LOADING MSCS WITH TREHALOSE BY ENDOCYTOSIS

serum (Hyclone, Logan, UT) at 37°C with 5% CO 2 and 90% relative humidity. The cells were used up through passage number 3 at a level of 90–95% confluence. Cells were harvested by washing once with Dulbecco’s phosphatebuffered saline (DPBS) and incubating for 5–7 min with trypsin-EDTA (0.05% trypsin and 0.53 mM EDTA-4Na). This cell suspension was pelleted at 167 g for 10 min and resuspended in medium or the specified buffer. Unused cells were counted before freezing and were frozen in mixture of 10% DMSO, 5% human serum albumin, and 70% Plasma Lyte A (the latter two from Baxter Healthcare Corp., Deerfield, IL) until they were needed. Fourier transform infrared (FTIR) spectroscopy MSCs harvested by trypsinization were resuspended in 2 mL of fresh medium, and the cells were allowed to settle for 30 min. The cell pellet was applied as a thin film between two CaF2 windows and scanned by FTIR spectroscopy on a Perkin Elmer Spectrum 2000 equipped with a mercury cadmium telluride detector. Data were collected from 3,600 to 900 cm21 every 2°C between 27 and 50°C using a ramp rate of 2°C/min. Temperature was controlled by a Peltier device and accurately measured by placement of a thermocouple directly on the windows. The frequency of the CH2 symmetric stretching band was plotted as a function of temperature, and phase transition temperatures were obtained by first derivative analysis.9,30 LYCH loading and detection The fluorescent dye LYCH was used to monitor the temperature dependence of solute uptake, as it can be employed over a shorter incubation time of 5 h than is required for trehalose. This was necessary, as longer incubation times at the extreme temperatures resulted in a large decrease in cell number and viability. For this experiment, 66,000 cells were plated per well in six-well plates, and grown until they reached 90–95% confluence. Cells were incubated at various temperatures in the presence of 10 mM LYCH in MSC growth media for 5 h, in the presence of atmospheric relative humidity and CO2. Following incubation,

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the cells were washed once with 10 mL of DPBS, and harvested by trypsinization. The cells were then washed an additional three times with 10 mL of DPBS each and collected by centrifugation (167 g). The pellet was resuspended in 1 mL of DPBS. Cell counts and viability were assessed by trypan blue exclusion using five counts of 50–100 cells per 1-mm2 hemocytometer grid square for each sample. In control experiments, a difference of only 1–2% was found between viability assessments using trypan blue and the fluorescent live/dead pair Hoechst 33342 and propidium iodide (data not shown). Cells were suspended in 1% (wt/vol) Triton X-100 (Calbiochem) in DPBS overnight at 4°C. LYCH fluorescence was measured on a Perkin Elmer LS50B luminescence spectrometer, using an excitation wavelength of 428 nm and emission wavelength of 530 nm. Final intracellular LYCH concentration was calculated by comparison with a standard curve for LYCH, and assuming an intracellular volume of 14,000 fL. This estimate was based on a 15 mm cellular radius, as measured on trypsinized cells using a hemocytometer grid, and a free volume of 50%, as has been used previously.7 However, the data presented are approximations, as small variations in the cell radius have a large effect on cell volume. Data are shown from three independent measurements. Trehalose loading and detection Trehalose uptake in MSCs was measured as a function of time and extracellular concentration. For these experiments, cells were grown in T-75 flasks to 90–95% confluence. For the time course, cells were incubated in MSC growth medium with 100 mM trehalose for 0, 1, 4, 7, or 24 h at 37°C. For the concentration series, cells were incubated at 37°C for 24 h in MSC growth medium with the addition of 0, 25, 50, 100, or 125 mM trehalose. Following incubation, the cells were washed once with 10 mL of DPBS, and harvested by trypsinization. The cells were then washed an additional three times with 10 mL of DPBS each and collected by centrifugation (167 g). The pellet was resuspended in 1 mL of DPBS. Cell counts and viability were assessed by trypan blue exclusion using five counts of 50–100 cells per 1-mm2 he-

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mocytometer grid square for each sample. The cells were extracted by incubating in 80% methanol at 80°C for 1 h. The trehalose enters the supernatant, which was collected after centrifuging the suspension at 200 g for 10 min. The supernatant was evaporated under a stream of N2 at 40°C, and the dry residue was dissolved in 3 mL of Nano-pure water. For trehalose quantitation, the anthrone reaction was used according to Umbreit et al.31 Briefly, the samples (3 mL) were mixed with 6 mL of anthrone reagent [2% anthrone (Sigma-Aldrich) in sulfuric acid], heated to 100°C for 3 min, and allowed to cool. Absorbance at 620 nm was read on an Amersham-Pharmacia Biotech Ultrospec 3300 Pro spectrophotometer at room temperature and compared with a standard curve. In control experiments, the last wash solution was assayed for residual trehalose. The resulting anthrone absorbance was negligible and fell within the range of experimental error for control samples containing DPBS buffer only without sugar. As the anthrone method detects all sugars, unloaded control cells were always treated in parallel. These values, normalized for cell count, were subtracted from the trehalose-loaded samples in order to evaluate trehalose specifically and to avoid artifact due to endogenous sugars. Data are shown for three independent measurements. In an important control experiment, gas chromatographic analysis revealed an essentially identical intracellular trehalose concentration to the anthrone technique (data not shown) in cells loaded with 100 mM trehalose for 24 h at 37°C. AlamarBlue™ (BioSource International, Camarillo, CA) was used to monitor cell health following a 24-h incubation with 100 mM trehalose. Each day the medium was changed and replaced with fresh medium containing 10% (vol/vol) alamarBlue. The fluorescence of the medium was measured daily (after being in contact with the cells for 24 h) on a Perkin Elmer LS50B luminescence spectrometer, using an excitation wavelength of 530 nm and emission wavelength of 585 nm. Inhibitor experiments To monitor trehalose loading in the presence of inhibitors, MSCs were plated in T-75 tissue

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culture flasks and grown until they reached 90–95% confluence. The cells were incubated in MSC medium with 100 mM trehalose and the desired concentration of inhibitor for 24 h at 37°C. For each experiment, a positive control (100 mM trehalose, no inhibitor) and a negative control (no trehalose, no inhibitor) were performed to give the maximal and zero trehalose values for the anthrone analysis, respectively. Following the incubation, the cells were washed and harvested, and the viability, cell count, and trehalose concentration were quantified as described above. Inhibitor concentrations were applied in the range shown to be useful in the literature: 20 mM colchicine,32,33 5 mg/mL (5 16.6 mM) nocodazole,34 300 mM amiloride,35 and 5 mM cytochalasin B36; for DMSO, 0–10% (vol/vol) is commonly used,37–39 but cellular toxicity mandated our concentrations not exceed 2% for the MSCs. Data shown represent the means (6 standard deviation) for three independent measurements. One-way analysis of variance (ANOVA, Tukey’s test) statistical analysis was performed with SigmaStat software (Jandel Scientific, San Rafael, CA). Fluorescence microscopy To visualize LYCH uptake, cells were plated in two-well LabTek CC2 glass slides, and grown for 5–7 days until they reached ,60% confluence. They were then incubated in MSC medium with 10 mM LYCH for 3 or 24 h. Following the incubation, cells were washed three times with 1.5 mL of DPBS and were fixed in 1% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in DPBS for 1 h at 22°C. Cells were mounted with AquaPoly/Mount (Polysciences, Inc., Warrington, PA), and observed and photographed using a BX30 (Olympus, Melville, NY) microscope equipped with a Zeiss Axiocam running Axio Vison 3.1 software. To visualize the effect of nocodazole on microtubule structure, the cells were grown as described above. They were then treated with 0 or 5 mg/mL of nocodazole in MSC growth medium for 1 h at 37°C. The cells were washed and fixed as described above, and permeabilized with absolute methanol for 10 min at 4°C. They were blocked with a 1% solution of sheep


serum (ICN, Costa Mesa, CA) for 1 h at 37°C. Monoclonal anti-beta-tubulin antibody (Amersham, Piscataway, NJ) was used to stain betatubulin (1:100 dilution for 1 h at 37°C). Alexa Fluor 488 Fab fragment of goat anti-mouse IgG (Molecular Probes, Eugene, OR) was used to label the primary antibody (1:800 dilution for 1 h at 37°C). Cells were coverslipped with AquaPoly/Mount, and observed and photographed using an Olympus BX30 microscope as described above. To visualize the effect of cytochalasin B on actin organization of MSCs, cells were incubated with 0 or 5 mM cytochalasin B for 1 h at 37°C. The cells were grown and treated as described above, except the cells were permeabilized with 0.1% Triton X-100 (wt/vol) and Texas Red-X phalloidin (Molecular Probes) which specifically binds filamentous actin40 (1:40 dilution at 37°C for 1 h). The cells were also treated with 30 nM 4,6-diamino-2phenylindole dihydrochloride (DAPI) for 10 min to stain the nuclei. Cells were mounted on slides using Aqua Poly/Mount and photographed as detailed above.

RESULTS Time, temperature, and concentration dependence of uptake The standard dye for monitoring FPE, LYCH, was used to determine the temperature dependence of solute uptake from the extracellular environment by MSCs. Cells were treated for 5 h with 10 mM LYCH in growth medium, following which they were washed, trypsinized, and lysed with 1% Triton X-100 in DPBS, as described in Materials and Methods. The cellular LYCH uptake was quantified by comparison with a standard curve and is shown in Figure 1A. LYCH uptake was minimal at 2, 5, 10, and 15°C, but increased dramatically at 20°C. The viability was high (.80%) at all temperatures tested, but some cells were lost (,30%) at the lowest temperatures (data not shown). The biphasic nature of this curve and the sharp increase in LYCH loading at 20°C correlate well with the membrane phase behavior of MSCs. As shown in Figure 1B, FTIR spectroscopy reveals two

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phase transitions in MSCs, one at ,15°C and another at ,35°C, a pattern similar to that reported in human platelets and red blood cells.30,41 The minimal uptake of LYCH at the lowest temperatures tested can be explained by the finding that the cell membranes are more ordered at those temperatures. Endocytosis is known to be inhibited in cells with membranes in an ordered state.28 Since some melting events are still occurring above 20°C, the sharp increase in loading at this temperature suggests that a threshold of membrane fluidity may be required for endocytotic vesicle formation. Thus, it is likely that the physical state of the membrane is of crucial importance in the functioning of the endocytotic mechanism. Activation energies were calculated for the LYCH uptake by plotting the natural log of the rate of internalization against reciprocal temperature (Fig. 1A, inset), according to the Arrhenius equation: ln(k) 5 ln (A) 1 (2Ea/RT), where K is a rate constant, Ea is the activation energy, and A is a frequency factor. Evidently, the LYCH uptake doesn’t show Arrhenius behavior throughout the temperature region between 4 and 33°C, as the data can be fit best by two intersecting lines. Below the inflection point of 15°C (the midpoint of the phospholipid transition), the activation energy was 11.6 (6 0.6) Kcal/mol, whereas above the inflection point, an activation energy of 15.3 (6 1.5) Kcal/mol was obtained. These values are in reasonable agreement with those found for other cell types. For instance, Madin–Darby canine kidney cells had activation energies for endocytosis of 8.7 (6 0.6) and 15.6 (6 3.2) Kcal/mol28 for temperatures below and above the phase transition of 27°C,42 respectively. This dependence of endocytotic rate on the physical state of the membrane has been seen also in human and pig platelets at critical temperatures of 22 and 15°C, respectively.7,43 We conclude that the endocytotic process is much more temperature sensitive when the membranes have reached a threshold membrane fluidity, which for the MSCs occurs above ,15°C. Although LYCH and trehalose are of similar size (molecular mass of 457 and 378 Da, respectively), and uptake of the two molecules from the extracellular space is likely to be similar, the molecule we are actually interested in

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A

B

FIG. 1. A: Dependence of LYCH uptake on incubation temperature. MSCs in six-well plates were incubated at 2, 10, 15, 23, or 33°C with 10 mM LYCH in MSC medium for 5 h. The cells were then washed, trypsinized, and suspended in 1% Triton X-100. LYCH was quantified by fluorescence spectroscopy by comparison with a standard curve (Ex 428, Em 530). Viability was measured by trypan blue exclusion. Data are shown for three independent samples. Inset: An Arrhenius plot of LYCH uptake: Natural log of the rate of internalization versus reciprocal temperature. B: Phase transitions of human MSCs. Two data sets are shown for a frequency versus temperature plot of the symmetric CH2 band. The first derivative plot (solid line) shows the phase transitions as the regions of greatest slope.


loading into the MSCs is trehalose. Therefore, the rest of the uptake experiments described were done with trehalose itself. Time dependence of trehalose uptake at 37°C is shown in Figure 2A. A linear relationship of trehalose uptake with incubation time (r2 5 0.96) was seen over the first 24 h, while the viability remained essentially unchanged. Control experiments measuring cellular metabolism with the fluorescent dye alamarBlue showed a small difference in the reproductive capability of control cells versus cells incubated in 100 mM trehalose for 24 h (see Fig. 2A, inset). Although the fluorescence in the two samples increased at the same rate after the third day, we note that lyophilization of these cells prior to that time will be conducted on cells in a less than optimal state of health. This will have implications for the protocols used in the drying experiments themselves. FPE is characterized by a nonsaturable increase in intracellular concentration as extracellular concentration increases. Trehalose uptake in MSCs follows this pattern, and the linear dependence of uptake on extracellular trehalose concentration is shown in Figure 2B (r2 5 0.94). The anthrone method used to quantify trehalose in these experiments was corroborated with gas chromatography, as described in Materials and Methods (data not shown), and indicated that after subtraction of endogenous glucose, the trehalose values were not obscured by breakdown products. Although the viability values remained high, even at 125 mM external trehalose, morphological differences indicating a decline in cell health (i.e., loss of normal stellate morphology) indicated that an external concentration of 100 mM trehalose was likely to be the limit for maintaining cell health and encouraging a high internal trehalose concentration simultaneously. As shown in Figure 2A and B, a 24-h incubation of the MSCs with 100 mM external trehalose at 37°C produces an internal trehalose concentration in the range of 20–30 mM. This trehalose concentration is similar to that shown to be successful for preserving human platelets during freeze-drying.7,8 As solute uptake follows the characteristic features of FPE (i.e., time- and temperature-dependent, and increasing in a nonsaturating way with external concentration), we conclude

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that FPE is most likely the mechanism of solute uptake in the MSCs. We used fluorescence microscopy to visualize this process in LYCHloaded MSCs, and the results are shown in Figure 3. At initial time points (Fig. 3A), the staining is primarily restricted to bright punctate vesicles, suggesting that the dye is localized within endosomes, whereas after longer incubation times, the LYCH staining tends to become more diffuse, suggesting the dye may be released into the cytosol (Fig. 3B). Microtubule depolymerization inhibits uptake In order to characterize more fully the endocytotic mechanism, we investigated several types of inhibitors with various effects on cellular microstructure. Colchicine and nocodazole both depolymerize microtubules, 44,45 and both inhibited trehalose uptake. ANOVA testing indicated a level of significance of p # 0.001 for inhibition by both compounds. Colchicine caused a 50% inhibition of trehalose uptake at 0.5 mM, and this effect was not increased by higher concentrations of colchicine (Fig. 4). Viability decreased only from 92% to 87% between 0 and 20 mM colchicine, so cell death could not account for the inhibition. Nocodazole had an even more dramatic effect on trehalose uptake. A strong and dose-dependent inhibition was seen between 0.5 and 5 mg/mL of nocodazole, resulting in an inhibition between 74% and 97%, respectively (Fig. 5). Again, viability remained high in this concentration range. To verify the effect of nocodazole on the microtubule organization, fluorescence micrographs were obtained of cells treated with nocodazole and stained with antibodies to tubulin. The microtubules are evident in control cells (Fig. 6A) but absent in the samples treated with 5 mg/mL of nocodazole (Fig. 6B). In these cases, morphology of the cells is abnormal, and the antibody staining of tubulin is diffuse, indicating that depolymerization of microtubules has occurred. DMSO has a different effect on the microtubule array, causing a greater number of shorter microtubules, 38 but not resulting in complete depolymerization. Although the results to be shown here are consistent with an effect on microtubules, DMSO is known to

A

B

FIG. 2. A: Dependence of trehalose uptake in MSCs on incubation time. MSCs were incubated at 37°C in the presence of MSC medium with 100 mM trehalose for 0–24 h. Inset: A comparison between MSCs incubated in 100 mM trehalose for 24 h and control MSCs. Cellular metabolism was measured with the fluorescent dye alamarBlue each day for 5 days following the incubation with trehalose. B: Dependence of trehalose uptake on extracellular trehalose concentration. MSCs were incubated for 24 h at 37°C in the presence of MSC medium with 0–125 mM trehalose. After the respective treatments, the cells were washed, trypsinized, and extracted as described in Materials and Methods. Trehalose was quantified by anthrone analysis and compared with a standard curve. Viability was measured by trypan blue exclusion. Data are shown for three independent samples.

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stock solutions of the other inhibitors were dissolved in DMSO. The highest concentration of DMSO achieved in any of the other experiments was 0.1%; thus Figure 7 shows that DMSO cannot account for the strong levels of inhibition seen with the other inhibitors. Further, DMSO was also the vehicle of delivery for cytochalasin B (described below), and no inhibition was seen in that case. Cytochalasin B and amiloride do not inhibit uptake of trehalose

FIG. 3. LYCH uptake over time in MSCs. MSCs were grown in two-well LabTek slides and treated for 3 h (A) or 24 h (B) with 10 mM LYCH in medium. The cells were then fixed, mounted, and examined by fluorescence microscopy as detailed in Materials and Methods. Note that in both micrographs, bright punctate staining of LYCH is visible, suggesting dye enclosed within endosomic vesicles. However, at the later time point (B) diffuse staining also becomes visible in the cytosol.

have other effects as well, so interpretation of these data strictly in terms of microtubules is not possible. The result of DMSO treatment during trehalose loading is shown in Figure 7. The effect is not as dramatic as is the case for nocodazole, but there was a dose-dependent decrease in trehalose uptake between 0.5% and 2% DMSO (p , 0.001), giving approximately 50% inhibition at 2% DMSO. Again, viability remained high in this concentration range, decreasing by less than 10% between the control cells and those treated with 2% DMSO. This experiment can also serve as a control for those described above, as the

Cytochalasin B depolymerizes actin filaments, 46 and cytochalasin B-treated cells contained punctate foci of fluorescently conjugated phalloidin labeling (Fig. 6D), indicating the loss of a normal actin array (Fig. 6C). However, this treatment had no effect on trehalose uptake in MSCs (Fig. 8A) (p . 0.5). This finding indicates that F-actin is not required for the endocytosis of extracellular trehalose and eliminates the possibility that macropinocytosis is the mechanism responsible for uptake. Macropinocytosis is characterized by actin-driven membrane protrusions that engulf a relatively large volume of the extracellular milieu (endosome diameter .1 mm47) and is frequently found in cells that undergo membrane ruffling in response to stimulation by growth factors. In addition, the inability of cytochalasin B to block trehalose uptake indicates that caveolae-mediated endocytosis is also not the responsible mechanism, as disruption of actin assembly has been shown to inhibit this process as well.48 The sodium channel blocker amiloride, which has been used to inhibit clathrin-independent pinocytosis,49,50 also does not cause a decrease in trehalose loading (Fig. 8B). ANOVA testing found no significant inhibition at any of the amiloride concentrations (p . 0.5). Taken together with the results described above, these data indicate that trehalose can be taken up by MSCs from the extracellular environment, probably by clathrin-dependent FPE that requires intact microtubules. DISCUSSION As a first step toward preserving human MSCs by dehydration, we have investigated

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FIG. 4. Inhibition of trehalose uptake by colchicine. MSCs were incubated at 37°C for 24 h in medium with 100 mM trehalose with the addition of 0–20 mM colchicine, following which they were washed, trypsinized, and extracted in methanol. Trehalose was quantified by anthrone analysis and compared with a standard curve. Viability was measured by trypan blue exclusion. Data shown are the means (6 standard deviation) for three independent samples.

the introduction of trehalose into the intracellular space by utilizing the cells’ own endocytotic machinery. Many adherent cell types, such as fibroblasts, endocytose a large portion of their plasma membrane and cellular volume every hour. This results in compensatory exocytosis to maintain a constant cell size.51,52 MSCs appear to be no exception to this rule, and we have found that they can be efficiently

loaded with solutes from the extracellular milieu, including LYCH and trehalose, by this mechanism. There is, of course, some variability in the measured values for trehalose uptake. This will result from the calculation, because small differences in cell size have significant effects on cell volume (as noted in Materials and Methods) and also from normal biological variability between samples. Thus, the data reported

FIG. 5. Inhibition of trehalose uptake by nocodazole. MSCs were incubated at 37°C for 24 h in medium with 100 mM trehalose with the addition of 0–5 mg/mL (0–16.6 mM) nocodazole, following which they were washed, trypsinized, and extracted in methanol. Trehalose was quantified by anthrone analysis and compared with a standard curve. Viability was measured by trypan blue exclusion. Data shown are the means (6 standard deviation) for three independent samples.


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FIG. 6. Effect of nocodazole on microtubule arrays and cytochalasin B on filamentous actin organization in MSCs. To visualize the effect of nocodazole on microtubule organization, cells were grown in LabTek slides and treated for 1 h at 37°C with 0 (A) or 5 mg/mL (B) of nocodazole. The cells were then washed, fixed, permeabilized, stained with antibodies to tubulin, and imaged by fluorescence microscopy. Note diffuse staining in the nocodazole-treated sample. To visualize the effect of cytochalasin B on actin organization, cells were grown in LabTek slides and treated for 1 h at 37°C with 0 (C) or 5 mM (D) cytochalasin B, following which they were washed, fixed, permeabilized, and stained with Texas red-X phalloidin.

are intended as estimates and not absolute values for internal trehalose concentrations. Assuming a radius for the endocytotic vesicles of ,150 nm,53 we can estimate a lower limit

to the endocytotic rate that would be necessary to deliver the measured amounts of trehalose found in the MSCs. Using the 1 h time point, we find an estimated 2 mM internal trehalose

FIG. 7. Inhibition of trehalose uptake by DMSO. MSCs were incubated at 37°C for 24 h in medium with 100 mM trehalose with the addition of 0–2% DMSO (vol/vol), following which they were washed, trypsinized, and extracted in methanol. Trehalose was quantified by anthrone analysis and compared with a standard curve. Viability was measured by trypan blue exclusion. Data shown are the means (6 standard deviation) for three independent samples.

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A

B

FIG. 8. A: Effect of cytochalasin B on trehalose uptake. MSCs were incubated at 37°C for 24 h in medium with 100 mM trehalose with the addition of 0–5 mM cytochalasin B. B: Effect of amiloride on trehalose uptake. MSCs were incubated at 37°C for 24 h in medium with 100 mM trehalose with the addition of 0–300 mM amiloride. After the respective treatments, the cells were washed, trypsinized, and extracted in methanol. Trehalose was quantified by anthrone analysis and compared with a standard curve. Viability was measured by trypan blue exclusion. Data shown are the means (6 standard deviation) for three independent samples.

concentration. To deliver this amount of trehalose in 1 h, it would take 20,000 vesicles, or an estimated uptake of 2% of the cell’s total volume. This is roughly 50% of the rate of internalization seen in fibroblasts,51 and thus is in a reasonable range, especially as this estimate does not include trehalose loss due to exocytosis. The lower limit, then, would be roughly 350 vesicles per minute in order to deliver the measured amount of trehalose. However, during that time, trehalose is being continually returned to the extracellular space through con-

comitant exocytosis. Thus, the actual value is probably significantly larger. It has been estimated that constitutive endocytosis results in the formation of 1,500–3,000 clathrin-coated vesicles per minute per cell54 in baby hamster kidney cells. Thus, the rate of endocytotic vesicle formation required to deliver the measured amount of trehalose into the MSCs is within the range seen in other cell types, and suggests that endocytosis is sufficient for this process. LYCH experiments demonstrated the uptake of solutes depended on the phase state of the

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cell membrane. This has been shown repeatedly7,28,43,55 and makes intuitive sense as well. In order for invagination and pinching-off of vesicles to occur normally, a threshold level of membrane fluidity must be reached. Thus, negligible endocytosis was seen at temperatures below 20°C, when the membrane lipids become increasingly ordered with decreasing temperature. Further, the highest levels of endocytosis were seen at 33°C, when the cell membranes have passed through the main transition at 15°C and are in the liquid crystalline state. This temperature is also in the range of the higher phase transition at ,35°C. This higher temperature transition has been linked in platelets to membrane rafts, rich in sphingomyelin and cholesterol,41,56 and may represent a similar phenomenon in the MSCs. All subsequent experiments were performed at 37°C, and showed timedependent trehalose internalization as well as a nonsaturating dependence on extracellular concentration, characteristic of FPE. The inhibitors colchicine and nocodazole, which depolymerize microtubules, and DMSO, which causes a shift to more numerous shorter microtubules, were all shown to decrease trehalose uptake. In contrast, cytochalasin B, which depolymerizes actin filaments and thus blocks both macropinocytosis and caveolae-dependent endocytosis, did not inhibit trehalose uptake. These results are consistent with clathrindependent endocytosis, because although endocytosis in yeast is actin-dependent,57 in mammalian cells actin-disrupting compounds have shown little or no effect on clathrin-coated vesicle formation.58 Further, amiloride, which blocks clathrin-independent pinocytosis, also had no effect on trehalose uptake. We were not able to work in the 1 mM range for amiloride used by some researchers for this purpose, 59 because of cellular toxicity and precipitous decrease in viability. However, Tujulin et al.35 showed clear effects on endocytosis in the range of 0–500 mM. Taken together, these data suggest that MSCs can take up solutes from the extracellular milieu by clathrin-mediated endocytosis. As it is highly unlikely that there are receptors for LYCH or trehalose on the plasma membrane of MSCs, however, the movement of these solutes into the cell is likely to be a collateral result of the constitutive clathrin-medi-

ated endocytosis responsible for the uptake of essential nutrients, which is a continuous process in all mammalian cells.60,61 It has previously been noted that coated vesicle formation can be responsible for both receptormediated endocytosis and FPE.62 Further, as recent results indicate that novel and as yet uncharacterized pathways of pinocytosis also exist,47 the present findings cannot rule out the possibility that one or more of these may also contribute to trehalose uptake in MSCs. In summary, these findings suggest that trehalose can be successfully loaded into MSCs in the range of 20–30 mM internal concentration by FPE that requires microtubules for proper functioning but not filamentous actin. This is an important first step in the effort to preserve human MSCs by dehydration for future use in the field of regenerative medicine.

ACKNOWLEDGMENTS We would like to thank Osiris Therapeutics (Baltimore, MD) for the gift of human mesenchymal stem cells. In addition, we thank Sarah Cole for excellent administrative assistance, Drs. Willem Wolkers and Nelly Tsvetkova for critical reading of the manuscript, and Dr. Jeffrey Norris for consultation regarding statistical analysis. This work was supported by DARPA grant N66001-02-C-8055.

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Address reprint requests to: Ann E. Oliver, Ph.D. Center for Biostabilization University of California Davis, CA 95616 E-mail: [email protected]