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TISSUE-SPECIFIC STEM CELLS |
aLaboratory of Experimental Immunology, Department of Medical Anatomy, Panum Institute, University of Copenhagen, Copenhagen, Denmark;
bDepartment of Ophthalmology, Third Hospital of Peking University, Peking University, Beijing, China;
cSingapore Eye Research Institute, Singapore
Key Words. Retinal precursor cells • Aqueous humor • Ascorbic acid • Proliferation • Low molecular mass protein
Correspondence: Jing Yang, M.D., Laboratory of Experimental Immunology, Department of Medical Anatomy, Panum Institute, University of Copenhagen Blegdamsvej 3C, Copenhagen DK-2200, Denmark. Telephone: 45-35327277; Fax: 45-35327269; e-mail: yangjingbell{at}hotmail.com
Received February 21, 2006;
accepted for publication August 4, 2006.
First published online in STEM CELLS EXPRESS August 10, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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One species that has been studied extensively with respect to retinal pathology is the rat, despite a number of evident dissimilarities, including the absence of a specialized fovea. Notable among the rat models of retinal degeneration is the Royal College of Surgeons (RCS) rat [1–5], although others include the light damage model [6], as well as the S334ter transgenic rat [7]. These models all exhibit photoreceptor degeneration and visual deficits that to some extent mimic the changes seen in patients with conditions such as retinitis pigmentosa. In addition, there is a rat model of optic nerve damage that to some extent resembles the changes seen in glaucoma [8]. Interventions that have shown promise in the rat include intraocular delivery of cytokines [9–11] and transplantation of various cell types, including retinal pigment epithelium (RPE) cells [12–15], Schwann cells [16], and, recently, cultured neural precursor cells [17–22].
Initial work involving the transplantation of adult hippocampal progenitor cells (AHPCs) to the rat retina showed widespread integration of donor cells after grafting to neonatal recipients but not in the mature retina of normal syngeneic adults [22]. When dystrophic RCS rats were used, however, widespread integration of AHPCs was again obtained, but only during the active phase of the degenerative process [18]. Grafted AHPCs expressed a number of neural markers normally present in the mature retina, but not rhodopsin, suggesting an inability of AHPCs to differentiate into rod photoreceptors. Although this apparent phenotypic restriction of brain-derived precursors may not be absolute [23], the need for substantial production of rod photoreceptors has stimulated the search for similar cells from within the neural retina.
In the mouse, retinal precursor cells (RPCs) have been isolated and propagated for extended periods. These cells passage continuously for over a year, express the retinal markers recoverin, rhodopsin, and cone opsin following subretinal transplantation, and are associated with rescue of host photoreceptors and improved light sensitivity compared with control animals [24]. In the rat, RPCs have also been isolated but have proven difficult to propagate in culture for extended periods [25, 26]. Although the reason for this lack of sustained proliferation of rat RPCs is not clear, it is possible that essential factors normally present during development are not supplied under the standard culture conditions used.
One way to explore the above possibility is to modify culture conditions in an attempt to better reflect the native microenvironment of the developing retina. Among the most accessible strategies of this type would be to supplement cultures with aqueous humor. In the adult eye, fluid secreted from the ciliary body not only gives rise to the aqueous humor in the anterior segment but also saturates the vitreous body and passes through the intercellular compartment of the neural retina as it leaves the posterior segment, partly through the action of cells of the RPE layer [27, 28]. Although size constraints make the composition and physiology of the extracellular fluid present in the embryonic rat retina difficult to determine, aqueous humor is readily obtainable from the eyes of large domesticated mammals. Given the importance of the rat as an experimental model of retinal pathology, the current study was directed toward exploring the potential effects of adult porcine aqueous humor on the proliferation and survival of rat RPCs in culture.
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MATERIALS AND METHODS |
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Cell Isolation and Culture
E19 embryos were harvested from timed-pregnant Sprague-Dawley rats. The eyes were dissected from the embryos with care taken to minimize the amount of adherent mesenchymal tissue. Enucleated eyes were collected in Hanks' balanced salt solution (HBSS) and then transferred to fresh HBSS in a separate culture dish. The optic nerve and remaining mesenchymal tissue were removed before isolating the retina. This approach was taken to avoid possible contamination of retinal isolates with brain-derived cells. The retina was carefully teased away from the RPE, and the central portion of the retina and the optic nerve head was removed and discarded. The isolated neural retina was collected in a dish, dissociated into small pieces, digested in 0.05% trypsin (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 5 minutes at 37°C, and then gently triturated using a 1-ml fire-polished glass Pasteur pipette to release single cells. Samples were then passed through a 40-µm filter. The resulting cell suspension was centrifuged at 300g for 6 minutes, and the pellet was resuspended in fresh serum-free culture medium composed of Dulbecco's modified Eagle's medium/Ham's F-12 medium with 1 mM glutamine (Gibco, Copenhagen, Denmark, http://www.invitrogen.com), N2 supplement (1%; Invitrogen), and 20 ng/ml epidermal growth factor (EGF) (Invitrogen). Eosin red was used to evaluate viability, and cell density was adjusted to 0.4 x 106 cells per milliliter for plating. Cultures were incubated at 37°C under 5% CO2.
Every 3 days, the medium was changed completely, and after 6–8 days in culture primary cellular aggregates were collected and incubated in cell dissociation buffer (Gibco) for 5 minutes at 37°C. These cellular aggregates characteristically displayed a roughly spherical shape, analogous to those cellular clusters elsewhere described as "neurospheres." To avoid potential overinterpretation, they are referred to here simply as "spheres." These spheres were then gently triturated using a fire-polished Pasteur pipette to release single cells, which were then plated at the same density as before and cultured in the identical manner. To establish the extent and efficiency with which the sphere-forming cells retained their ability to form secondary (first passage [P1]) spheres, their numbers were counted before plating.
Preparation of Aqueous Humor
Samples of aqueous humor were obtained from enucleated pig eyes by anterior chamber paracentesis within approximately 1 hour of death. The eyes were transported on ice and kept at 4°C until use. The samples were collected using a 25-gauge needle with great care taken to ensure that the needle did not touch the iris, lens, or corneal endothelium during collection. Samples were centrifuged for 5 minutes at 700g, and supernatants were collected. These were either used in cell culture, stored at –80°C, or fractionated by gel filtration chromatography (GFC).
Proliferation Assay
Fresh retinal isolates were plated into 96-well flat-bottomed plates at 0.4 x 106 cells per milliliter as a single-cell suspension in the experimental media of various compositions being tested. To these plates was added [3H]thymidine (1.1 Ci per well; 2 Ci/mmol), and the cells were incubated for 15 hours before [3H]thymidine incorporation was determined using a Microplate Scintillation and Luminescence Counter (PerkinElmer, Wellesley, MA, http://www.perkinelmer.com) to generate a proliferation curve for the time period from day 0 to day 4.
Morphological Analysis
Images of the primary cell isolates and subsequent cultures were captured using a Leica DM IRB microscope equipped with a Leica DC 300 camera and computer running IM 50 image analysis software (Leica, Heerbrugg, Switzerland, http://www.leica.com). Images of the central field of RPC cultures were recorded daily at a magnification of x50. Measuring and counting were performed using a Nikon 600 Imagepro cell statistics program (Nikon, Tokyo, http://www.nikon.com). Spheres were counted, and cross-sectional areas were measured. Many "spheres" were actually oblong in shape; hence, diameter frequently varied within a given sphere. For this reason, cross-sectional area was considered a more accurate assessment of sphere size than diameter. The threshold parameters for a sphere to be counted were defined as cross-sectional area greater than 800 µm2 and minimum diameter greater than 30 µm.
Statistical Analysis
Analysis of variance was performed to statistically evaluate differences in the proliferation of cells grown in media of different compositions.
Flow Cytometry
Either primary or cultured rat RPCs were pelleted by centrifugation (500g; 5 minutes; room temperature) and resuspended as a single-cell suspension of approximately 106 cells in 100 µl of phosphate-buffered saline (PBS) (without serum) using a fire-polished glass Pasteur pipette. Cell membranes were permeabilized by saponin for 15 minutes prior to labeling, followed by a PBS wash. Cells were then incubated with 1 µg of anti-nestin primary antibody, or isotype control, for 30 minutes on ice. Unbound antibody was removed with two washes in PBS of 5 minutes each. Cells were then incubated with 1 µg/106 cells of the secondary antibody PE for 30 minutes on ice. Washes were again performed as described previously. Cells were then fixed in 1% paraformaldehyde. Automated analysis and sorting were performed using a FACS Caliber (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).
Gel Filtration Chromatography
These experiments used an AKTA Explorer system (Amersham Biosciences, Hillerod, Denmark, http://www.amershamhealth.com). Separations were performed using a Superose 12HR 10/30 gel filtration column (Amersham Biosciences) with optimal separation range between 1,000 and 300,000 Da. Potassium phosphate buffer (50 nM, pH 7.4), containing 100 mM KCl and 0.6 mM sodium azide, was used in all separations with a constant flow rate of 0.5 ml/minute. This buffer was also used for reconstitution and dilution of protein standards. The volume of aqueous humor applied to the column was 250 µl. A gel filtration calibration kit for molecular mass proteins was purchased from Sigma and included blue dextran (2,000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), ß-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome C (12.4 kDa), and cytidin (243 Da). The elution volume of each standard protein was determined, and the logarithms of their molecular masses were plotted against elution time. This calibration curve was used to determine the approximate molecular mass of the protein peaks seen in the chromatograms.
Evaluation of the Proliferative Activity of Aqueous Fractions
Primary rat retinal cells in a volume of 160 µl and at a density of 0.15 x 106 cells per milliliter were plated into individual wells of flat-bottomed 96-well plates containing 40 µl (20% by volume) of a specific aqueous humor fraction, previously separated from fresh aqueous humor by GFC. Three wells were used for each fraction. In addition, control wells containing 160 µl of cells and 40 µl (20%) of either unfractionated aqueous humor or PBS were also plated at this time. Cellular proliferation was assayed by [3H]thymidine incorporation on day 2, as described previously.
Spectrophotometric Analysis of Aqueous Humor
To examine the molecular composition of aqueous humor, specifically with respect to the presence of ascorbic acid, UV absorption spectra were obtained on three separate samples, and the data were compared by using a UV spectrophotometer (Beckman DU 650 spectrophotometer; Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). These included unfractionated porcine aqueous humor (fresh, diluted 10-fold in PBS), the maximum aqueous fraction exhibiting peak absorbance for UV light of 280 nm (corresponding to an elution volume of 
18 ml), as well as pure ascorbic acid (Merck & Co., Whitehouse Station, NJ, http://www.merck.com; as a 0.1 mg/ml solution in PBS). The length of the light path in the quartz cells used was 1 cm.
Evaluation of the Proliferative Effect of Ascorbic Acid on Rat RPCs
Primary rat retinal cells in a volume of 160 µl of standard EGF-containing growth media were plated into individual wells of flat-bottomed 96-well plates at a density of 0.24 x 106 cells per milliliter. To each well was also added 40 µl (20% by volume) of either ascorbic acid (0.1 mg/ml in PBS), porcine aqueous humor, or additional standard EGF-containing growth media. Three wells were used for each sample type. Cellular proliferation was assayed by [3H]thymidine incorporation from day 1 to day 3, as described previously. In an additional experiment, cells were plated as before and the proliferative effects of ascorbic acid were assayed, in this case over a range of concentrations (each 20% supplement contained from 0.03125 to 2 mg/ml ascorbic acid in 40 µl of PBS; hence, the total concentration in each well varied from 0.00625 to 0.4 mg/ml).
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RESULTS |
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Aqueous Humor Enhances Proliferation of Rat RPCs in Culture
Image analysis of rat E19 retinal precursors cultured with and without 20% aqueous supplementation showed evidence of increased sphere formation in the aqueous-supplemented medium (Fig. 1). Beginning on day 0, and continuing to day 4, it was apparent that more spheres reached threshold criteria in the aqueous-supplemented medium at each time point and that these spheres were larger in size. This impression was confirmed by quantitative analysis, with both number (Fig. 2A) and size (Fig. 2B) of spheres being significantly higher in the aqueous-supplemented cultures, as compared with growth factor-containing medium alone.
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Having obtained morphological evidence suggesting that 20% aqueous supplementation promotes the proliferation of rat RPCs beyond the level seen with growth factor-containing medium alone, we sought to re-examine this finding using a quantitative measure of cellular proliferation. [3H]Thymidine incorporation assays confirmed increasing proliferation of E19 rat retinal precursor cells in medium containing the growth factor EGF up to days 2–3 in culture, followed by a subsequent decline (Fig. 3). Twenty percent aqueous supplementation resulted in significantly higher proliferation at all time points; however, the overall growth pattern again showed slowing of proliferation from days 3 to 4 (Fig. 3). Controls in which EGF-containing growth medium was supplemented with PBS showed slightly less benefit than growth medium alone, perhaps resulting from a dilutional effect on EGF concentration (Fig. 3A). As expected, the use of medium lacking the mitogenic growth factor EGF resulted in an immediate decline in proliferative activity, dropping to undetectable by day 3 (Fig. 3A). Supplementation of mitogen-free medium with 20% aqueous humor helped to sustain proliferative activity, albeit at levels less than that seen when aqueous was combined with EGF (Fig. 3A). Culturing rat RPCs in 100% aqueous humor resulted in an immediate decline in proliferative activity, dropping to negligible levels by day 3 (Fig. 3B).
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Comparison of Activity in Fresh and Freeze-Thawed Aqueous Samples
The protein content of freeze-thawed aqueous humor could potentially be altered through the presence of endogenous proteolytic activity. To rule out this possibility, proliferation results for rat RPCs obtained using fresh aqueous humor were compared with those obtained using previously frozen aqueous humor (stored at –80°C for less than 1 month). No difference was found between these conditions, whereas a decrease in stimulation by frozen-thawed aqueous humor would be expected if proteolytic activity were present in these samples.
Stability of Marker Expression with Exposure to Aqueous Humor
Flow cytometry was used to evaluate the expression of phenotypic markers. The percentage of cells positive for the primitive neuroepithelial marker nestin was 23% in primary E19 retinal isolates (Fig. 4B). Markers associated with more mature retinal cell types were not evident. In particular, the RGC-associated marker Thy-1 (CD90), the astroglial marker GFAP, the bipolar marker PKC
, and the amacrine marker syntaxin were not detected using this method (data not shown). After 4 days in culture in the presence of 20% aqueous supplementation, the percentage of nestin-expressing cells was again 23% (Fig. 4D), and the other markers were again not detected. Taken together, these data are most consistent with stable neurodevelopmental marker expression and give no indications that aqueous humor induces RPCs to differentiate, although this latter possibility cannot be ruled out.
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Of note, in the experiments shown in Figure 7A and 7B, cells were plated at lower densities than is optimal for proliferation (0.24 x 106 cells per milliliter, as opposed to 0.4 x 106 cells per milliliter) in an effort to better delineate any proproliferative activity present. This accounts for the progressive decline in proliferative activity over time observed in all samples, versus that seen when cells were cultures at higher density (Fig. 3).
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DISCUSSION |
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Aqueous humor has been reported to stimulate the proliferation of a variety of cell types in culture, including those derived from the Tenon capsule, lens epithelium, corneal endothelium, and iris epithelium [29–34]. Other published reports indicate that aqueous humor contains many growth inhibitory factors [35–38]. In those studies, cow [36], rabbit [29, 30, 39], human [29, 30, 37, 39], and monkey [38] aqueous samples were tested for growth effects on cultured cells or explants from various tissue sources. In general, the collective observations of these studies indicate that in the presence of high concentrations of aqueous humor, there is significantly less growth than in parallel aqueous-free control cultures. Since the opposite conclusion was drawn from our experiments, it is important to consider whether different concentrations of aqueous humor have different affects on the cells' proliferation.
In the present study, supplementation of proliferation medium with 20% aqueous humor induced a statistically significant increase in thymidine uptake by rat RPCs over that seen with standard proliferation medium alone. Furthermore, 10% and 50% aqueous humor also appeared to promote proliferation. Aqueous humor appeared to support, to some extent, the proliferation of rat RPCs in the absence of any added growth factors, although proliferation and survival were poor under these conditions. In contrast, growth in 100% aqueous humor led to decreased proliferation of rat RPCs as compared with using proliferation medium alone. Thus aqueous humor cannot entirely substitute for proliferation medium; however, the combination of aqueous and proliferation medium appears to be superior to proliferation medium alone. This suggests that aqueous humor contains a proproliferative activity that acts synergistically with EGF-containing proliferation medium.
The aqueous humor supplies nutrients to avascular structures in the anterior eye, including the cornea, lens, and trabecular meshwork, as well as contributing to the bulk flow of fluid through the vitreous compartment. In terms of composition, albumin (0.596 µg/µl) is the major protein component in human aqueous humor [40]. High molecular mass proteins are present at low levels in aqueous fluid, at least in part because of a molecular sieve effect related to the blood-aqueous barrier, corresponding to a theoretical pore size of 104 Å [41]. Proteins that are present include IgA (0.011 µg/µl), IgG (0.011 µg/µl or less) [42, 43], and lysozyme (0.064 µg/µl) but not IgM. Other compounds include myocilin, transferrin, basic fibroblast growth factor (bFGF), cystatin, superoxide dimutase, glutathione peroxidase, glutathione reductase, lactic acid, latent collagenase, tumor necrosis factor-ß, -tocopherol, glutathione, and ascorbic acid.
Particularly well studied is the composition of primate aqueous humor compared with serum [44]. In aqueous humor, the concentrations of phosphates, calcium, urea nitrogen, creatinine, glucose, cholesterol, glutamic-pyruvic transaminase, glutamic-oxaloacetic transminase, lactic dehydrogenase, creatinine phosphokinase, and alkaline phosphatase were lower than in serum. Conversely, aqueous humor contained higher concentrations of chloride, bicarbonate, uric acid, lactate, and ascorbic acid than did serum. The concentrations of sodium, potassium, and magnesium were nearly equal in the two fluids. Despite a several-hundred-fold lower protein concentration, aqueous humor osmolality was equal to serum osmolality. Aqueous humor was deficient in a number of amino acids compared with serum. Two amino acids—cysteine and valine—were of equal concentration in both fluids. Serine, methionine, leucine, phenylalanine, and tyrosine appeared to be more concentrated in the pooled aqueous humor than in the corresponding serum.
Aqueous composition has also been evaluated for the presence of immunomodulatory molecules. Various ocular microenvironments have long been recognized as sites of relative immune privilege, and immunosuppressive neuropeptides found in aqueous humor are central to this immunoregulation [45]. These neuropeptides include -melanocyte-stimulating hormone, vasoactive intestinal peptide, calcitonin gene-related peptide, and somatostatin. Along with transforming growth factor-ß2, the neuropeptides target specific cells and pathways in innate and adaptive immunity. These aqueous humor factors prevent pathogen-induced inflammation and activation of Th1 cells, while promoting induction of regulatory T-cells. Therefore, the ocular microenvironment, through the constitutive production of immunosuppressive factors found in aqueous humor, maintains immune privilege by manipulating regional innate and adaptive immunity away from inflammatory responses. These factors could also potentially influence the proliferation of stem or precursor cells.
The separated polypeptide components of aqueous humor appear to have differential effects on the proliferation of RPCs. Although aqueous fractions did not generally stimulate as well as unfractionated aqueous, this could relate to dilutional effects resulting from the fractionation process. With regard to potential species-related effects, in this study porcine aqueous humor was used to stimulate the proliferation of rat RPCs. Although these two species differ greatly from one another, many previous studies have indicated that cytokines frequently produce effects across species lines. For instance, recombinant human growth factors can be used to culture rodent CNS progenitor cells. Indeed, the porcine aqueous used here was seen to have a growth-promoting effect on the rat cells in this study. This is not to say that porcine aqueous humor might not have an even greater proliferative effect on porcine RPCs, although this possibility would need to be examined.
Ascorbic acid, as opposed to peptides, exhibits no such species differences. This molecule, widely recognized for its role as an antioxidant, is not routinely included in standard proliferation protocols used for culturing progenitor cells from the developing central nervous system. Although the role of ascorbic acid in the eye might seem reasonable based on the concentration of this molecule in intraocular fluid, there is evidence that it can also play a role in the ontogenetic status of precursor cells in other systems, notably, enhancing the differentiation of a variety of more primitive cells into osteoclasts [46] and osteoblasts [47, 48]. Although the influence of ascorbic acid on osteogenesis would appear to be opposite to that seen in the present study, it is interesting to note that elsewhere in the nervous system the redox state has been proposed as an important determinant of ontogenetic status in mitotic glial precursor cells, with less oxidized states favoring self-renewal [49]. Nevertheless, it has been reported that ascorbic acid does not enhance the proliferation of rat cortical precursor cells but instead promotes their differentiation [50]. Whether this disparity in results as compared with the present study reflects a biological difference between retinal and forebrain precursors as opposed to specific culture conditions remains to be elucidated.
Conclusions
Supplementation of EGF-containing growth medium with 20% aqueous humor clearly increased the proliferation of rat RPCs in culture. In addition, it appears that EGF was not responsible for the proproliferative activity of aqueous. First, aqueous supplementation somewhat augmented proliferation in the absence of EGF but not at similar levels. In addition, the activity of aqueous fractions did not suggest a prominent benefit at 6 kDa, which would be consistent with EGF. These same data did not support a role for bFGF either. Thus, the proproliferative activity of EGF, present in the growth medium, and the activity present in aqueous appear to result from different molecules and act synergistically to promote proliferation of rat RPCs.
It will be of considerable interest to know whether EGF- and aqueous-/ascorbic acid-based activities are mediated through different pathways and whether this finding can be extended to RPCs of different species. This is of considerable potential interest in the setting of human RPCs, which exhibit limited ability to passage in culture [51]. In the case of rat cells, the synergy achieved through the use of aqueous supplementation was insufficient to sustain the long-term viability of the cells in culture. Although extended proliferation of rat retinal cultures has been reported following supplementation with FCS [26], a hazard of this approach is that it is known to alter cell fate decisions [52]. Of equal importance is the question of whether exposure of RPCs to aqueous and/or ascorbic acid influences the maturation or fate decisions of the cells when cultured over longer time periods than were possible under current experimental conditions.
It will also be of interest to determine whether vitreous liquid exerts a proliferative effect on precursor cells as well and, if so, whether such an effect can be attributed to the same constituents as those present in aqueous humor. Studies of the relationship between progenitor cells and intraocular fluids should lead to a better understanding of ocular development and assist in the development of novel therapeutic modalities based on cell transplantation. Furthermore, the demonstration of potentially novel regulatory factors in the fluid component of the ocular microenvironment may stimulate the search for analogous soluble factors in other stem cell-containing compartments of the body.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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REFERENCES |
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