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TRANSLATIONAL AND CLINICAL RESEARCH |
aCell Engineering Laboratory,
cPlastic and Reconstructive Surgery Department, and
dPathology Department, La Paz University Hospital, Madrid, Spain;
bVissum Ophthalmological Institute of Alicante and Miguel Hernandez University, Alicante, Spain
Key Words. Cornea • Cell therapy • Adipose-derived stem cells • Mesenchymal stem cells • Transplant • Keratocan Stem cell transplantation • Keratocyte
Correspondence: Correspondence: Maria P. De Miguel, Ph.D., Cell Engineering Laboratory, La Paz University Hospital, Maternity Building, Paseo Castellana 261, Madrid 28046. Spain. Telephone: 34-91-2071458; Fax: 34-91-7277050; e-mail: maria.demiguel{at}uam.es
Received on August 7, 2007;
accepted for publication on November 27, 2007.
First published online in STEM CELLS EXPRESS December 6, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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To overcome the current disadvantages of corneal transplantation, particularly primary immune rejection and a shortage of corneas [3, 4], cell-based therapy of the cornea is a promising therapeutic approach [5]. Human corneal epithelial stem cells [6] and human corneal endothelial cells have been successfully cultured [7] and transplanted into animal and human hosts [8–14]. Recently, nonocular cells have also been used to reconstruct corneal epithelium, mainly using autologous oral mucosal epithelium [15–17].
However, most corneal diseases primarily or secondarily involve the corneal stroma, which accounts for 90% of the corneal thickness, and include immune, infectious, and ecstatic diseases; corneal dystrophies; and corneal failure [18]. Laser refractive surgery complications preferentially affect the stroma as well. Our purpose is to search for a cell source to repopulate and restore corneal stroma.
Keratocytes, which are mesenchymal-derived cells, are the principal cells of the corneal stroma. In adult tissue, keratocytes are mitotically quiescent, with a flat, dendritic morphology, and are positive for CD34 and aldehyde-3-dehydrogenase (ALDH) [19]. They secrete collagens and keratan sulfate proteoglycans, such as keratocan [20], which is used as a keratocyte-specific marker [21]. The unusual proteoglycan composition of the corneal stroma is essential for corneal transparency [22]. During corneal wound healing, keratocytes are activated and transform into fibroblasts and/or myofibroblasts, losing their dendritic morphology [23, 24] and resulting in a reduction in ALDH [25], keratan sulfate [26], and corneal transparency.
Tissue engineering of functional corneal equivalents has been developed. Keratocytes are embedded into a scaffold that can support cell growth [27–29]. However, there are well-known drawbacks to the use of scaffold-based designs. Strong inflammatory responses are induced upon their biodegradation, and nearly all polymer materials cause a nonspecific inflammatory response [30]. Furthermore, it is often observed that on the scaffolds, peripheral cells are generally healthy and closely resemble native tissues, whereas central cells form a necrotic core, often resulting in unsatisfactory tissue regeneration. Cell-based therapies using single-cell suspensions could be a better option.
Ex vivo expansion of keratocytes is possible [29, 31–33], but obtaining high numbers by subculturing has been difficult. In addition, keratocytes are neither numerous nor easy to obtain, and autologous extraction is not feasible without damaging the donor cornea.
Bone marrow mesenchymal stem cells (MSC) have been shown to have multilineage potential to differentiate into adipogenic, chondrogenic, myogenic, and osteogenic cells [34], all of which are mesoderm-derived lineages, as is the corneal stroma. MSC are also present in periosteum, muscle, synovial membrane, and adipose tissue [35]. The most important features of adipose tissue as a stem cell source are the relative expendability of this tissue and the ease with which processed lipoaspirate derived (PLA) cells with MSC differentiation potential can be obtained in large quantities with minimal risk [36–38]. We investigated the ability of human PLA cells to be delivered to and to repair/regenerate the corneal stroma of experimental animals.
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MATERIALS AND METHODS |
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In Vitro and In Vivo Collagen Detection by Immunofluorescence
First to fourth passage cultured PLA cells were cultured on slides and examined for collagen expression by immunofluorescence. Also, rabbit mock- and PLA-transplanted corneas (described below) were examined for collagen expression in the same way. The expression of collagens type I, III, IV, and VI was investigated. Primary antibodies mouse anti-collagen I (Sigma-Aldrich), mouse anti-collagen III (Abcam, Cambridge, U.K., http://www.abcam.com), mouse anti-collagen IV (Sigma-Aldrich), and rabbit anti-collagen VI (Abcam) were used at 1:100; 1:500, 1:20, and 1:50 dilutions, respectively. Fluorescein isothiocyanate-conjugated (Sigma-Aldrich) or biotin-conjugated (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) anti-mouse secondary antibodies were used at a 1:100 dilution in collagen I, III and IV cultures, whereas rhodamine-conjugated (Boehringer Ingelheim GmbH, Ingelheim, Germany, http://www.boehringer-ingelheim.com) or biotin-conjugated (Vector Laboratories) anti-rabbit secondary antibodies were used at 1:50 and 1:100 dilutions, respectively, in the collagen VI slides. When secondary anti-mouse or anti-rabbit biotinylated antibodies were used, incubation with avidin-aminomethylcoumarin-3-acetic acid (Vector Laboratories) at a 1:100 dilution was performed to detect expression by fluorescence microscopy. The cultures and paraffin sections were then mounted with Vectashield (Vector Laboratories) and examined with fluorescence optical microscopy. Primary antibody-omitted cultures were used as negative controls.
Induction of Chondrogenic and Osteogenic Differentiation
Chondrogenic Induction. Cells were plated using the micromass culturing technique [39]. In brief, a 10-µl culture medium drop containing 8 x 106 cells per milliliter of suspension was plated in normal medium. Five hours later, the culture medium was replaced by a chondrogenic differentiation culture medium [34]: DMEM, 1x insulin-transferrin-selenium (Sigma-Aldrich), 0.1 µM dexamethasone (Merck, Darmstadt, Germany, http://www.merck.com), and 50 µg/ml 2-phosphate ascorbic acid (Fluka, Ronkonkoma, NY, http://www.sigmaaldrich.com). Medium changes were done 3 days a week for 4 weeks. Chondrogenic differentiation was confirmed using Alcian Blue staining at acidic pH to show production of sulfate proteoglycans [40].
Osteogenic Induction. Cells were plated at 2 x 104 cells per cm2 and cultured in normal medium for 24 hours. Afterward, the medium was changed to an osteogenic induction medium [34]: DMEM, 10% FBS, 0.1 µM dexamethasone (Merck), and 50 µg/ml 2-phosphate ascorbic acid (Fluka).
The medium was changed 3 days a week for 2 weeks. Osteogenic differentiation was confirmed by alkaline phosphatase activity detection using 1 mg/ml Fast Red-TR (Sigma-Aldrich) and 0.04% Naphthol AS-MX (Sigma-Aldrich).
Cell Labeling and Harvest
Cultured PLA cells at passage 2 were incubated with a 1:200 dilution of dialkylcarbocyanine fluorescent solution Vybrant chloromethylbenzamido (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) in D-phosphate-buffered saline (D-PBS) for 4, 8, 16 or 20 minutes and then rinsed and cultured in the medium until harvest. In this way, all intracytoplasmic membranes were fluorescently labeled, and transplanted cells could be easily identified under fluorescence optics (described below). Labeled PLA cells were trypsinized and resuspended in Hanks' balanced salt solution (HBSS; Gibco-BRL) half an hour before the transplant.
Induction of Quiescence and Fluorescence-Activated Cell Sorting Analysis
Quiescence was induced by using fasting medium (DMEM with 0.5% FBS). After 20, 40, 60, and 80 hours, cells were trypsinized and centrifuged, and the remaining pellet was resuspended in 0.1% glucose (BDH Chemicals Ltd., Poole, U.K., http://www.merck.de) in PBS. A solution of 69 µM propidium iodide (Sigma-Aldrich) in 38 mM sodium citrate at pH 7.4 and 10 mg/ml RNase (Molecular Probes) was added to the cells and incubated for 45 minutes at 37°C. Labeled cells were detected by fluorescence-activated cell sorting (FACS), and the analysis was done using the CellQuest Pro program (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).
Experimental Model 1: Eye Surgery and Cell Transplant of Intact Corneas for Biosafety and Immune Rejection Studies
Animal studies were performed in accordance with guidelines set forth by the animal research committees at Vissum Ophthalmological Institute of Alicante, Spain, and La Paz Hospital, Spain. To evaluate the biosafety and possible immune rejection of human cells transplanted into rabbit's cornea, a first set of experiments was performed using seven adult New Zealand White rabbits (Granja San Bernardo Tulebros, Navarra, Spain). The animals were anesthetized with a combination of intramuscular ketamine (25 mg/kg) and medetomidine (0.5 mg/kg). An intrastromal corneal pocket of 6 mm in diameter was created by femtosecond laser technology at the central cornea to allow space for the cell suspension fluid. In vivo survival of PLA cells in the corneal stroma was assessed.
The rabbits were placed under an IntraLase femtosecond 30 KHz laser [41] (IntraLase Corp., Irvine, CA, http://www.intralase.com). Each eye was fixed by a suction unit, and the laser pulses were guided by a computer-controlled scanner toward the treated eye. When the laser beam was focused at the desired corneal depth (120 µm), laser-induced optical breakdown occurred at low energy, vaporizing small volumes of corneal stroma without creating thermal or shockwave damage to the surrounding tissue, creating a horizontal cleavage plane along the corneal stroma, with an accuracy of ±4 µm in depth, without compromising the cornea above the horizontal plane. The laser began at the hinge (nasally) and progressed temporally. When the deep resection plane was completed, the laser then cut vertically to the cornea, at the margins of the horizontal plane, to make a complete flap.
Under x10 magnification, a Sinskey hook (Katena Products Inc., Denville, NJ, http://www.katena.com) was used to enter the laser wound in the upper quadrant near the hinge, creating an opening as small as possible. Since the femtosecond laser does not produce a loose flap separation, an AlióRodríguez spatula (Katena Products) was used to break down the microadhesions, moving back and forth gently until it dissected the interface 5 mm, keeping out of the vertical cut edge, except in the initial opening where the dissection begun. In this way, an intrastromal pocket with minimal opening to avoid leakage was created.
A 100-µl Hamilton microsyringe with a 22-gauge Hamilton needle (Fisher Hamilton, Pittsburgh, http://www.fishersci.com) was used to inject a total of 3 x 105 cells (10 µl of HBSS) of PLA cells into the stromal pocket of the right eye. The same procedure was repeated in the left eye, injecting only HBSS, to be used as a mock-injected negative control. The main outcome measure was the presence of viable human adipose-derived cells at the rabbit corneal stroma interface during follow-up.
Experimental Model 2: Eye Surgery and Cell Transplant of Damaged Corneas for Regeneration Studies
To demonstrate the regeneration of corneal stroma by transplanted PLA cells, a second set of experiments in which corneal damage was inflicted to six adult New Zealand White rabbits (Granja San Bernardo) was performed. Rabbits were anesthetized as described above. Rabbits were placed under the operating microscope of a Schwind Esiris excimer laser (Schwind, Frankfurt, Germany, http://www.eye-tech-solutions.com). A Moria M2 suction ring was placed on the eye, and suction was applied. A Moria M2 microkeratome (Moria, Antoni, France, http://www.moria-surgical.com) was used to create a flap at an estimated depth of 120–140 µm. The flap was retracted, an ablation of 50 µm in thickness of the corneal stroma was then completed in a routine manner (as in laser in situ keratomileusis [LASIK]), and the flap was floated into its primary position. To avoid flap displacement and to maintain a relatively watertight intrastromal space, three 10/0 nylon sutures were fixed in the flap borders. A total of 3 x 105 PLA cells in HBSS were delivered to the stromal pocket of one randomly selected eye with a 100-µl Hamilton microsyringe with a 22-gauge Hamilton needle (Fisher Hamilton). As a negative control, the contralateral eye was treated using the same procedure, but injecting HBSS alone. PLA cells were previously labeled with CM-DiI as described above. Twelve weeks after the surgery, rabbits were euthanized, and the corneas were embedded in paraffin and observed under fluorescence microscopy or frozen in liquid nitrogen for mRNA analysis.
Clinical Observation
Each treated eye was examined with a portable slit lamp 1 week after surgery and then every 2 or 3 weeks before sacrifice, to look for corneal inflammation, opacities, or any other ocular surface or anterior chamber complication. Haze was evaluated by an external expert ophthalmologist on a masked basis [42], on a scale of 0–4, according to severity of the haze: 0, total transparency; 1, mild haze; 2, moderate haze; 3, severe haze; 4, very severe haze making it impossible to observe the eye's internal structures.
Tissue Procurement
For the biosafety and rejection experiment, three rabbits were euthanized after 3 weeks, and the remaining four rabbits were euthanized 10 weeks after cell transplant. For the corneal regeneration experiments, one rabbit was euthanized at 8 weeks and the remaining rabbits at 12 weeks after surgery. Sacrifice was performed by i.v. administration of T-61 euthanasia solution, a combination of embutramide, mebezonium iodide, and tetracaine hydrochloride. The eyes were enucleated, formalin-fixed, and paraffin-embedded or snap-frozen in liquid nitrogen for reverse transcription (RT)-polymerase chain reaction (PCR) analysis.
Histological Examination and Localization of PLA Cells in the Stroma
Several sections of each cornea were stained with hematoxylin and eosin for light microscopy examination. DAPI stain (2 µg/ml; Sigma-Aldrich) was used to label all cells' nuclei in the corneas in the biosafety experiment. Localization of CM-DiI-labeled human PLA cells was achieved using an epifluorescence microscope filtered for excitation/emission at 546/590 nm.
ALDH and Keratocan Immunohistochemistry
To demonstrate differentiation into functional keratocytes, human ALDH and stromal cornea-specific proteoglycan human keratocan were detected by immunohistochemistry on PLA cultures and in paraffin sections. Rabbit anti-human ALDH (Abcam) was incubated at 1:100 overnight. Goat anti-human keratocan (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) was incubated at 1:25 overnight. Secondary antibodies anti-rabbit or anti-goat conjugated with biotin (Vector Laboratories) were respectively incubated at 1:100 for 30 minutes, and then slides were incubated for 30 minutes with avidin-AMCA (Vector Laboratories), which fluoresces in blue, so CM-DiI-labeled PLA cells could be identified in red in the same sections. Expression of human ALDH and keratocan was confirmed by fluorescence microscopy. Contralateral mock-injected eye sections were used as negative controls. Sections of a human cornea from an autopsy performed at La Paz Hospital served as positive controls.
RT-PCR
To confirm the differentiation of human PLA cells into keratocytes, expression of human keratocan was detected by RT-PCR and sequencing. Total RNA was isolated by standard methods using RNA-bee (Tel-Test, Friendswood, TX, http://www.tel-test.com) from both injected and noninjected rabbit corneas. First-strand cDNA was obtained by RT reaction using the Superscript first-strand system for RT-PCR kit (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and random hexamers. Human keratocan was amplified using the following primers, designed to amplify part of exons 2 and 3 of human keratocan and thus amplify only cDNA sequences: hKERf, 5'-GCCTCCAAGATTACCAGCCAA-3'; and hKERr, 5'-ACGGAGGTAGCGAAGATGAGGT-3' [43]. For control of amplification of a housekeeping gene, β-actin amplification was performed using the primers hBactinF (5'-AACCGCGAGAAGATCACCCAGATCATGTTT-3') and hBactinR (5'-AGCAGCCGTCATCTCTTGCTCGAAGTC-3'). PCR conditions were 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. For negative controls, reactions with no cDNA, cDNA from noninjected rabbit cornea, and DNA from human and rabbit tissues were used. For positive control, cDNA from a human cornea donated after cornea transplant at Vissum Ophthalmological Institute was used. The 358-base pair (bp) amplified fragments of human keratocan or 350 bp of human β-actin were separated on a 1% agarose gel.
To confirm the specificity of amplification, PCR products were extracted from the agarose gel using the QIAquick gel extraction kit (Qiagen, Valencia, CA, http://www1.qiagen.com) and sequenced by the big dye method and the automatic sequencer ABI Prism 377 (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). The sequences obtained were compared with the human and rabbit keratocan sequences from GenBank.
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RESULTS |
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Differentiation of PLA Cells into Chondrogenic and Osteogenic Lineages
To ensure that we were using PLA cells capable of multipotent lineage differentiation, we differentiated our harvested cells toward the chondrogenic (Fig. 1A, 1B) and osteogenic (Fig. 1C) lineages, confirming their differentiation potential.
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Induction of Quiescence
As keratocytes in the corneal stroma are quiescent, PLA-cultured cells were serum-deprived to promote quiescence and therefore theoretically improve adherence before transplantation. After 60 hours or more of serum deprivation, approximately 90% of cells were withheld at the G1 phase of the cell cycle as confirmed by propidium iodide labeling and FACS analysis (not shown), and thus the cells subsequently used for cell transplant were serum-starved for at least 60 hours.
Biosafety and Immune Rejection Experiment
Surgery and Clinical Observation. All seven animals tolerated the procedure without complications. In the 1st week of follow-up, one rabbit's right eye and two rabbits' left eyes had a partial lateral flap displacement in the stromal bed, so that there was an area with preserved stromal interface and an area where the interface was cover by epithelium. This displacement led to stromal haze of the flap and the interface as a complication of the technique. No corneal opacity or haze was observed in the rest of the rabbits' corneas with direct illumination at any point in time after the transplant (Fig. 2A, 2B).
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Histological Analysis of Transplanted Corneas. No histological differences were found between control and transplanted corneas. No inflammatory reaction was observed in the interface, and no immune rejection developed even though it was a xenotransplant and the rabbits were not immunosuppressed (Fig. 2C, 2D).
Human PLA cells were identified in the flap interface of the host tissue, as verified by localization of fluorescent CM-DiI labeling. Three weeks after injection, PLA cells were visible exclusively at the flap-stromal interface, in a fashion similar to that of corneal keratocytes, scattered in the interface between collagen lamella, and they displayed a flat elongated morphology (Fig. 3A–3C).
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As mentioned above, three eyes showed lateral flap displacement the day after surgery, one PLA-injected eye and two mock-injected eyes. Early flap displacement resolved later with vascularization over the flap bed, as seen macroscopically and in histological sections (not shown). However, PLA-stained cells were still visible in the interface of the PLA-injected cornea on histological sections (not shown).
Corneal Regeneration Experiment
Surgery and Clinical Observation. Surgery was successful for the six rabbits. During surgery, rabbit 4 suffered a buttonhole flap on its PLA-injected eye, so corneal ablation was peripheral instead of centered. Animals were checked at 2, 5, 8, and 12 weeks after surgery. At the 8-week check, rabbit 3 showed a very severe haze in the mock-injected eye and mild haze in the PLA-injected eye; therefore, it was euthanized, and the corneas were used for RT-PCR analysis. The rest of the animals were euthanized at 12 weeks. At that time, two rabbits showed moderate haze in the PLA-injected eye, and one rabbit showed moderate haze in both eyes. The rest of the animals showed complete corneal transparency in both eyes (Table 1). Thus, no significant differences in haze were seen in mock- versus PLA-injected corneas.
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Histological Analysis of Transplanted Damaged Corneas. As in the case of nonablated corneas, no differences were observed in the stromal pattern of the mock- versus PLA-injected corneas (not shown). Human PLA cells were also identified in the flap interface of the host tissue, as verified by localization of fluorescent CM-DiI labeling, and displayed a multilayered pattern more frequently than monolayered (not shown).
Collagen Production by Transplanted PLA Cells. To characterize collagen production by the PLA cells after transplant, immunohistochemistry for collagens I, III, IV, and VI was performed in transplanted ablated corneas. As expected, collagens type I and type VI were expressed in the rabbits' corneal stroma and were also detected in transplanted PLA cells (Fig. 4A–4C, 4J–4L). Collagens III and IV, not expressed normally in corneal stroma, were detected neither in the transplanted corneas (Fig. 4D–4F) nor in transplanted human PLA cells (Fig. 4G–4I).
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ALDH and Keratocan Production by Transplanted PLA Cells. To confirm differentiation into functional keratocytes, the expression of the keratocyte marker ALDH and the corneal stroma-specific proteoglycan keratocan by PLA cells was studied in paraffin sections 12 weeks after surgery. Using immunofluorescence, ALDH was found to be slightly positive in keratocytes of mock-injected rabbit corneas (reflecting a cross-reaction of the anti-human ALDH antibody with rabbit ALDH) (Fig. 5A) and strongly positive in human corneal sections (Fig. 5B). In the injected rabbit cornea, ALDH expression was also strongly detected in some of the CM-DiI-labeled human cells (Fig. 5C, 5D).
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To confirm that the keratocan expressed in transplanted rabbit corneas was in fact human, RT-PCR analysis was performed. Human keratocan was amplified using primers that span two exons to ensure that the detected keratocan was actually from mRNA. Confirming the immunofluorescence results, human keratocan was amplified from injected rabbit cornea cDNA and the donated human cornea cDNA exclusively (Fig. 6). The 358-bp band was extracted from the gel, sequenced, and compared with the published human and rabbit keratocan sequences, confirming that it corresponded exactly to the human sequence (not shown).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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In this study, we found that human PLA-derived mesenchymal stem cells can be procured easily and transplanted into the corneal stroma of rabbits. Femtosecond corneal laser technology allows for delivery of the cell suspension at the exact desired depth of the corneal stroma.
When cultured, PLA cells spontaneously express collagens found in corneas, such as collagens type I and VI, consistent with a recent study [48] that showed expression of collagens I, VI, and XIV by these cells. When PLA cells are transplanted into the corneal stroma, they acquire a morphology similar to that of keratocytes, with a dendritic shape, and arrange scattered within the collagen lamella. Moreover, they not only resemble keratocytes but also behave as them, as they produce collagens I and VI but not other collagens, such as type III and IV, not usually found in the corneal stroma. Thus, the pattern of collagen expression in PLA cells does not change after the transplant.
Our main finding is that human PLA cells differentiate into functional keratocytes when injected into an ablated corneal stroma (creating a niche) after 8 and 12 weeks, as assessed by the expression of the cornea-specific proteoglycan, keratocan, and ALDH. However, we observed moderate haze in several of the PLA-injected corneas. This could be due in part to the facts that not all transplanted cells were positive for these two markers and that keratocan is essential for preservation of transparency [21]. Although we cannot rule out the expression of other extracellular matrix proteins not determined in this study, the fact that common collagens such as III and IV are not expressed suggests that poor keratocan expression is mainly responsible for the observed haze. In vitro predifferentiation of PLA cells toward keratocytes might improve transparency in future studies. The finding of a slight corneal opacity, similar to that seen in patients after old-technique photorefractive keratectomy, indicates that collagen expression, probably on an immature basis and disorganized, is occurring in the area. It is too early to know the significance of this finding for human corneal stroma regeneration, but this should indeed be interpreted as new tissue, and thus, its main significance is that there is some degree of regeneration and anatomic restoration.
The observed differentiation of PLA cells into keratocytes is the response of multipotent PLA cells to the damaged corneal microenvironment, and it is probably mediated by paracrine growth factors. In fact, both ALDH and keratocan expression were induced by the corneal stromal microenvironment, as none of these proteins were expressed in vitro by PLA cells. It is interesting to note that this differentiation was only achieved in the ablated corneas, suggesting that the creation of a niche for proper PLA cell homing and the paracrine secretion of unknown cytokines are important for stem cell-mediated corneal regeneration.
In our study, although using immune-competent animals, the human-derived cells did not elicit an immune response in the corneal stromal pocket, even though we were using a xenograft. Free of vessels, the cornea is a well-known immune-privileged site. However, immune rejection is observed sometimes after allograft. Another exciting explanation is that PLA cells have immunosuppressive properties as bone marrow MSC, as previously described in the literature for both types of cells [49–51].
We observed that PLA cells survive up to 12 weeks after the transplant, maintain their shape, and remain intermingled between the corneal stroma without disrupting its histological pattern. PLA cells did not migrate from the flap interface, where the cells had been deposited. Interestingly, transparency was preserved throughout the biosafety and immune rejection experiment, even 10 weeks after the transplant, when PLA cells formed a discontinuous layer in the corneal stroma. Other studies have shown similar in vivo survival rates for PLA cells, for instance, up to 12 weeks in the bladder and urethra of rats and mice [52]. We found cells in six of seven injected rabbits in the biosafety and rejection experiment and in five of six rabbits in the regeneration experiment. A single case per experiment showed no human cells, we believe as a result of unsuccessful cell delivery to the stromal pocket, since there is a relative risk of leakage from the corneal opening in the experimental model if the flap is not dissected carefully. In addition, the optimal amount of cells may vary depending on the degree of damage, and further studies are needed to address this issue.
In the biosafety experiment, the three eyes that showed flap displacement after the surgery showed signs of chronic inflammation and vascularization unrelated to the injection of PLA cells, since it affected two mock-injected eyes and one cell-transplanted eye. This complication was seen in the first rabbits transplanted because of excessive opening of the flap, and insufficient apposition as we tried not to press onto the flap to avoid leaking of the injected solution. Smaller opening and sufficient apposition of the flap was achieved in the following cases, so this complication was not seen thereafter.
Both femtosecond laser (first set of experiments) and LASIK (second set of experiments) techniques are associated with a proper wound-healing response preserving corneal transparency and not eliciting differentiation of keratocytes into fibroblasts [53]. Whether or not perfect optic transparency is achieved, obtaining a cell source that can be committed to keratocytes and able to repopulate and regenerate cornea-like tissue, as found in our study, could be very valuable in situations compromising the stroma, such as corneal thinning diseases, traumatic loss of stromal tissue, or corneal damage after infection or immune response. Particularly in the latter situation, the immunosuppressive properties of PLA cells render them very suitable for transplant.
We have also described a new model system based on state-of-the-art techniques in refractive surgery using femtosecond laser technology to assess survival and functionality of transplanted adult stem cells. This model produces a favorable wound-healing response, preventing excessive inflammatory reaction [53], which may distort the stem cell behavior. The cornea is an external tissue suitable to stem cell therapy because of its easy accessibility by surgical maneuvers. In addition, survival and functionality can be easily monitored by noninvasive methods such as microscopic observation. Moreover, growth factors, cytokines, and other molecules can be delivered to corneal cells when applied topically. Genetically modified cells could also be delivered in this way to the stroma at the desired depth as a genetic therapy for corneal diseases or disorders such as allograft rejection, laser-induced postoperative haze, herpes simplex keratitis, and wound healing in animal models.
To conclude, based on our results, which demonstrate longevity and functionality of the transplanted cells, we believe that adipose-derived adult stem cells can be a cell source for stromal repopulation and repair in diseased corneas or for tissue engineering of corneal equivalents. The low health impact of the surgical procedure performed to obtain the PLA cells provides this cell source with an additional beneficial feature for their possible future autologous use in human patients.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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