XWH - 07 - 2 - 0038 TITLE : Military Vision Research Program

Lucas KG, Qiao H, Stein-Streilein J. Retinal laser burn interferes with immune privilege of the eye Abstract 40th Annual Meeting of the Society for Leukocyte Biology, Cambridge, MA, 2007. appendix. TASK 3: James Zieske, Leader Task: To develop an anti-infective corneal bandage. A. Subtasks 1. Subtask (1): Produce a self-assembled collagen gel comprising a natural, strong, clear collagenous matrix that is generated de novo. 2. Subtask (2): Investigate the bio-stability of the corneal bandage. 3. Subtask (3): Determine whether human umbilical cord mesenchymal stem cells (MSCs) can be differentiated into corneal endothelial cells. 4. Subtask (4): To determine the basis for S. aureus adherance to corneal epithelial cells. B. Hypothesis Currently, there is little that can be done to treat eye injuries on the battlefield. We propose to develop a corneal bandage consisting of naturally occurring matrix materials. Ideally, this bandage will adhere to the wound, provide protection, promote maintenance of the globe, and inhibit detrimental immune responses. We will use this corneal bandage to study wound healing and infection, toward the ultimate goal of designing a second-generation bandage that prevents infection. We envision this as a multi-step project, first attempting to create a temporary bandage and secondly attempting to generate a more permanent bandage that would be incorporated into the cornea. C. Results Subtask (1): Produce a self-assembled collagen gel comprising a natural, strong, clear collagenous matrix that is generated de novo. (Jeffrey Ruberti, PhD) Dr. Ruberti’s laboratory has been addressing two aims intended to produce an organized collagenous matrix, which can be used as a mechanically strong clear substrate into which cells and anti-inflammatory agents may be seeded. Our specific approach involves the development of two cell-free methods capable of controlling collagen assembly on the nanoscale: 1) Influenced self-assembly in bulk thin shear film (Spin Coating) 2) Local control of collagen fibril assembly in nanoreactors (Collagen Nanoloom) Each of these approaches has the potential to generate aligned sheets of collagen fibrils similar to those found in the normal corneal stroma. Progress in Year 2 Aim1: Influenced self-assembly in bulk thin shear film (Spin Coating) A graduate student, Nima Saeidi, has been focusing on developing optimal parameters, which will allow shear and confinement to control the fibrillogenesis in thin films. In his work he has developed a shear chamber, which has been used to inform the work in the spin coating system. With the shear chamber, we are able to directly observe the polymerization of collagen on surfaces over which solution is flowing. From this work, we have found that the shear rate which produces aligned collagen can be as low as 7 sec. Spin-coating of collagen produces even better alignment, but is very difficult to control (due to film instability). However, although shear-alignment produces reasonable alignment of fibrils, it was determined that the collagen fibril morphology was poor (Figure 1). Nima has been funded principally by the NIH, but some of the TATRC funds were used, as proposed, on this aspect of his work. Figure 1. Quick-Freeze Deep Etch image of polymerized collagen on glass. The fibril has poor morphology (likely due to surface effects). Note clear resolution of the orientation of individual collagen monomers. Bar is 50 nm Future work for Aim 1: Because of this difficult to address problem, the remainder of year 2 has focused on further development of the nanoloom. Aim 2: Local control of collagen fibril assembly in nanoreactors (Collagen Nanoloom) Nanoloom construction/design: During year 1, a device was constructed by Northeastern University undergraduates, which will allow us to test the hypothesis that collagen synthesis and organization can be controlled by simulating cell fibropositors. (The construction cost was funded by an NSF SGER award). Testing of the nanoloom has been carried through year 2 of the TATRC funding period by Katie Portale and Dr. Ericka Bueno. Katie has focused on nanoloom design and testing while Ericka has focused on transport of collagen. 0 10 5 10 1 10 1.5 10 2 10 2.5 10 2.49 10 4.98 10 J s (μ m ol /c m 2 s ) ∆P (dynes/cm) untreated membrane, 2.8 mg/ml untreated membrane, 7.0 mg/ml PEG-modified membrane, 2.8 mg/ml PEG-modified membrane, 7.0 mg/ml 0 10 5 10 1 10 1.5 10 2 10 2.5 10 3 10 3.5 10 2.49 10 4.98 10 J v (c m /s ) ∆P (dynes/cm) untreated membrane, 2.8 mg/ml untreated membrane, 7.0 mg/ml PEG-modified membrane, 2.8 mg/ml PEG-modified membrane, 7.0 mg/ml Figure 2. Flux of collagen (Left) and solution (Right) across polycarbonate track-etched membranes for various experimental conditions. There are two pressures (10 or 20 inH2O), two concentrations (2.8 or 7.0 mg/ml) and two membrane surface conditions (PEGylated or untreated). The data demonstrate a potential maximum transport window at 10 inH2O, 2.8 mg/ml using PEGylated polycarbonate membranes. (See next page for description of experiments) Collagen transport. Dr Ericka Bueno has concluded a series of transport experiments designed to produce optimal parameters (pressure, concentration, membrane treatment) to elicit maximum collagen transport across the Nanoloom reactor arrays. The data indicate that collagen is a very complex macromolecule which will self-associate at nominal concentrations. Transport of solution and collagen was examined with a Ussing chamber designed to test flux across membranes (low-volume NaviCyte® single-channel vertical diffusion chamber Harvard Apparatus, Holliston MA). The two graphs above summarize the results of the experiments which suggest that a window of maximum transport across the membrane occurs at 2.8 mg/ml and 10 inH2O. Because the maximum porosity available in commercial track-etched membranes is relatively small (~2%), we are attempting to use newly developed (at NEU) aluminum oxide membranes which can be created with high aspect ratio pores and with nearly hexagonal close packing values of porosity. These membranes are also treatable. The results of this transport work have been compiled in a manuscript (near final draft attached) which will be submitted within two weeks of this progress report. The funding provided for this project has largely been applied to the work that was done by Dr. Bueno. Figure 3. Collagen Nanoloom Collagen fibrillogenesis in the Nanoloom. A graduate student, Kathryn Portale, has been investigating methods by which the polymerization of the collagen can be confined to one side of the track etched membrane (Nanoloom reactor array). By an accidental discovery, we found that collagen may be extruded in alignment into a bath with high ionic strength. Our explanation for this phenomenon is that the high strength salt solution causes the collagen monomers to collapse onto one another due to cononsolvency effects. This fortuitous discovery has led us to postulate that any solution which “steals” water from the collagen will make collapse. We have thus begun a series of experiments to test whether or not this is the case. The benefit of this effect is the fact that a steep temperature gradient may not be needed. Attempts were made to print collagen without fine temperature control, which encouraging results. In figure 4, what we believe are our first few lines of printed collagen are shown. However, we have not proven that this is the case. Figure 4. DIC images of strip of “collagen” printed by the nanoloom. The solution into which the collagen was printed was 10x PBS and scale bar is 30 microns. Future work for Aim 2: Our intent is to continue to optimize the parameters which will make this device work. Changes include development of simple, directly observable test bed to enable dynamic views of collagen extrusion, use of high porosity alumina membranes, and printing into solutions with high osmotic pressures including polyethylene glycol (PEG) and hyaluronic acid. Subtask (2): Investigate the bio-stability of the corneal bandage. (James Zieske, PhD) Introduction: The goal of this subtask is to determine the response of the host to the corneal bandage by applying the bandage (artificial stroma) to mice using two experimental models. In the first model, a sheet of artificial stroma was implanted inside the recipient’s stroma (intralamellar corneal transplantation model). In the second model, a sheet of the artificial stroma was placed on the surface of the recipient’s stroma (onlay model). Methods: Primary human fibroblasts were isolated from human donors, cultured, propagated in vitro, and characterized. The cells were seeded in Transwell inserts and then allowed to grow for four weeks. During this time the cells stratified and produced an extracellular matrix to form a 3-D construct. In the intralamellar model, cells were labeled with Q-tracker (fluorescent red) before transplant. In this model the construct was transplanted into BALB/c mice. The original protocol had called for the use of immunodeficient scid mice, but these proved to be unnecessary. To generate this model, a pouch was made in the stroma and the construct was placed in the pouch. Graft acceptance was indicated by the continuing presence of the Qtracker fluorescence. Graft rejection was indicated by neovascularization and loss of fluorescence. In the onlay model, a 2-mm superficial keratectomy was made in the central cornea of BALB/c mice to expose the stroma. Rose Bengal (0.05%) was applied to the exposed corneal stromal surface, and the corneal bandage was laid down on the stroma and irradiated with a green laser (532nm) for 180 seconds. Prior to transplantation, the artificial stroma was labeled with DTAF. The attachment of the corneal bandage was examined by the presence of the DTAF. The mice were sacrificed and the eyes were processed and examined by immunofluorescence microscopy for the presence of infiltrating immune cells with antiCD45. Results: The isolated corneal cells expressed keratan sulfate proteoglycan (KSPG) Ki67 and CD90. After forming the 3-D construct, the KSPG positive cells laid down extracellular matrix components including collagen VI, fibronectin, and thrombospondin 1. Pouch Model: In the intralamellar model, 75% of the constructs were maintained for 4 weeks and 50% were maintained for 8 weeks. The constructs did not appear to generate an immune response. Loss of fluorescence was accompanied by neovasculariazation. Onlay Model: In the onlay model, the Rose Bengal and the green laser irradiation made a tight bond between the bandage and the corneal stroma. The DTAF labeled collagen stayed on the corneal surface for at least 14 days (one mouse maintained the bandage for 140 days). The construct was covered by epithelium by day 3. CD45 positive immune cells were found on day 1 in the limbus and central cornea in eyes +/the artificial bandage. However, by day 3 the number of immune cells decreased in the corneas with the bandage; whereas, the corneas without bandage still maintained numerous CD45 positive cells. Conclusions: The corneal bandage is accepted by the wounded recipient and provides a good barrier for protecting the injured cornea stroma. Subtask (3): Determine whether human umbilical cord blood mesenchymal stem cells (MSCs) can be differentiated into corneal endothelial cells. (Nancy Joyce, Ph.D.) INTRODUCTION: Corneal endothelium is the single layer of cells at the posterior of the cornea that is responsible for maintaining corneal clarity. Preparation of a clear, anti-infective corneal bandage therefore requires the presence of a functional layer of corneal endothelial cells. Although our laboratory has had consistent success in culturing human corneal endothelial cells (HCEC), the proliferative capacity of these cells is limited and decreases with age. Since the majority of donor corneas are obtained from older individuals, there is a limited ability of endothelial cells isolated from these corneas to be expanded in culture. Thus, the ability to develop a corneal bandage containing normal endothelium may be compromised. During eye development, corneal endothelium is formed from neural crest-derived mesenchymal cells that migrate beneath primary stromal material. Because of this origin, HCEC share characteristics with neuronal cells, such as expression of neuron-specific enolase, S-100, and Ncadherin, while at the same time expressing the mesenchymal intermediate filament protein, vimentin. Multipotent mesenchymal stem cells obtained from post-partum human umbilical cord blood have the capacity to differentiate into several cell types, including neurogenic cells. As such, we hypothesize that human umbilical cord blood mesenchymal stem cells (MSCs) can be differentiated into HCEC to provide a ready supply of cells for tissue bioengineering. This is a NOVEL APPROACH, which could have a significant impact on the development of a clear, anti-infective corneal bandage, as well as on treatment of corneal blindness in patients with corneal endothelial dysfunction. SPECIFIC AIM: The single Specific Aim of this sub-project is to determine whether human umbilical cord blood mesenchymal stem cells (MSCs) can be differentiated into corneal endothelial cells. RESEARCH ACCOMPLISHMENTS: We obtained six clones of human umbilical cord blood MSCs from Dr. Biagio Saitta, Coriell Institute for Medical Research, Camden, NJ. Umbilical cord blood was obtained after full-term deliveries. Donor confidentiality was maintained in accordance with the requirements of the Internal Review Boards at Coriell and Schepens. 1) Expansion of MSC: All MSC cloned cultures were passaged under basal conditions (DMEM with low glucose, 10% FBS) to expand cell numbers. Cells were frozen in liquid nitrogen to maintain their stem-like characteristics. Two of these cultures (MSC 34b and MSC 44) were used to conduct initial tests. Images of these cultures are presented in Figure 1. Figure 1. Two clones of human umbilical cord blood mesenchymal stem cells (MSCs) showing somewhat flattened morphology. Original magnifications are indicated. PLEASE NOTE: There is no known specific marker for human corneal endothelial cells, so the ability to differentiate MSCs to corneal endothelium will be determined by a combination of morphological characteristics, Western blot studies of proteins known to be expressed by corneal endothelial cells, and, eventually, by in vivo opacity tests to analyze endothelial cell function. 2) Test of Effect of HCEC Culture Medium on MSC Growth Characteristics: An initial series of experiments was conducted to determine whether MSCs would grow and form a confluent monolayer by incubation in a culture medium developed in our laboratory that supports growth of HCECs. This medium contains OptiMEM-1, selected nutrients, 8% fetal bovine serum (FBS), and growth factors, including epidermal growth factor (EGF) and nerve growth factor (NGF). Figure 2. Effect of culture medium on MSC morphology. (A) MSCs in basal medium containing DMEM, low glucose, 10% FBS as a control. (B) MSCs cultured for 8 days in complete HCEC medium. Note the elongated cell shape and lack of cell-cell contacts. (C) MSCs cultured for 26 days in HCEC medium containing 8% FBS without additional growth factors. (D) Confluent HCECs cultured from a 75yo donor. Original magnification: 20X. MSCs were grown for 8 days under basal conditions as a negative control (Fig. 2A) or in the medium described above that supports the growth of HCECs (Fig. 2B). Culture in this medium promoted growth of MSCs and cells remained as a single layer; however, cell morphology changed in this medium from a somewhat rounded shape to a highly elongated, neuronal-like shape with long, sometimes branching cell processes. MSCs grown for 26 days in HCEC culture medium containing 8% FBS, but no additional growth factors, formed an apparently confluent monolayer of cells (Fig.2C). A confluent monolayer of HCECs is shown in Fig. 2D for comparison. Results indicate that MSCs are capable of growing in HCEC culture medium in both the presence and absence of additional growth factors. Growth in medium containing FBS plus additional growth factors, such as NGF, causes a significant shape change, with cells having a neuronal appearance. Growth in HCEC medium containing only 8% FBS caused cells to have a rounder morphology and to form an apparently confluent monolayer, suggesting that MSC might be able to differentiate into HCEC-like cells. 3) Western Blot Detection of Mesenchymal Neural Crest Markers. Initial studies were conducted to compare the relative expression of several marker proteins in an embryonic stem cell line (ESCs), MSCs, and HCEC cultured from young and older donors. Figure 3 provides evidence that MSCs and HCECs both express neuron-specific enolase (NSE), N-cadherin, and vimentin. As expected, expression of these mesenchymal neural crest marker proteins was generally lowest in ESCs. Figure 3. Western blots showing relative expression of neuron-specific enolase (NSE), N-cadherin, and vimentin in ESCs, MSCs, and HCECs cultured from young and older donors. Beta-actin or non-muscle myosin wasused a loading control based on the expected molecular weight of the test protein. Western blots were also used to compare the relative expression of the neuronal marker, S-100, and stem cell markers, nestin, Sox2, and Musashi. Inconsistent results were obtained for these proteins, so the blots will be repeated. Overall, results indicate that MSCs express higher levels of mesenchymal neural crest marker proteins than do embryonic stem cells. MSCs and HCECs expressed similar marker proteins, showing a close relationship between these cells and providing suggestive evidence that it should be possible to find methods to differentiate MSCs into functional HCECs. REFERENCES: Should go at the very end of the task. 1. Chen K-H, Azar D, Joyce NC. Transplantation of adult human corneal endothelium ex vivo. Cornea. 2001;20:731. 2. Zhu CC, Joyce NC. Proliferative response of corneal endothelial cells from young and older donors. Invest Ophthalmol Vis Sci. 2004;45:1743. 3. Joyce NC, Zhu CC. Human corneal endothelial cell proliferation: Potential for use in regenerative medicine. Cornea 2004;23 (Suppl. 1):S8. 4. Senoo T, Joyce NC. Cell cycle kinetics in corneal endothelium from old and young donors. Invest Ophthalmol Vis Sci. 2000;41:660. 5. Hay ED, Revel JB. Fine structure of the developing avian cornea. Monogr Dev Biol. 1969;1:1. 6. Bard JB, Hay ED, Meller SM. Formation of the endothelium of the avian cornea: a study of cell movement in vivo. Dev Biol. 1975;42:334. 7. Hayashi K, et al. Immunohistochemical evidence of the origin of human corneal endothelial cells and keratocytes. Graefes arch Clin Exp Ophthalmol. 1986;224:452. 8. Bohnke M, Vogelberg K, Engelmann K. Detection of neurone-specific enolase in long-term cultures of human corneal endothelium. Graefes Arch Clin Exp Ophthalmol. 1998;236:522. 9. Foets B, et al. A comparative immunohistochemical study of human corneotrabecular tissue. Graefes Arch Clin Exp Ophthalmol. 1992;230:269. 10. Beebe DC, Coats JM. The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye. Dev Biol. 2000;220:424. 11. Lee OK, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood. 2004;103:1669. 12. Hou L, et al. Induction of umbilical cord blood mesenchymal stem cells into neuron-like cells in vitro. Int J Hematol. 2003;78:256 REPORTABLE OUTCOMES: Additional studies need to be conducted prior to submitting a paper for publication. Importantly, the data obtained in these studies will be used in an R21 application to the National Eye Institute for additional funding of this project. Reportable outcomes are also needed from Zieske, Ruberti, and Gilmore. CONCLUSIONS: 1) The morphology of MSCs can be altered depending on culture medium conditions; and 2) MSCs and HCECs share expression of a number of markers of neural crest-derived mesenchymal cells, indicating a close relationship between the two cell types. Overall, additional studies are needed to find specific conditions that will more fully differentiate MSCs to form functional HCECs; however, progress has been made toward this goal. Subtask (4): To determine the basis for S. aureus adherance to corneal epithelial cells. (Michael Gilmore, PhD.) Introduction: Staphylococcus aureus is a major cause of bacterial keratitis linked with non-surgical trauma to the eye, including contact lens wear. The pathology results from a combination of bacterial toxins and the host immune response. Interestingly, some S. aureus toxins can also modulate cellular behavior at sublytic concentrations by altering receptor processing and intracellular signaling events. Most of these toxins are modulated by global regulators Agr and Sar. The ocular surface plays an important role in innate immunity by acting as a barrier to infectious agents as well as secreting anti-microbial peptides, cytokines and chemokines following exposure to bacteria. Little is known regarding the global effects of S. aureus and its toxins on human corneal epithelial cells in the early phases of infections. The main goal of this study was to determine the genetic program expressed by corneal epithelial cells in response to exposure to S. aureus and its products at the earliest step in infection – a point where intervention might prevent subsequent pathology. Materials and Methods: Human corneal epithelial cells : Monolayers of Araki-Sasaki cells (hCEC) and primary human corneal epithelial cells (hCEC-3, Cascade BiologicsTM) were grown at 37oC under 5% CO2 in defined serum-free keratinocyte media. S. aureus strains : RN6390 (wt; toxigenic) and ALC135 (isogenic agr-/sarmutant; non-toxigenic) were cocultured with the hCEC’s Infection Conditions : Late-logarithmic phase S. aureus cultures were washed and resuspended in defined SF-DKM to a multiplicity of infection (MOI) of 20 bacteria/epithelial cell. Infections were conducted at 37oC under 5% CO2 for 6 hrs. Viability of the infected epithelial monolayer was noted to be ≥ 90%. The corresponding RN6390 culture supernatants contained hemolytic activity; whereas, ALC135 supernatants did not. Alternatively, monolayers were pretreated with cytochalasin D (0.5mg/ml) for 1hrs prior to infection or Pam3Cys (10mg/ml) only for 12hrs. Microarray Analysis : RNA was extracted with a RNeasy Mini kit (QIAGEN), and cDNA quantification was assessed by hybridization with Affymetrix Gene Chip Human Genome U133 Plus 2.0 Array Cytokine Analysis : Bio-Plex Human Cytokine 8A Assay (BIO-RAD) and DuoSet ELISA human CL20/MIP3a (R&D Systems) Results: Changes in gene expression by hCEC’s resulting from exposure to S. aureus are shown in Table 1.