AU2006200140B2 - Stem cell cultivation devices and methods - Google Patents

Abstract

P:\WPDOCS\CRN\AMM\Coronco\12646221 abstract.doc-13/01/06 The present invention relates to a device and methods for culturing stem cells, and in particular, for culturing ocular stem cells and the use of stem cells cultured using the 5 devices and methods of the invention for the treatment of diseases. 20 Figure 1

Description

AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S):: Minas Theodore Coroneo ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys Level 10, 10 Barrack Street, Sydney, New South Wales, Australia, 2000 INVENTION TITLE: Stem cell cultivation devices and methods The following statement is a full description of this invention, including the best method of performing it known to me/us: 11 A') STEM CELL CULTIVATION DEVICES AND METHODS FIELD OF THE INVENTION The present invention relates to a device and methods for culturing stem cells, and in 5 particular, for culturing ocular stem cells and the use of stem cells cultured using the devices and methods of the invention for the treatment of diseases. BACKGROUND OF THE INVENTION Stem cells are relatively undifferentiated cells that possess the ability to self-renew, 10 and to generate one or more types of differentiated progeny or daughter cells, in adult or embryonic tissue. Adult stem cells continually replace lost cells in healthy and damaged tissue types. There are a wide variety of different types of stem cells that are resident in the adult, 15 human body, including in the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, eye, skin and liver. A number of different stem-cell populations have been identified to date in the mammalian eye. Adult corneal and conjunctival stem cells are a small subpopulation 20 of epithelial cells that make up the ocular surface, with corneal epithelial stem cells residing in the basal region of the limbus and playing a role in the regeneration of the corneal epithelium (Ang & Tan, 2004, Ann Acad Med Singapore, 33:576-80). Corneal stem cells, which are slow-cycling cells that self renew, mature to form transient, amplifying cells, which themselves give rise to progressively more differentiated, 25 non-dividing cells of the anterior corneal epithelium (Boulton & Albon, Int Jour Biochem & Cell Biol, 2004, 36:643-657). Conjunctival stem cells, which belong to a separate lineage from corneal stem cells, reside in the conjunctival fomiceal region but can be found throughout other regions 30 of the conjunctiva (Ang & Tan, above). In three areas in particular, stem cell depletion has been recognized as underlying the etiology of eye disease. These three areas include: 1. the comeal-scleral limbus - a site of corneal epithelial stem cells; 2 2. the conjunctival fornices - a site of conjunctival epithelial stem cells; and 3. The retina/ciliary body junction - a site of retinal stem cells. It is well recognized that the corneo-scleral limbus is important in regeneration of 5 corneal epithelium and perhaps bulbar conjunctiva. The limbus is a specialized transitional zone where sclera (with overlying conjunctiva) merges with the cornea. The superficial limbus is a narrow elliptical strip of tissue which extends circumferentially around the corneal periphery (where Bowman's membrane ends), 10 the cornea having a superficial horizontal diameter of approximately 11.5mm and a vertical diameter of 10.6mm. In general terms, its posterior limit is a line running parallel to the corneal periphery, 2mm beyond this limit vertically and 1.5mm horizontally. Underling this, the flat curve of the sclera, meets the steeper curve of the cornea, resulting in a surface groove, the external scleral sulcus in which an implant 15 could be placed without causing significant protrusion. Maintaining the "smoothness" of the ocular surface is important since "overhangs" can result in local drying of the surface of the eye which can cause devitalization of tissue (dellen) and even perforation of the eye. 20 The corneal epithelial stem cells are located in the palisades of Vogt, which are corregations that greatly increase limbal surface area and consequently, the number of cells present. The palisades are composed of epithelial rete pegs and the interpalisades are stromal papillae richly, vascularized (including lymphatics) and innervated. 25 The palisades of Vogt commence in the peripheral corneal epithelium and extend into the limbus as spoke-like radiations arranged at right angles to the peripheral cornea. The palisades measure 0.5mm in width by 2-4mm in length. Other types of ocular stem cells that have been found in various anatomical locations 30 in the eye include palpebral conjunctival epithelial stem cells, retinal stem cells and lens epithelial stem cells. In relation to stem-cell related eye disease, stem cell depletion can occur in any of a number of stem cell populations, which usually occur in protected niches. Depletion 3 of stem cells in these locations has serious consequences for eye health. Ocular stem cells are essentially pluri-potential but importantly, they are able to maintain tissue mass and normality of function by appropriated division and migrations as part of normal tissue turnover or in response to injury. Stem cells may also be critical to 5 maintaining tissue borders, so that cell types do not overgrow boundaries. This is critical in maintaining the precise optical functions of the eye. Ocular disorders that are a result of deficiencies include hereditary and acquired diseases or conditions, including aniridia keratitis and keratitis caused by endocrine 10 deficiency, Steven-Johnson syndrome, ocular cicatricial pemphigoid, contact lens induced keratopathy, neurotrophic keratopathy and peripheral ulcerative keratitis. To date, the treatment of ocular stem cell deficiencies has included grafting tissue from a patient's healthy eye into the diseased eye. For example, in limbal stem cell 15 deficiency, a lamellar removal of limbal segments from a patient's healthy eye can be transplanted to the limbal-deficient eye, following a complete, superficial keratectomy and conjunctival peritomy for removal of diseased surface epithelium in the affected eye (Ang & Tan, above). However, the use of autologous stem cells is not possible where a patient has bilateral stem cell deficiency, necessitating the use of allogenic 20 stem cells, which can be associated with a graft-rejection response. Immunosuppressant medications can be administered to minimise the risk of such a response (Boulton & Albon, above), however, such drugs are not without substantial side effects. 25 More recently, ex vivo expansion of ocular stem cells has been attempted, where a small biopsy from the patient's eye provides a small population of the appropriate stem cell type, which is then cultured on planar substrates, such as human amniotic membranes or fibrin-based substrates (see eg. Burman et al., Cell Tissue Bank. 2004;5(3):161-75). In a study of the use of cells derived in this manner, however, ex 30 vivo, allograft limbal stem cells that were used to treat limbal stem cell deficient patients were found to have a considerably short lifetime in the eye. In particular, PCR analysis showed no donor DNA present just three months after the procedure was performed (Daya, American Academy of Ophthalmology Annual Meeting, 2003, Abstract).

4 A number of diseases and conditions have been, and may potentially be, treated or prevented using stem-cell therapy, that is, the replacement of a patient's damaged or abnormal stem cells with healthy populations of stem cells, or the implantation of 5 replacement cell types, that have been derived (differentiated) from stem cells, to replace differentiated cells that have been damaged by disease or other trauma. For example, the growth and differentiation of dopaminergic neural cells from neural stem cells has been proposed as a means of replacing damaged dopamine-producing neurons in Parkinson's disease, as has the replacement of insulin-producing pancreatic 10 cells in type-I diabetes from multipotent pancreatic stem cells. Other conditions that may benefit from stem cell supplementation, or replacement of diseased or damaged differentiated cells with differentiated, stem-cell progeny include spinal cord injuries, incontinence, cardiovascular diseases, bums, arthritis and various liver and kidney diseases. 15 Adult stem cells are usually grown in culture dishes in incubators at body temperature (ie. 37"C), under conditions of high humidity and with nutrients that are specific for a given stem-cell type. A commonly encountered difficulty is obtaining adequate numbers of stem cells in an undifferentiated state. This is because adult stem cells are 20 very difficult to isolate and culture as they are few in number (eg. only 1 in 10 billion bone marrow cells are stem cells), and the cells have a tendency to stop dividing in culture after a time. They also tend to lose their ability to differentiate when they are grown in culture, and it can thus be difficult to promote the differentiation of the cells when the need arises. 25 Given the potential of stem cells and their differentiated progeny for therapeutic purposes, there is thus a need for improved techniques for stem-cell proliferation. SUMMARY OF THE INVENTION 30 The present invention is based on the finding that stem cell proliferation can be promoted by providing the cells with a specific micro-environment, which encourages proliferation and/or differentiation of stem cells.

C:\NRPorIb\DCC\GR5 9112670 1. DOC.2M)9/2l Il - 4a According to a first aspect of the invention there is provided an ocular scaffold device for promoting ocular stem cell proliferation and/or differentiation in a patient comprising: (a) a biocompatible material having an external surface and an internal compartment and being sized and shaped to be implanted into the patient's eye, 5 wherein the external surface comprises a plurality of external folds configured to promote neovascularization, and wherein the internal compartment is defined by an internal surface comprising a plurality of micron-sized internal folds configured to create niches that protect and promote proliferation of ocular stem cells, 10 wherein the plurality of internal folds increase the surface area of the internal compartment by at least two orders of magnitude compared to the surface area of the internal compartment without folds; and (b) a plurality of ocular stem cells seeded in the internal compartment. 15 According to a second aspect of the invention there is provided a method for promoting ocular stem cell proliferation and/or differentiation comprising: seeding the ocular stem cells into an ocular scaffold device according to the first aspect; and providing the internal compartment with an appropriate nutrient supply capable of 20 inducing proliferation and/or differentiation of the ocular stem cells.

5 Accordingly, in a first broad form, the present invention relates to a method for promoting stem cell proliferation and/or differentiation comprising seeding stem cells into a scaffold device comprising an internal compartment having a plurality of folds, and providing the internal compartment with an appropriate nutrient supply capable of 5 inducing proliferation and/or differentiation of the stem cells. In one preferred form, the stem cell proliferation and/or differentiation is performed in vitro. 10 In a further preferred form, the stem cell proliferation and/or differentiation is performed in vivo. In another preferred form, the scaffold device is shaped on an external and/or internal surface to mimic an anatomical environment. 15 In one embodiment, the anatomical environment is a source of the stem cells. In another embodiment, the anatomical environment is the desired transplantation site of the stem cells and/or their differentiated progeny. 20 In a preferred form, the stem cells are ocular stem cells. Preferably, the ocular stem cells are limbal stem cells. 25 In a further preferred form, the ocular stem cells are conjunctival epithelial stem cells. In yet another preferred form, the ocular stem cells are retinal progenitor cells. In a further preferred form, the ocular stem cells are corneal epithelial stem cells. 30 In yet a further preferred form, the ocular stem cells are palpebral conjunctival epithelial stem cells. In another preferred form, the ocular stem cells are epithelial stem cells.

6 Preferably, the scaffold device further comprises an external surface having a plurality of external folds. 5 In a preferred form, the external surface and external folds promote neovascularisation. Preferably, neovascularisation occurs prior to seeding the stem cells in the scaffold device. 10 In a particularly preferred form, the scaffold device is implanted in a patient's eye. In yet another preferred form, the scaffold device is implanted under a patient's limbus. 15 In a one embodiment, the scaffold device is an oval shaped scaffold device. Preferably, the oval shaped scaffold device has a vertical diameter of between about 8 mm and 12mm, a horizontal diameter of between about 9mm and 14 mm, and a width 20 of about 0.5mm to 4 mm. In another preferred form, the plurality of folds of the internal compartment of the scaffold device increase an internal surface area of the scaffold device by two orders of magnitude. 25 In this preferred form, the plurality of folds have dimensions of about 30-50 microns in width and the length of about 36-90 microns, and an interfold spacing of about 70 150 microns. 30 Preferably, the scaffold device is implanted at the fornices of the conjunctiva. In another preferred form, the scaffold device is implanted at a lid margin. In yet another preferred form, the scaffold device is a rod-shaped scaffold device.

7 Preferably, the rod-shaped scaffold device has a diameter of about 0.2 to 3mm and a length of about 20-50 mm. 5 Even more preferably, in this embodiment, the scaffold device is implanted at the patient's mucocutaneous junction. In another preferred form according to this embodiment, the scaffold device is implanted at the patient's ciliary margin, between the patient's retina and ciliary body. 10 Preferably, the stem cells that are seeded in the scaffold devices of the invention are autologous stem cells. In another preferred form, the stem cells that are seeded in the scaffold devices of the 15 invention are allogenic stem cells. Preferably, the scaffold device is made of a biocompatible material. Even more preferably, the biocompatible material is bio-absorbable. 20 In a further preferred form, the biocompatible material is poly(e-caprolactone). In yet another preferred form, the biocompatible material is a biosynthetic hybrid scaffold comprised of a fibrinogen backbone and crosslinked with difunctional 25 polyethylene glycol side chains. In another broad form, the present invention provides a scaffold device for promoting stem cell proliferation and/or differentiation comprising an internal compartment having a plurality of folds. 30 Preferably, the scaffold device is shaped on an external and/or internal surface to mimic an anatomical environment. In a preferred form, the anatomical environment is a source of the stem cells.

8 In another preferred form, the anatomical environment is the desired transplantation site of the stem cells and/or their differentiated progeny. 5 Preferably, the scaffold device of the invention further comprises an external surface having a plurality of external folds. In one preferred form, the plurality of external folds promotes neovascularisation. 10 In a further preferred form, the scaffold device is an oval shaped scaffold device. Preferably, the oval shaped scaffold device has a vertical diameter of between about 8 mm and 12mm, a horizontal diameter of between about 9mm and 14 mm, and a width of about 0.5mm to 4 mm. 15 In a particularly preferred form, the plurality of folds of the internal compartment of the scaffold device increase an internal surface area of the scaffold device by two orders of magnitude. 20 In a preferred form, the plurality of folds have dimensions of about 30-50 microns in width and the length of about 36-90 microns, and an interfold spacing of about 70-150 microns. In a further preferred form, the scaffold device is a rod-shaped scaffold device. 25 Preferably, the rod-shaped scaffold device has a diameter of about 0.2 to 3mm and a length of about 20-50 mm. In a particularly preferred form, the scaffold device of the invention is made of a 30 biocompatible material. Preferably, the biocompatible material is bio-absorbable. In another preferred form, the biocompatible material is poly(e-caprolactone).

9 In a further preferred form, the biocompatible material is a biosynthetic hybrid scaffold comprised of a fibrinogen backbone and crosslinked with difunctional polyethylene glycol side chains. 5 In another broad form, the present invention relates to an ocular scaffold device for promoting ocular stem cell proliferation and/or differentiation comprising an internal compartment having a plurality of internal folds and an external surface comprising a plurality of external folds for promoting neovascularisation, the internal compartment 10 for seeding with stem cells, the ocular scaffold device being made from poly(e caprolactone). Preferably, the ocular scaffold device is implantable into an eye of a patient. 15 In a further form, the present invention relates to a method of treating or preventing a disease in a patient comprising administering to the patient stem cells and/or their differentiated progeny, obtained according to any of the methods of the invention. In yet another form, the invention relates to a method of treating or preventing a 20 disease in a patient comprising in vivo growth in the patient of stem cells and/or their differentiated progeny, according to any of the methods for in vivo stem cell proliferation of the invention. In a further form, the present invention relates to a method of treating or preventing a 25 disease in a patient comprising implantation into the patient of a scaffold device for promoting in vivo stem cell proliferation and/or differentiation in the patient.

10 BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a diagramatic side view of an example of an ocular scaffold device. 5 FIGURE 2 is a schematic representation of a mammalian eye showing the location of ocular stem cell populations. FIGURE 3 is an illustration of injury to an eye of a patient caused by chemical insult. 10 FIGURE 4 is an illustration showing normal Palisades of Vogt of the limbus.

11 DETAILED DESCRIPTION OF THE INVENTION Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion 5 of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In a first broad form, the present invention relates to a scaffold device having an internal compartment with plurality of internal folds, which can be seeded with stem 10 cells, and to methods of proliferating and/or differentiating stem cells using the scaffold device. The internal folds of the scaffold device provide an increased surface area for supporting the stem cells that are seeded into the scaffold device for proliferation and/or differentiation. 15 The internal compartment is provided with an adequate nutrient supply capable of inducing proliferation and/or differentiation of the stem cells therein. For example, the nutrient supply may be provided by fashioning holes and/or tubing in an external surface of the scaffold device, the holes permitting the flow of cell culture broth or blood through to the internal compartment of the device. Alternatively, the nutrient 20 supply may be injected into the internal compartment. In a preferred form, the scaffold device has an external surface having a number of external infoldings, which permit neovascularisation of the scaffold device, thus also providing a nutrient supply to the internal compartment of the scaffold device. 25 Preferably, the scaffold device may be used in vivo or in vitro for the proliferation of stem cells. Preferably, stem cells that are seeded in the scaffold device are autologous stem cells. 30 Sources of autologous stem cells include multipotent adult progenitor stem cells, also known as MAPCs, isolated from bone marrow or other tissues in which they reside, mesenchymal stem cells derived from bone marrow, chord blood-derived stem cells, chord matrix (Wharton's jelly)-derived stem cells, and tissue-specific stem cells, for example, ocular stem cells.

12 In one embodiment, the scaffold device is shaped on its external and/or internal surface to mimic an anatomical environment, which may be an anatomical environment that the stem cells being grown in the device are isolated from. For 5 example, the internal surface of the scaffold device, which has a plurality of folds in its internal compartment, may mimic spongy bone, which consists of trabeculae and bars of bone adjacent to small, irregular cavities that contain red bone marrow. A further example is where the foldings of the internal compartment of the scaffold device may mimic the shape of liver lobules, and thus promote the proliferation 10 and/or differentiation of stem cells into hepatic stem cells or hepatocytes. Alternatively, the anatomical environment is a site where the stem cells and/or their differentiated progeny will be transplanted in a patient. For example, it is known that stem cells from adult brain when transplanted into sublethally irradiated mice will 15 colonize the host bone marrow and differentiate into granulocytes, macrophages, and B cells. Accordingly, stem cells from one tissue may be placed in a scaffold device that is shaped on its external and/or internal surface to mimic the new anatomical microenvironment and thus encourage the proliferation and differentiation of stem cells derived from a different tissue. The shape of the scaffold structure can thus 20 exploit the pluripotent nature of a variety of stem cells. The term "patient" refers to patients of human or other mammal and includes any individual it is desired to examine or treat using the methods and devices of the invention. However, it will be understood that "patient" does not imply that symptoms 25 are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes). 30 Preferably, the patient is human. In a preferred embodiment, the scaffold device is implantable in a patient.

13 In a particularly preferred form, the implantable device mimics the anatomy of the ocular stem cell niches of a patient in areas including, but not restricted to, the three areas of corneal and conjunctival epithelial as well as retinal stem cells. 5 In this form, the ocular scaffold device will conform to the normal anatomy of the three areas, where the foldings of the internal compartment, greatly increase tissue surface area. In this embodiment, the ocular scaffold device thus has two compartments: an 10 external compartment and an internal compartment. The external compartment comprises a surface that has a plurality of external foldings, which, after implantation is invaded by blood vessels. After this has occurred an internal compartment and its foldings will be populated by cultured stem cells, which can be injected into the internal compartment by a few openings to the exterior. An example of an ocular 15 scaffold device according to this embodiment is shown in Figure 1, which shows an external schematic view of an example of an ocular implantable scaffold device of the invention. The complex inner foldings of the internal compartment are not shown in this representation. 20 Preferably, the scaffold devices of the invention are manufactured from material that is chosen so that it minimises the risk of causing an inflammatory or toxic response. Suitable scaffold materials include, but are not limited to, type I collagen; glycosaminoglycans such as hyaluronic acid, dermatan sulphate, chondroitin sulphate, heparin, heparan sulphate, and keratan sulphate; chitosan; polyhydroxyalkanoates 25 polyesters; poly-caprolactone and poly (glycolic acid, poly (lactic acid) and their copolymers. In a particularly preferred form, the scaffold material is poly(e-caprolactone), or a biosynthetic hybrid scaffold comprised of a fibrinogen backbone and crosslinked with 30 difunctional polyethylene glycol side chains. In one embodiment, the scaffold device is manufactured from a material that is bio absorbable, that is, it is metabolised in the body after fulfilling its purpose, leaving substantially no trace.

14 Preferably, the rate of matrix regeneration is close to the biodegradability rate of the scaffold. 5 In a further broad form, the present invention relates to methods of treating or preventing a disease or condition in a patient comprising administering to the patient stem cells obtained according to any of the methods of the invention, or in vivo growth in the patient of stem cells according to any of the methods of the invention for in vivo stem cell proliferation and/or differentiation. 10 In another form, the present invention relates to a method of treating or preventing a disease or condition in a patient comprising implantation into the patient of a scaffold device for promoting in vivo stem cell proliferation and/or differentiation in the patient. 15 The terms "disease" and "condition" as used herein are interchangeable, and refer to an impairment of health or a state of health characterised by abnormal functioning in a patient. 20 Diseases that may be treated according to the methods of the invention include, but are not limited to, Parkinson's disease, Alzheimer's disease, spinal cord injury, stroke, bums, cancer, incontinence, heart disease, certain inherited blood disorders, type 1 diabetes, osteoarthritis, rheumatoid arthritis, muscular dystrophies, liver diseases, alopecia, certain liver and kidney disorders, and ocular diseases. 25 In relation to a number of ocular diseases and conditions, it is now recognized that a primary cause is the depletion of stem cell populations, resulting in failure to maintain the normal features of that tissue. This can be the result of trauma, such as chemical injury to the surface of the eye. It may also be that stem cell depletion is part of the 30 aging process. Inability to maintain normal tissue characteristics impacts on vision, typically because transparency of tissue is affected, causing image degradation. Certain ocular stem cell populations are shown in Figure 2. In most instances, the stem cells are located in relatively protected niches and usually the tissue in these 15 areas are thrown into folds, greatly increasing tissue surface area and consequently the number of cells that can be located there. This may be critical given the limited space in and on the eye. 5 An example of chemical injury causing comeal and conjunctival stem cell depletion is shown in Figure 3. Figure 3 demonstrates comeal opacity, conjunctival scarring and lack of eyelashes and a well-defined, eyelid margin as a result of deficiencies in corneal and conjunctival stem cell populations caused by chemical injury, which has depleted stem cell populations including those of the limbus. As part of limbal stem 1o cell failure, the infoldings of the limbus (Palisades of Vogt, see Figure 4) are lost and these infoldings greatly increase tissue surface area and therefore cell numbers. Without being limited to any single theory or mode of action, it is postulated that stem cells, when placed in culture or following attempts to replace a patient's own stem cell 15 populations, suffer a loss of "stemness", which may, in part, be caused by a failure to recreate a "niche" or an appropriate, pseudo-anatomical environment that protects and promotes proliferation of stem cells. In the absence of such an environment, stem cells will not survive in sufficient numbers of cells present to last long term, particularly when placed in the body of a patient, or will not differentiate sufficiently or 20 appropriately. In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non limiting examples. 25 Persons skilled in the art will appreciate that numerous variations and modifications will become apparent. All such variations and modifications which become apparent to persons skilled in the art, should be considered to fall within the spirit and scope that the invention broadly appearing before described.

16 EXAMPLES EXAMPLE 1 5 Limbal Stem Cell Scaffold Device This scaffold device is designed to conform to standard limbal dimensions and is oval in shape - (normal corneal dimensions: vertical diameter: 10.6mm, horizontal diameter: 11.75 mm) with a width of 1-2 mm. 10 The ocular scaffold devices consist of a biocompatible polymer scaffold which will, after the vascular and stem cell components are established will dissolve. This substance, in the first instance is poly(e-caprolactone) or a biosynthetic hybrid scaffold comprised of a fibrinogen backbone and crosslinked with difunctional 15 polyethylene glycol (PEG) side chains. In this regard, it has recently been demonstrated that autologous human conjunctival epithelial stem cells can be grown on polycaprolactone and successfully transplanted back onto deficient human ocular surfaces. 20 The under surface (that surface which lies on the globe at the limbus is infolded) and the external surface, smooth. Internally the structure is infolded to mimic the normal limbal anatomy, so that the surface area is increased by approximately 2 orders of magnitude. The normal limbal palisades of Vogt are 30-50 microns wide, the interpalisade width is 70-150 microns and the length of each palisade, 36-90 microns. 25 This internal cavity is sealed but is accessible after implantation (after the basal surface of the implant is vascularized). Corneal epithelial stem cells are then injected into this cavity and after they have colonized the scaffold device, the scaffold dissolves, leaving behind a new "pseudo-limbus" that mimics the original. 30 The implantation procedure is carried out under local anaesthetic. The conjunctiva is detached from the limbus (ie. peritomy), and the implant laid on the limbal "groove"(ie. the external scleral sulcus, where the disparate curvatures of the cornea and sclera meet). The implant can be secured in place with an absorbable suture such 17 as 10-0 Biosorb (polyglycolic acid) and the conjunctiva is closed over the implant with the same suture material. EXAMPLE 2 5 Conjunctival Epithelial Stem Cell Scaffold Device Conjunctival epithelial stem cell populations exist in the conjunctival fornices, the mucocutaneous junction of the eyelid and perhaps in the limbus. The device for 10 implantation in this context is ideally rodlike for insertion in the fornix and/or lid margin. The implants are thus rod like and of 0.5 to 2mm diameter and 30-40 mm in length. For the fornices (superior and inferior), small incisions are made in the conjunctiva laterally. By using a blunt probe, a space is opened along the length of each fornix and the implant inserted. Similarly, for the eyelid margins, a small 15 incision is made laterally and a blunt probed used to open a passage the length of the eyelid margin. EXAMPLE 3 20 Retinal Stem Cell Scaffold Device Retinal stem cells have been identified at the junction of the ciliary body and peripheral retina. This region is relatively thin and importantly avascular. The region 25 is 6-9 mm posterior to the limbus and has a diameter of 10-24 mm. Using standard vitrectomy/retinal surgery techniques, instruments are inserted into the eye, and this junctional zone identified and incised. A probe is then inserted and a subretinal space created into which the implant is inserted. 30

Claims (21)

1. An ocular scaffold device for promoting ocular stem cell proliferation and/or differentiation in a patient comprising: (a) a biocompatible material having an external surface and an internal compartment and being sized and shaped to be implanted into the patient's eye, wherein the external surface comprises a plurality of external folds configured to promote neovascularization, and wherein the internal compartment is defined by an internal surface comprising a plurality of micron-sized internal folds configured to create niches that protect and promote proliferation of ocular stem cells, wherein the plurality of internal folds increase the surface area of the internal compartment by at least two orders of magnitude compared to the surface area of the internal compartment without folds; and (b) a plurality of ocular stem cells seeded in the internal compartment.

2. The ocular scaffold device according to claim 1, wherein the plurality of internal folds have dimensions of about 30-50 microns in width, a length of about 36-90 microns, and an interfold spacing of about 70-150 microns.

3. The ocular scaffold device according to claim 1, wherein the external and/or internal surface of the scaffold device is shaped to mimic an anatomical environment.

4. The ocular scaffold device according to claim 3, wherein the anatomical environment is a source of the ocular stem cells.

5. The ocular scaffold device according to claim 3, wherein the anatomical environment is the desired transplantation site of the ocular stem cells and/or their differentiated progeny.

6. The ocular scaffold device according to claim 1, wherein the ocular scaffold device is oval shaped or rod-shaped C:\NRPorb\DCC\GRS\392670_1.DOC-29/0/201I - 19

7. The ocular scaffold device according to claim 6, wherein the oval shaped ocular scaffold device has a vertical diameter of between about 8 mm and 12 mm, a horizontal diameter of between about 9 mm and 14 mm, and a width of about 0.5 mm to 4 mm.

8. The ocular scaffold device according to claim 6, wherein the rod-shaped ocular scaffold device has a diameter of about 0.2 to 3 mm and a length of about 20-50 mm.

9. The ocular scaffold device according to claim 1, wherein the biocompatible material is bio-absorbable.

10. The ocular scaffold device according to claim 9, wherein the biocompatible material is poly(e-caprolactone).

11. The ocular scaffold device according to claim 1, wherein the biocompatible material is a biosynthetic hybrid scaffold comprised of a fibrinogen backbone and crosslinked with difunctional polyethylene glycol side chains.

12. The ocular scaffold according to claim 1, wherein the ocular scaffold device is implantable into an eye of a patient.

13. A method for promoting ocular stem cell proliferation and/or differentiation comprising: seeding the ocular stem cells into an ocular scaffold device according to any one of claims I to 12; and providing the internal compartment with an appropriate nutrient supply capable of inducing proliferation and/or differentiation of the ocular stem cells.

14. The method according to claim 13, wherein the plurality of internal folds of the internal compartment of the ocular scaffold device increase an internal surface area of the ocular scaffold device by two orders of magnitude. C :NRPOrbl\DCC\GRS%902670_ .DOC-29A)9/211 - 20

15. The method according to claim 13, wherein the ocular stem cells are selected from the group consisting of limbal stem cells, conjunctival epithelial stem cells, retinal progenitor cells, corneal epithelial stem cells and palpebral conjunctival epithelial stem cells.

16. The method according to claim 13, wherein the ocular stem cell proliferation and/or differentiation occurs in vitro.

17. The method according to claim 13, wherein the ocular stem cell proliferation and/or differentiation occurs in vivo.

18. The method according to claim 13, further comprising implanting the ocular scaffold device into a patient's eye.

19. The method according to claim 13, wherein the ocular stem cells that are seeded in the ocular scaffold device are autologous or allogeneic to the patient.

20. The method according to claim 13, wherein the ocular scaffold device is oval shaped or rod-shaped.

21. The method according to claim 13, further comprising implanting the ocular scaffold device under a patient's limbus, at the fornices of the conjunctiva, at a lid margin, at a patient's mucocutaneous junction, or at a patient's ciliary margin, between the patient's retina and ciliary body.