Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
1. The amendment filed 02/16/2026 has been entered. Claims 1, 13, and 19 – 21 and new claims 22 – 24 are pending. Claims 1, 13, and 21 – 24 are under consideration.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 and 12 – 18) in the reply filed on 11/29/2023 is acknowledged.
3. Claims 19 and 20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 11/29/2023.
Priority
4. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in parent Application No. JP 2018-098954, filed on 05/23/2018.
Information Disclosure Statement
5. The information disclosure statement (IDS) submitted on 12/04/2025 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Maintained Claim Rejections
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
6. Claim(s) 1 remains rejected under 35 U.S.C. 103 as being unpatentable over Hayashi (Hayashi, Ryuhei, et al. Nature Protocols 12.4 (2017): 683-696; previously cited), hereinafter Hayashi which is cited on the IDS filed 11/19/2020 in view of Hayashi-2016 (Hayashi, Ryuhei, et al. Nature 531.7594 (2016): 376-380; previously cited), hereinafter Hayashi-2016 which is cited on the IDS filed 11/19/2020 in view of Devi (US-8338175-B2; Filed 02/23/2007; Published 12/25/2012; previously cited), hereinafter Devi in view of Kurosawa (Kurosawa, Hiroshi. Journal of bioscience and bioengineering 114.6 (2012): 577-581; previously cited), hereinafter Kurosawa.
Regarding step (1)(a), Hayashi teaches maintenance culture of pluripotent stem cells on laminin 511E8 for 10 days in serum-free medium (page 683, right col. para. 1; Figure 2; page 685, right col. para. 1; page 689, steps 1 – 7; page 690).
Regarding step (1)(b), Hayashi teaches culturing the pluripotent stem cells in serum-free medium (DM) for 4 weeks to form a SEAM (Figure 2; page 683, right col. para. 1; page 685, right col. para. 2; page 689, right col. “Differentiation Medium”; page 691, steps 27 – 34).
Regarding step (2), Hayashi does not teach a “conjunctival differentiation medium” comprising EGF signaling activator and ROCK inhibitor or “conjunctival epithelial maintenance medium” where neither media contains KGF. However, Hayashi teaches culturing SEAM cells in differentiation media for 4 weeks followed by culturing in CEM media for 2 to 8 weeks (“culturing the colonies of SEAM cell population in a conjunctival differentiation medium for 3 to 5 weeks, and then inducing differentiation by culturing the colonies of SEAM cell population in a conjunctival epithelial maintenance medium for 2 to 6 weeks”) to obtain corneal epithelial cells where the culture techniques to no require serum (page 683, right col. para. 2; Figure 2). Hayashi teaches cells in zone 3 have characteristics of ocular-surface ectoderm and are cultured in media containing growth factors and the ROCK inhibitor Y-27632 (“ROCK inhibitor”) for an additional 4 weeks (page 686, left col. and right col.; Table 1). Hayashi teaches the protocol is distinct because it generates a range of cellular anlages that have characteristics of several quite distinct tissues in the eye (page 683, right col. para. 2). Hayashi teaches the method comprises purification of cells by FACS using a combination of antibodies to only obtain corneal iPS-derived epithelial cells free of impurities (page 684, left col. para. 1). Hayashi teaches obtaining other cell types from the iPS cell-derived SEAM will likely require modifications to the protocol (page 685, left col. para. 1). Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development (page 685, left col. para. 2). Hayashi teaches SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise (page 685, left col. para. 2).
Regarding “conjunctival” cells, Hayashi-2016 teaches the SEAM formation from iPS cells method of Hayashi in Figure 1a (page 376, left col. para. 1). Hayashi-2016 teaches cells in SEAM zone 3 most closely resembles those of the presumptive ocular surface and these zone 3 cells were subjected to FACS to isolate a specific ocular surface lineage – corneal or conjunctival (page 377, left col. para. 2; Figure 3a). Hayashi teaches further differentiation of the SEAM for 4 weeks in differentiation media followed by 2 – 7 weeks in maintenance media resulted in both corneal and conjunctival epithelial cells in Figure 3a where the differentiation media containing a serum replacement (KSR), FGF2, and Y-27632 and the maintenance media contained KGF and Y-27632 and the serum-free supplement B27 (page 381, left col. para. 1 – 3). Hayashi-2016 teaches FGF2 in differentiation medium was not essential for corneal epithelial induction (page 381, left col. para. 3). Hayashi-2016 teaches conjunctival epithelial cells, conjunctival goblet cells, and conjunctival progenitor cells were found in the SEAM (Figure 3e – f; page 378, left col.; Extended Data Figure 4a). Hayashi-2016 teaches cells in zone 3 could be developed for surgical use (page 378, left col.). Hayashi-2016 does not teach an EGF signaling activator in the differentiation and maintenance media or that both media do not contain KGF. One would have been motivated to combine the teachings of Hayashi and Hayashi-2016 regarding a method of SEAM formation because both teach the same method and Hayashi-2016 teaches the method produces conjunctival cells.
Regarding an EGF signaling activator and the media does not contain KGF, Devi teaches a method for culturing and expanding and differentiating conjunctival progenitor cells obtained from a tissue biopsy in Example 1 where conjunctival progenitor cells were cultured in media containing human EGF (col. 17, lines 8 – 19 and 40 – 50). Devi teaches it was surprising that the method which uses a single step media for the proliferation and differentiation of the conjunctival cells could successfully produce a tissue system for transplant (col. 17, lines 51 – 54). Devi teaches the cells expressed MUC4 and MUC5AC (col. 19, lines 17 – 21). Devi teaches transplanting the conjunctival tissue on a rabbit model of conjunctival injury in Example 2 (col. 17, lines 63 – 66; col. 19, lines 57 – 67). Devi teaches the efficiency of the transplant was quantified by re-epithelization and reappearance of goblet cells at the site of injury in comparison to control animals (col. 20, lines 15 – 18; col. 21, lines 6 – 15; Table 2). Devi teaches there were no allergic reactions or signs of rejection and that the observation of human cells interspersed at the site of transplant suggests the cells play an important role in healing the injury (col. 20, lines 47 – 57; col. 21, lines 20 – 26). Devi teaches stem cells that continually replace conjunctival epithelium have been shown to reside in the conjunctival fornix and are known as conjunctival stem cells or progenitor conjunctival cells (col. 2, lines 15 – 22). Devi teaches the multiple layers of stratified conjunctival epithelium along with the interspersed goblet cells together are continuously regenerated maintaining the functional integrity of the ocular surface (col. 2, lines 32 – 36). Devi teaches a major role for the conjunctiva is to provide ocular surface hydration and lubrication through the production of tears by mucin-producing goblet cells (col. 2, lines 23 – 28). Devi teaches the conjunctiva supports the health of the corneal epithelium (col. 2, lines 43 – 47). Devi teaches that because the corneal epithelium is dependent on a healthy conjunctival surface to maintain clear vision, many ocular surface diseases initiate with conjunctival damage followed by secondary limbal and corneal damage (col. 2, lines 47 – 51). Devi teaches mucin deficiency is detected in alkali burns, chemical and thermal burns, Stevens-Johnson Syndrome, neurotrophic keratitis, and Ocular Cicatricial Pemphigoid (col. 2, lines 38 – 40). Devi teaches in grade 4 – 6 ocular burns in which the surviving corneal and conjunctival epithelium is thin to zero, the desirable ocular management may be to attempt a restorative and reconstructive intervention by re-establishing both corneal and conjunctival epithelial cover one after another (col. 3, lines 12 – 17). Devi teaches there is a need for more desirable conjunctival tissue equivalent (col. 4, lines 18 – 19). Therefore, Devi teaches a media for obtaining conjunctival cells containing EGF but does not teach the media contains a ROCK inhibitor. One would have been motivated to combine the teachings of Hayashi and Hayashi-2016 regarding a method of SEAM formation that produces conjunctival cells with the teachings of Devi regarding differentiation and expansion of conjunctival progenitor cells in media containing EGF because Hayashi-2016 teaches the method produces conjunctival cells from pluripotent cells thus eliminating the need for obtaining a tissue biopsy as taught in the method of Devi.
Kurosawa teaches ROCK inhibitor could promote the survival of dissociated human stem cells and is a useful tool in the cultivation of pluripotent stem cells (page 577, right col. para. 2). Kurosawa teaches pluripotent stem cells are susceptible to dissociation induced apoptosis and Y-27632 ROCK helps to control this and improve yield (page 578, left col. para. 2 and right col. para. 1). Kurosawa teaches Y-27632 has been used in various routine manipulations in stem cell research including (page 578, left col. para. 2; Figure 2). Kurosawa teaches Y-27632 increases the efficiency of colony formation of pluripotent stem cells in serum-free and feeder-free culture conditions and thus Y-27632 removes the problem of xenogeneic contaminants from serum and feeder layers and will encourage the use of stem cells for clinical applications (page 579, left col. para. 2). Kurosawa teaches Y-27632 enhances pluripotent stem cell differentiation (page 579, left col. para. 3 – 4).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Hayashi regarding a method of obtaining a SEAM population from pluripotent stem cells with differentiation media and maintenance media where the maintenance media contains a serum replacement with the teachings of Hayashi-2016 regarding the method of Hayashi produces conjunctival cells where desired cells can be isolated by FACS with the teachings of Devi regarding a method of differentiating conjunctival progenitor cells in media containing EGF with the teachings of Kurosawa regarding a ROCK inhibitor promotes survival of pluripotent stem cells in serum-free media and enhances differentiation to arrive at the claimed method for inducing differentiation into conjunctival epithelial cells, conjunctival goblet cells, and conjunctival epithelial stem and progenitor cells, comprising: (1) obtaining colonies of a self-formed ectodermal autonomous multi-zone (SEAM) cell population by (a) culturing pluripotent stem cells in the presence of laminin 511E8 for 8 to 10 days in maintenance culture; and (b) culturing the pluripotent stem cells in a serum-free medium appropriate for animal cell culture for 4 weeks to induce autonomous differentiation to form the
colonies of SEAM cell population; and (2) culturing the colonies of SEAM cell population in a conjunctival Differentiation medium for 3 to 5 weeks, and then inducing differentiation by culturing the colonies of SEAM cell population in a conjunctiva! epithelial maintenance medium for 2 to 6 weeks, wherein the conjunctival differentiation medium contains an epidermal growth factor (EGF) signaling activator and a ROCK inhibitor, wherein the conjunctival epithelial maintenance medium contains the EGF signaling activator, the ROCK inhibitor and serum replacement, and wherein neither the conjunctival differentiation medium nor the conjunctival epithelial maintenance medium contain a keratinocyte growth factor (KGF). One would have been motivated to combine the teachings of Hayashi, Hayashi-2016, Devi, and Kurosawa in a method of generating a conjunctival tissue from pluripotent stem cells as Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development and SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise and Hayashi-2016 teaches cells in zone 3 could be developed for surgical use and Devi teaches there is a need for more desirable conjunctival tissue equivalent. One would have a reasonable expectation of success in combining the teachings as Hayashi-2016 teaches SEAM contains conjunctival cells and Devi teaches media containing EGF but not KGF formed conjunctival tissue that could repair ocular injury in vivo.
7. Claim(s) 13 remains rejected under 35 U.S.C. 103 as being unpatentable over Hayashi (Hayashi, Ryuhei, et al. Nature Protocols 12.4 (2017): 683-696; previously cited), hereinafter Hayashi which is cited on the IDS filed 11/19/2020 in view of Hayashi-2016 (Hayashi, Ryuhei, et al. Nature 531.7594 (2016): 376-380; previously cited), hereinafter Hayashi-2016 which is cited on the IDS filed 11/19/2020 in view of Devi (US-8338175-B2; Filed 02/23/2007; Published 12/25/2012; previously cited), hereinafter Devi in view of Kurosawa (Kurosawa, Hiroshi. Journal of bioscience and bioengineering 114.6 (2012): 577-581; previously cited), hereinafter Kurosawa in view of Hayashi (WO-2016114285-A1; previously cited), hereinafter Hayashi-285. A machine translation of WO-2016114285 was previously provided. The translation was performed on December 20, 2023 of pages 1 - 30 of the original document.
Regarding step (1)(a), Hayashi teaches maintenance culture of pluripotent stem cells on laminin 511E8 for 10 days in serum-free medium (page 683, right col. para. 1; Figure 2; page 685, right col. para. 1; page 689, steps 1 – 7; page 690).
Regarding step (1)(b), Hayashi teaches culturing the pluripotent stem cells in serum-free medium (DM) for 4 weeks to form a SEAM (Figure 2; page 683, right col. para. 1; page 685, right col. para. 2; page 689, right col. “Differentiation Medium”; page 691, steps 27 – 34).
Regarding step (2), Hayashi does not teach a “conjunctival differentiation medium” comprising EGF signaling activator and ROCK inhibitor or “conjunctival epithelial maintenance medium” where neither media contains KGF. However, Hayashi teaches culturing SEAM cells in differentiation media for 4 weeks followed by culturing in CEM media for 2 to 8 weeks (“culturing the colonies of SEAM cell population in a conjunctival differentiation medium for 3 to 5 weeks, and then inducing differentiation by culturing the colonies of SEAM cell population in a conjunctival epithelial maintenance medium for 2 to 6 weeks”) to obtain corneal epithelial cells where the culture techniques to no require serum (page 683, right col. para. 2; Figure 2). Hayashi teaches cells in zone 3 have characteristics of ocular-surface ectoderm and are cultured in media containing growth factors and the ROCK inhibitor Y-27632 (“ROCK inhibitor”) for an additional 4 weeks (page 686, left col. and right col.; Table 1). Hayashi teaches the protocol is distinct because it generates a range of cellular anlages that have characteristics of several quite distinct tissues in the eye (page 683, right col. para. 2). Hayashi teaches the method comprises purification of cells by FACS using a combination of antibodies to only obtain corneal iPS-derived epithelial cells free of impurities (page 684, left col. para. 1). Hayashi teaches obtaining other cell types from the iPS cell-derived SEAM will likely require modifications to the protocol (page 685, left col. para. 1). Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development (page 685, left col. para. 2). Hayashi teaches SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise (page 685, left col. para. 2).
Regarding step (3), Hayashi teaches isolation of cells from the SEAM cell population using antibodies specific for SSEA-4 and ITGB4 that are weakly SSEA-4 positive and ITGB4 positive (Figure 4; page 687, right col. para. 1; page 688, left col. Reagents; page 692, steps 42 – 53; page 693, steps 54 – 56; Figure 4). Hayashi teaches ITGB4 is used to select basal cells of the stratified epithelium, rich in epithelial stem/progenitor cells and SSEA4 is expressed in corneal epithelial cells as well as in a significant proportion of stem and progenitor cells in the corneal epithelial stem cell niche at the edge of the cornea at the limbus of the eye (page 687, right col. para. 1). Hayashi does not teach CD200 antibody or CD200-negative cells of step (3). However, Hayashi teaches isolation of TRA-1-60 cells that are SSEA-4 positive and ITGB4-positive in Figure 4c -d where TRA-1-60 is a pluripotent stem cell marker and is used to remove undifferentiated iPS cells (page 687, right col. para. 1).
Regarding “conjunctival” cells, Hayashi-2016 teaches the SEAM formation from iPS cells method of Hayashi in Figure 1a (page 376, left col. para. 1). Hayashi-2016 teaches cells in SEAM zone 3 most closely resembles those of the presumptive ocular surface and these zone 3 cells were subjected to FACS to isolate a specific ocular surface lineage – corneal or conjunctival (page 377, left col. para. 2; Figure 3a). Hayashi teaches further differentiation of the SEAM for 4 weeks in differentiation media followed by 2 – 7 weeks in maintenance media resulted in both corneal and conjunctival epithelial cells in Figure 3a where the differentiation media containing a serum replacement (KSR), FGF2, and Y-27632 and the maintenance media contained KGF and Y-27632 and the serum-free supplement B27 (page 381, left col. para. 1 – 3). Hayashi-2016 teaches FGF2 in differentiation medium was not essential for corneal epithelial induction (page 381, left col. para. 3). Hayashi-2016 teaches conjunctival epithelial cells, conjunctival goblet cells, and conjunctival progenitor cells were found in the SEAM (Figure 3e – f; page 378, left col.; Extended Data Figure 4a). Hayashi-2016 teaches conjunctival cells are SSEA4 weakly positive in Extended Data Figure 4b and functional ocular surface epithelial lineage cells are ITGB4+ (Extended Data Figure 4 legend). Hayashi-2016 teaches cells in zone 3 could be developed for surgical use (page 378, left col.). Hayashi-2016 does not teach an EGF signaling activator in the differentiation and maintenance media or that both media do not contain KGF or CD200 antibody or CD200-negative cells. One would have been motivated to combine the teachings of Hayashi and Hayashi-2016 regarding a method of SEAM formation because both teach the same method and Hayashi-2016 teaches the method produces conjunctival cells.
Regarding an EGF signaling activator and the media does not contain KGF of step (2), Devi teaches a method for culturing and expanding and differentiating conjunctival progenitor cells obtained from a tissue biopsy in Example 1 where conjunctival progenitor cells were cultured in media containing human EGF (col. 17, lines 8 – 19 and 40 – 50). Devi teaches it was surprising that the method which uses a single step media for the proliferation and differentiation of the conjunctival cells could successfully produce a tissue system for transplant (col. 17, lines 51 – 54). Devi teaches the cells expressed MUC4 and MUC5AC (col. 19, lines 17 – 21). Devi teaches transplanting the conjunctival tissue on a rabbit model of conjunctival injury in Example 2 (col. 17, lines 63 – 66; col. 19, lines 57 – 67). Devi teaches the efficiency of the transplant was quantified by re-epithelization and reappearance of goblet cells at the site of injury in comparison to control animals (col. 20, lines 15 – 18; col. 21, lines 6 – 15; Table 2). Devi teaches there were no allergic reactions or signs of rejection and that the observation of human cells interspersed at the site of transplant suggests the cells play an important role in healing the injury (col. 20, lines 47 – 57; col. 21, lines 20 – 26). Devi teaches stem cells that continually replace conjunctival epithelium have been shown to reside in the conjunctival fornix and are known as conjunctival stem cells or progenitor conjunctival cells (col. 2, lines 15 – 22). Devi teaches the multiple layers of stratified conjunctival epithelium along with the interspersed goblet cells together are continuously regenerated maintaining the functional integrity of the ocular surface (col. 2, lines 32 – 36). Devi teaches a major role for the conjunctiva is to provide ocular surface hydration and lubrication through the production of tears by mucin-producing goblet cells (col. 2, lines 23 – 28). Devi teaches the conjunctiva supports the health of the corneal epithelium (col. 2, lines 43 – 47). Devi teaches that because the corneal epithelium is dependent on a healthy conjunctival surface to maintain clear vision, many ocular surface diseases initiate with conjunctival damage followed by secondary limbal and corneal damage (col. 2, lines 47 – 51). Devi teaches mucin deficiency is detected in alkali burns, chemical and thermal burns, Stevens-Johnson Syndrome, neurotrophic keratitis, and Ocular Cicatricial Pemphigoid (col. 2, lines 38 – 40). Devi teaches in grade 4 – 6 ocular burns in which the surviving corneal and conjunctival epithelium is thin to zero, the desirable ocular management may be to attempt a restorative and reconstructive intervention by re-establishing both corneal and conjunctival epithelial cover one after another (col. 3, lines 12 – 17). Devi teaches there is a need for more desirable conjunctival tissue equivalent (col. 4, lines 18 – 19). Therefore, Devi teaches a media for obtaining conjunctival cells containing EGF but does not teach the media contains a ROCK inhibitor. One would have been motivated to combine the teachings of Hayashi and Hayashi-2016 regarding a method of SEAM formation that produces conjunctival cells with the teachings of Devi regarding differentiation and expansion of conjunctival progenitor cells in media containing EGF because Hayashi-2016 teaches the method produces conjunctival cells from pluripotent cells thus eliminating the need for obtaining a tissue biopsy as taught in the method of Devi.
Kurosawa teaches ROCK inhibitor could promote the survival of dissociated human stem cells and is a useful tool in the cultivation of pluripotent stem cells (page 577, right col. para. 2). Kurosawa teaches pluripotent stem cells are susceptible to dissociation induced apoptosis and Y-27632 ROCK helps to control this and improve yield (page 578, left col. para. 2 and right col. para. 1). Kurosawa teaches Y-27632 has been used in various routine manipulations in stem cell research including (page 578, left col. para. 2; Figure 2). Kurosawa teaches Y-27632 increases the efficiency of colony formation of pluripotent stem cells in serum-free and feeder-free culture conditions and thus Y-27632 removes the problem of xenogeneic contaminants from serum and feeder layers and will encourage the use of stem cells for clinical applications (page 579, left col. para. 2). Kurosawa teaches Y-27632 enhances pluripotent stem cell differentiation (page 579, left col. para. 3 – 4).
Regarding “CD200” of step (3), Hayashi-285 teaches isolation of CD200-negative, ITGB4-positive and SSEA4-positive cells from a SEAM (page 8, 0066; page 10, 0102 - 0104). Hayashi-285 teaches selection with a CD200 antibody is better at removing impurities compared to TRA-1-60 (page 10, 0104).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Hayashi regarding a method of obtaining a SEAM population from pluripotent stem cells with differentiation media and maintenance media where the maintenance media contains a serum replacement and ITGB4 is used to select basal cells of the stratified epithelium, rich in epithelial stem/progenitor cells and SSEA4 is expressed in corneal epithelial cells as well as in a significant proportion of stem and progenitor cells in the corneal epithelial stem cell niche at the edge of the cornea at the limbus of the eye with the teachings of Hayashi-2016 regarding the method of Hayashi produces conjunctival cells where desired cells can be isolated by FACS with the teachings of Devi regarding a method of differentiating conjunctival progenitor cells in media containing EGF with the teachings of Kurosawa regarding a ROCK inhibitor promotes survival of pluripotent stem cells in serum-free media and enhances differentiation with the teachings of Hayashi-285 regarding CD200 selection is better than TRA-1-60 selection to remove impurities to arrive at the claimed method for producing conjunctival epithelial stem and progenitor cells, comprising: (1) obtaining colonies of a self-formed ectodermal autonomous multi-zone (SEAM) cell population by (a) culturing pluripotent stem cells in the presence of laminin 511E8 for 8 to 10 days in maintenance culture; and (b) culturing the pluripotent stem cells in a serum-free medium appropriate for animal cell culture for 4 weeks to induce autonomous differentiation to form the colonies of SEAM cell population; and (2) culturing the colonies of SEAM cell population in a conjunctival Differentiation medium for 3 to 5 weeks, and then inducing differentiation by culturing the colonies of SEAM cell population in a conjunctiva! epithelial maintenance medium for 2 to 6 weeks, wherein the conjunctival differentiation medium contains an epidermal growth factor (EGF) signaling activator and a ROCK inhibitor, wherein the conjunctival epithelial maintenance medium contains the EGF signaling activator, the ROCK inhibitor and serum replacement, and wherein neither the conjunctival differentiation medium nor the conjunctival epithelial maintenance medium contain a keratinocyte growth factor (KGF), and (3) isolating cells from the SEAM cell population with antibodies specific for ITGβ4, SSEA-4, and CD200, and wherein the isolated cells are CD200-negative, weakly SSEA-4-positive and ITGβ4- positive cells. One would have been motivated to combine the teachings of Hayashi, Hayashi-2016, Devi, Kurosawa, and Hayashi-285 in a method of producing a conjunctival tissue from pluripotent stem cells as Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development and SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise and Hayashi-2016 teaches cells in zone 3 could be developed for surgical use and Devi teaches there is a need for more desirable conjunctival tissue equivalent. One would have a reasonable expectation of success in combining the teachings as Hayashi teaches ITGB4 is used to select basal cells of the stratified epithelium, rich in epithelial stem/progenitor cells, Hayashi-2016 teaches SEAM contains conjunctival progenitor cells and conjunctival cells that are SSEA4 weakly positive and desired cells can be isolated by FACS and Hayashi-285 teaches ITGB4 is a marker of corneal epithelial cells and selection with a CD200 antibody is better at removing impurities compared to TRA-1-60 and Devi teaches media containing EGF but not KGF formed conjunctival tissue that could repair ocular injury in vivo.
8. Claim(s) 21 remains rejected under 35 U.S.C. 103 as being unpatentable over Hayashi (Hayashi, Ryuhei, et al. Nature Protocols 12.4 (2017): 683-696; previously cited), hereinafter Hayashi which is cited on the IDS filed 11/19/2020 in view of Hayashi-2016 (Hayashi, Ryuhei, et al. Nature 531.7594 (2016): 376-380; previously cited), hereinafter Hayashi-2016 which is cited on the IDS filed 11/19/2020 in view of Devi (US-8338175-B2; Filed 02/23/2007; Published 12/25/2012; previously cited), hereinafter Devi in view of Kurosawa (Kurosawa, Hiroshi. Journal of bioscience and bioengineering 114.6 (2012): 577-581.), hereinafter Kurosawa in view of Hayashi (WO-2016114285-A1; previously cited), hereinafter Hayashi-285. A machine translation of WO-2016114285 was previously provided. The translation was performed on December 20, 2023 of pages 1 - 30 of the original document.
Regarding step (1)(a), Hayashi teaches maintenance culture of pluripotent stem cells on laminin 511E8 for 10 days in serum-free medium (page 683, right col. para. 1; Figure 2; page 685, right col. para. 1; page 689, steps 1 – 7; page 690).
Regarding step (1)(b), Hayashi teaches culturing the pluripotent stem cells in serum-free medium (DM) for 4 weeks to form a SEAM (Figure 2; page 683, right col. para. 1; page 685, right col. para. 2; page 689, right col. “Differentiation Medium”; page 691, steps 27 – 34).
Regarding step (2), Hayashi does not teach a “conjunctival differentiation medium” comprising EGF signaling activator and ROCK inhibitor or “conjunctival epithelial maintenance medium” where neither media contains KGF. However, Hayashi teaches culturing SEAM cells in differentiation media for 4 weeks followed by culturing in CEM media for 2 to 8 weeks (“culturing the colonies of SEAM cell population in a conjunctival differentiation medium for 3 to 5 weeks, and then inducing differentiation by culturing the colonies of SEAM cell population in a conjunctival epithelial maintenance medium for 2 to 6 weeks”) to obtain corneal epithelial cells where the culture techniques to no require serum (page 683, right col. para. 2; Figure 2). Hayashi teaches cells in zone 3 have characteristics of ocular-surface ectoderm and are cultured in media containing growth factors and the ROCK inhibitor Y-27632 (“ROCK inhibitor”) for an additional 4 weeks (page 686, left col. and right col.; Table 1). Hayashi teaches the protocol is distinct because it generates a range of cellular anlages that have characteristics of several quite distinct tissues in the eye (page 683, right col. para. 2). Hayashi teaches the method comprises purification of cells by FACS using a combination of antibodies to only obtain corneal iPS-derived epithelial cells free of impurities (page 684, left col. para. 1). Hayashi teaches obtaining other cell types from the iPS cell-derived SEAM will likely require modifications to the protocol (page 685, left col. para. 1). Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development (page 685, left col. para. 2). Hayashi teaches SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise (page 685, left col. para. 2).
Regarding step (3), Hayashi teaches isolation of cells from the SEAM cell population using antibodies specific for SSEA-4 and ITGB4 where TRA-1-60 negative cells are weakly SSEA-4 positive, SSEA-4 positive or ITGB4 negative, (Figure 4; page 688, left col. Reagents; page 692, steps 42 – 53; page 693, steps 54 – 56; Figure 4). Hayashi teaches ITGB4 is used to select basal cells of the stratified epithelium, rich in epithelial stem/progenitor cells and SSEA4 is expressed in corneal epithelial cells as well as in a significant proportion of stem and progenitor cells in the corneal epithelial stem cell niche at the edge of the cornea at the limbus of the eye (page 687, right col. para. 1). Hayashi does not teach CD200 antibody or CD200-negative cells of step (3). However, Hayashi teaches isolation of TRA-1-60 cells that are SSEA-4 positive and ITGB4-positive in Figure 4c -d where TRA-1-60 is a pluripotent stem cell marker and is used to remove undifferentiated iPS cells (page 687, right col. para. 1).
Regarding “conjunctival” cells, Hayashi-2016 teaches the SEAM formation from iPS cells method of Hayashi in Figure 1a (page 376, left col. para. 1). Hayashi-2016 teaches cells in SEAM zone 3 most closely resembles those of the presumptive ocular surface and these zone 3 cells were subjected to FACS to isolate a specific ocular surface lineage – corneal or conjunctival (page 377, left col. para. 2; Figure 3a). Hayashi teaches further differentiation of the SEAM for 4 weeks in differentiation media followed by 2 – 7 weeks in maintenance media resulted in both corneal and conjunctival epithelial cells in Figure 3a where the differentiation media containing a serum replacement (KSR), FGF2, and Y-27632 and the maintenance media contained KGF and Y-27632 and the serum-free supplement B27 (page 381, left col. para. 1 – 3). Hayashi-2016 teaches FGF2 in differentiation medium was not essential for corneal epithelial induction (page 381, left col. para. 3). Hayashi-2016 teaches conjunctival epithelial cells, conjunctival goblet cells, and conjunctival progenitor cells were found in the SEAM (Figure 3e – f; page 378, left col.; Extended Data Figure 4a). Hayashi-2016 teaches conjunctival cells are SSEA4 weakly positive in Extended Data Figure 4b (Extended Data Figure 4 legend). Hayashi-2016 teaches cells in zone 3 could be developed for surgical use (page 378, left col.). Hayashi-2016 does not teach an EGF signaling activator in the differentiation and maintenance media or that both media do not contain KGF or CD200 antibody or CD200-negative cells. One would have been motivated to combine the teachings of Hayashi and Hayashi-2016 regarding a method of SEAM formation because both teach the same method and Hayashi-2016 teaches the method produces conjunctival cells.
Regarding an EGF signaling activator and the media does not contain KGF of step (2), Devi teaches a method for culturing and expanding and differentiating conjunctival progenitor cells obtained from a tissue biopsy in Example 1 where conjunctival progenitor cells were cultured in media containing human EGF (col. 17, lines 8 – 19 and 40 – 50). Devi teaches it was surprising that the method which uses a single step media for the proliferation and differentiation of the conjunctival cells could successfully produce a tissue system for transplant (col. 17, lines 51 – 54). Devi teaches the cells expressed MUC4 and MUC5AC (col. 19, lines 17 – 21). Devi teaches transplanting the conjunctival tissue on a rabbit model of conjunctival injury in Example 2 (col. 17, lines 63 – 66; col. 19, lines 57 – 67). Devi teaches the efficiency of the transplant was quantified by re-epithelization and reappearance of goblet cells at the site of injury in comparison to control animals (col. 20, lines 15 – 18; col. 21, lines 6 – 15; Table 2). Devi teaches there were no allergic reactions or signs of rejection and that the observation of human cells interspersed at the site of transplant suggests the cells play an important role in healing the injury (col. 20, lines 47 – 57; col. 21, lines 20 – 26). Devi teaches stem cells that continually replace conjunctival epithelium have been shown to reside in the conjunctival fornix and are known as conjunctival stem cells or progenitor conjunctival cells (col. 2, lines 15 – 22). Devi teaches the multiple layers of stratified conjunctival epithelium along with the interspersed goblet cells together are continuously regenerated maintaining the functional integrity of the ocular surface (col. 2, lines 32 – 36). Devi teaches a major role for the conjunctiva is to provide ocular surface hydration and lubrication through the production of tears by mucin-producing goblet cells (col. 2, lines 23 – 28). Devi teaches the conjunctiva supports the health of the corneal epithelium (col. 2, lines 43 – 47). Devi teaches that because the corneal epithelium is dependent on a healthy conjunctival surface to maintain clear vision, many ocular surface diseases initiate with conjunctival damage followed by secondary limbal and corneal damage (col. 2, lines 47 – 51). Devi teaches mucin deficiency is detected in alkali burns, chemical and thermal burns, Stevens-Johnson Syndrome, neurotrophic keratitis, and Ocular Cicatricial Pemphigoid (col. 2, lines 38 – 40). Devi teaches in grade 4 – 6 ocular burns in which the surviving corneal and conjunctival epithelium is thin to zero, the desirable ocular management may be to attempt a restorative and reconstructive intervention by re-establishing both corneal and conjunctival epithelial cover one after another (col. 3, lines 12 – 17). Devi teaches there is a need for more desirable conjunctival tissue equivalent (col. 4, lines 18 – 19). Therefore, Devi teaches a media for obtaining conjunctival cells containing EGF but does not teach the media contains a ROCK inhibitor. One would have been motivated to combine the teachings of Hayashi and Hayashi-2016 regarding a method of SEAM formation that produces conjunctival cells with the teachings of Devi regarding differentiation and expansion of conjunctival progenitor cells in media containing EGF because Hayashi-2016 teaches the method produces conjunctival cells from pluripotent cells thus eliminating the need for obtaining a tissue biopsy as taught in the method of Devi.
Kurosawa teaches ROCK inhibitor could promote the survival of dissociated human stem cells and is a useful tool in the cultivation of pluripotent stem cells (page 577, right col. para. 2). Kurosawa teaches pluripotent stem cells are susceptible to dissociation induced apoptosis and Y-27632 ROCK helps to control this and improve yield (page 578, left col. para. 2 and right col. para. 1). Kurosawa teaches Y-27632 has been used in various routine manipulations in stem cell research including (page 578, left col. para. 2; Figure 2). Kurosawa teaches Y-27632 increases the efficiency of colony formation of pluripotent stem cells in serum-free and feeder-free culture conditions and thus Y-27632 removes the problem of xenogeneic contaminants from serum and feeder layers and will encourage the use of stem cells for clinical applications (page 579, left col. para. 2). Kurosawa teaches Y-27632 enhances pluripotent stem cell differentiation (page 579, left col. para. 3 – 4).
Regarding “CD200” of step (3), Hayashi-285 teaches isolation of CD200-negative, ITGB4-positive and SSEA4-positive cells from a SEAM (page 8, 0066; page 10, 0102 - 0104). Hayashi-285 teaches selection with a CD200 antibody is better at removing impurities compared to TRA-1-60 (page 10, 0104). Hayashi-285 teaches ITGB4 can be used to isolate corneal epithelial cells (page 8, 0066; page 9, 0089; page 10, 0102 – 0104).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Hayashi regarding a method of obtaining a SEAM population from pluripotent stem cells with differentiation media and maintenance media where the maintenance media contains a serum replacement and ITGB4 is used to select basal cells of the stratified epithelium, rich in epithelial stem/progenitor cells and SSEA4 is expressed in corneal epithelial cells as well as in a significant proportion of stem and progenitor cells in the corneal epithelial stem cell niche at the edge of the cornea at the limbus of the eye with the teachings of Hayashi-2016 regarding the method of Hayashi produces conjunctival cells where desired cells can be isolated by FACS with the teachings of Devi regarding a method of differentiating conjunctival progenitor cells in media containing EGF with the teachings of Kurosawa regarding a ROCK inhibitor promotes survival of pluripotent stem cells in serum-free media and enhances differentiation with the teachings of Hayashi-285 regarding CD200 selection is better than TRA-1-60 selection to remove impurities to arrive at the claimed method for producing conjunctival epithelial cells and conjunctival goblet cells, comprising: (1) obtaining colonies of a self-formed ectodermal autonomous multi-zone (SEAM) cell population by (a) culturing pluripotent stem cells in the presence of laminin 511E8 for 8 to 10 days in maintenance culture; and (b) culturing the pluripotent stem cells in a serum-free medium appropriate for animal cell culture for 4 weeks to induce autonomous differentiation to form the colonies of SEAM cell population; and (2) culturing the colonies of SEAM cell population in a conjunctival Differentiation medium for 3 to 5 weeks, and then inducing differentiation by culturing the colonies of SEAM cell population in a conjunctiva! epithelial maintenance medium for 2 to 6 weeks, wherein the conjunctival differentiation medium contains an epidermal growth factor (EGF) signaling activator and a ROCK inhibitor, wherein the conjunctival epithelial maintenance medium contains the EGF signaling activator, the ROCK inhibitor and serum replacement, and wherein neither the conjunctival differentiation medium nor the conjunctival epithelial maintenance medium contain a keratinocyte growth factor (KGF), and (3) isolating cells from the SEAM cell population with antibodies specific for ITGβ4, SSEA-4, and CD200, and wherein the isolated cells are selected from CD200-negative, weakly SSEA-4-positive and ITGβ4-negative cells and CD200-negative, SSEA-4-positive and ITGβ4-negative cells. One would have been motivated to combine the teachings of Hayashi, Hayashi-2016, Devi, Kurosawa, and Hayashi-285 in a method of producing a conjunctival tissue from pluripotent stem cells as Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development and SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise and Hayashi-2016 teaches cells in zone 3 could be developed for surgical use and Devi teaches there is a need for more desirable conjunctival tissue equivalent. One would have a reasonable expectation of success in combining the teachings as Hayashi teaches ITGB4 is used to select basal cells of the stratified epithelium, rich in epithelial stem/progenitor cells, Hayashi-2016 teaches SEAM contains conjunctival progenitor cells and conjunctival cells that are SSEA4 weakly positive and desired cells can be isolated by FACS and Hayashi-285 teaches ITGB4 is a marker of corneal epithelial cells and selection with a CD200 antibody is better at removing impurities compared to TRA-1-60 and Devi teaches media containing EGF but not KGF formed conjunctival tissue that could repair ocular injury in vivo.
Maintained Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
9. Claim 1, 13, and 21 remain rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 – 15 of U.S. Patent No. 12247219 in view of Hayashi-2016 (Hayashi, Ryuhei, et al. Nature 531.7594 (2016): 376-380; previously cited), hereinafter Hayashi-2016 which is cited on the IDS filed 11/19/2020.
Patent claim 1 recites a method for producing a colony consisting of concentric circular-like zones of different ectodermal cell lineages of ocular cells. Patent claim 1 lacks the steps of differentiating SEAM colonies and isolating cells of the SEAM colonies of the instant claims. The specification of the instant application defines SEAM (Self-formed Ectodermal Autonomous Multi-zone) as multi-zone concentric circular structure (page 4, 0012). Therefore, the patent claims and instant claims are both drawn to a method of producing SEAM. Hayashi-2016 teaches a method of SEAM formation from pluripotent stem cells and differentiation of zone 3 colonies into conjunctival cells and isolation of desired cell types by FACS (page 377, left col. para. 2; page 378, left col.; Figure 3; Extended Data Figure 4; page 381, left col. para. 3). Hayashi-2016 teaches the SEAM mimic whole-eye development because of the different ocular lineages present in the SEAM and thus represents a promising resource for new and ongoing studies of ocular morphogenesis and has translational potential (Abstract). Hayashi-2016 teaches that the method produces a construct capable of treating ocular disease in a rabbit model (page 379, right col.).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to have modified the method of producing a colony consisting of concentric circular-like zones of different ectodermal cell lineages of ocular cells of Patent claim 1 to include the method of differentiation of SEAM colonies to conjunctival cells and isolation of conjunctival cells of Hayashi-2016 to study ocular development and obtain a construct capable of treating ocular disease. One would have been motivated to make the combination as Hayashi-2016 teaches the SEAM mimics whole-eye development because of the different ocular lineages present in the SEAM and thus represents a promising resource for new and ongoing studies of ocular morphogenesis and has translational potential.
10. Claims 1, 13, and 21 remain provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 – 3 and 5 – 7 of copending Application No. 18852235 in view of Hayashi-2016 (Hayashi, Ryuhei, et al. Nature 531.7594 (2016): 376-380; previously cited), hereinafter Hayashi-2016 which is cited on the IDS filed 11/19/2020.
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims and reference claims are drawn to a method of producing conjunctival cells. Reference claim 1 broadly recites “differentiating pluripotent stem cells into conjunctival epithelial stem and progenitor cells” and “collecting cells expressing a conjunctival epithelial marker” and “culturing the cells expressing the conjunctival epithelial marker”. Reference claim 3 recites culturing in a medium containing an EGF receptor agonist and instant claim 1, 13, and 21 recite medium containing an EGF signaling activator. Reference claim 5 recites inducing SEAM from pluripotent stem cells and instant claim 1, 13, and 21 recite forming a SEAM from pluripotent stem cells. Instant claims 13 and 21 recite isolating conjunctival cells from the SEAM population with ITGB4, SSEA-4 and CD200 antibodies. Therefore, the reference and instant claims are drawn to a method of producing conjunctival cells from a SEAM obtained from pluripotent stem cells.
The reference claims lack a ROCK inhibitor in the culture medium as recited in instant claim 1. Hayashi-2016 teaches a method of SEAM formation from pluripotent stem cells and differentiation of zone 3 colonies into conjunctival cells and isolation of desired cell types by FACS where the culture medium contains the ROCK inhibitor Y-27632 (page 377, left col. para. 2; page 378, left col.; Figure 3; Extended Data Figure 4; page 381, left col. para. 3). Hayashi-2016 teaches the SEAM mimic whole-eye development because of the different ocular lineages present in the SEAM and thus represents a promising resource for new and ongoing studies of ocular morphogenesis and has translational potential (Abstract). Hayashi-2016 teaches that the method produces a construct capable of treating ocular disease in a rabbit model (page 379, right col.).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to have modified the method of producing conjunctival epithelial cell population of Reference claim 1 to include the method of differentiation of SEAM colonies to conjunctival cells in media containing a ROCK inhibitor and isolation of conjunctival cells of Hayashi-2016 to study ocular development and obtain a construct capable of treating ocular disease. One would have been motivated to make the combination as Hayashi-2016 teaches the SEAM mimics whole-eye development because of the different ocular lineages present in the SEAM and thus represents a promising resource for new and ongoing studies of ocular morphogenesis and has translational potential.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Rejections Necessitated by Amendment
11. Claim(s) 1 and 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hayashi (Hayashi, Ryuhei, et al. Nature Protocols 12.4 (2017): 683-696; previously cited), hereinafter Hayashi which is cited on the IDS filed 11/19/2020 in view of Hayashi-2016 (Hayashi, Ryuhei, et al. Nature 531.7594 (2016): 376-380; previously cited), hereinafter Hayashi-2016 which is cited on the IDS filed 11/19/2020 in view of Devi (US-8338175-B2; Filed 02/23/2007; Published 12/25/2012; previously cited), hereinafter Devi in view of Kurosawa (Kurosawa, Hiroshi. Journal of bioscience and bioengineering 114.6 (2012): 577-581; previously cited), hereinafter Kurosawa.
Hayashi in view of Hayashi-2016, Devi, and Kurosawa make obvious the limitations of claim 1 as set forth above.
Regarding claim 22, Hayashi-2016 teaches that the whole SEAM colony contains conjunctival cells in zone 3 that are PAX6+ (conjunctival progenitors) and K13+K12- (conjunctival epithelium) after 7 – 8 weeks and MUC5AC+K7+ conjunctival goblet cells after more than 12 weeks in culture (Figure 3b; page 378, left col.; Extended Data Figure 4a and 5d; page 381, left col. para. 3; Figure 3a; page 381, left col. para. 3). Hayashi-2016 teaches zone 3 cells were isolated for characterization but that these cells are present in the whole SEAM colony (page 377, left col. para. 2; page 378, left col.; Extended Data Figure 4a). Hayashi-2016 also teaches differentiation of the whole SEAM colony by a protocol which encourages retinal differentiation and RPE cells were formed in zone 2 after 7 weeks in culture (page 376, right col. para. 1; Figure 2c,d). Thus, Hayashi-2016 teaches that whole SEAM colonies can be differentiated into conjunctival epithelial, goblet, and progenitor cells where conjunctival epithelial, progenitors, and goblet cells are formed in culture media that contains Y-27632 and a serum replacement but lacks EGF.
Devi teaches conjunctival progenitors can be cultured, expanded, and differentiated in media containing EGF that does not contain KGF and after 20 days in this media, goblet cells were observed that expressed MUC5AC (col. 17, lines 8 – 62; col. 19, lines 14 – 21; Figure 1E). Devi teaches the method produces a tissue system for the treatment of an injury with distorted epithelial cells and lacking goblet cells (Table 2; col. 19, lines 64 – 65). Devi teaches one month after transplanting the tissue system at the site of injury, a multilayered epithelium was formed and goblet cells reappeared (Table 2; col. 20, lines 53 – 57 and 62 – 63; col. 21, lines 6 – 15).
Devi teaches cell therapy has the potential to treat any disease that is associated with cell dysfunction or damage, including the potential for manipulating stem cells, whether embryonic stem cells or adult stem cells to repair or replace diseased or damaged tissue (col. 1, lines 54 – 60). Devi teaches adult stem cells are essential for the maintenance of a healthy ocular surface (col. 2, lines 2 – 6). Devi teaches conjunctival stem or progenitor cells continually replace conjunctival epithelium (col. 2, lines 15 – 22). Devi teaches the multiple layers of stratified conjunctival epithelium along with the interspersed goblet cells together are continuously regenerated maintaining the functional integrity of the ocular surface (col. 2, lines 32 – 37). Devi teaches the conjunctiva supports the health of the corneal epithelium and many ocular surface diseases initiate with conjunctival damage followed by corneal damage (col. 2, lines 46 – 51). Devi teaches a need for a more desirable conjunctival tissue equivalent (col. 4, lines 18 – 19).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Hayashi regarding a method of obtaining SEAM colonies with the teachings of Hayahsi-2016 regarding a method of differentiating whole SEAM colonies in a culture medium containing Y-27632 to form conjunctival cells with the teachings of Devi regarding a method of culturing conjunctival progenitors in media that contains EGF for 20 days to form conjunctival goblet cells with the teachings of Kurosawa regarding a ROCK inhibitor promotes survival of pluripotent stem cells in serum-free media and enhances differentiation to arrive at the claimed method comprising step (2) of culturing whole SEAM colonies in a differentiation medium for 3 to 5 weeks and then culturing the whole SEAM colonies in a maintenance medium for 2 to 6 weeks wherein the differentiation medium contains EGF and Y-27632 and the maintenance medium contains EGF, Y-27632, and serum replacement and neither medium contains a KGF. One would have been motivated to combine the teachings of Hayashi, Hayashi-2016, Devi, and Kurosawa in a method of generating a conjunctival tissue from pluripotent stem cells as Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development and SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise and Hayashi-2016 teaches cells in zone 3 could be developed for surgical use and Devi teaches there is a need for more desirable conjunctival tissue equivalent. One would have a reasonable expectation of success in combining the teachings as Hayashi-2016 teaches SEAM contains conjunctival cells and the whole SEAM colonies can be differentiated to produce cells within a specific zone of the SEAM and Devi teaches media containing EGF but not KGF formed conjunctival tissue that could repair ocular injury in vivo.
12. Claim(s) 13 and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hayashi (Hayashi, Ryuhei, et al. Nature Protocols 12.4 (2017): 683-696; previously cited), hereinafter Hayashi which is cited on the IDS filed 11/19/2020 in view of Hayashi-2016 (Hayashi, Ryuhei, et al. Nature 531.7594 (2016): 376-380; previously cited), hereinafter Hayashi-2016 which is cited on the IDS filed 11/19/2020 in view of Devi (US-8338175-B2; Filed 02/23/2007; Published 12/25/2012; previously cited), hereinafter Devi in view of Kurosawa (Kurosawa, Hiroshi. Journal of bioscience and bioengineering 114.6 (2012): 577-581; previously cited), hereinafter Kurosawa in view of Hayashi (WO-2016114285-A1; previously cited), hereinafter Hayashi-285. A machine translation of WO-2016114285 was previously provided. The translation was performed on December 20, 2023 of pages 1 - 30 of the original document.
Hayashi in view of Hayashi-2016, Devi, Kurosawa, and Hayashi-285 make obvious the limitations of claim 13 as set forth above.
Regarding claim 23, Hayashi-2016 teaches that the whole SEAM colony contains conjunctival cells in zone 3 that are PAX6+ (conjunctival progenitors) and K13+K12- (conjunctival epithelium) after 7 – 8 weeks and MUC5AC+K7+ conjunctival goblet cells after more than 12 weeks in culture (Figure 3b; page 378, left col.; Extended Data Figure 4a and 5d; page 381, left col. para. 3; Figure 3a; page 381, left col. para. 3). Hayashi-2016 teaches zone 3 cells were isolated for characterization but that these cells are present in the whole SEAM colony (page 377, left col. para. 2; page 378, left col.; Extended Data Figure 4a). Hayashi-2016 also teaches differentiation of the whole SEAM colony by a protocol which encourages retinal differentiation and RPE cells were formed in zone 2 after 7 weeks in culture (page 376, right col. para. 1; Figure 2c,d). Thus, Hayashi-2016 teaches that whole SEAM colonies can be differentiated into conjunctival epithelial, goblet, and progenitor cells where conjunctival epithelial, progenitors, and goblet cells are formed in culture media that contains Y-27632 and a serum replacement but lacks EGF.
Devi teaches conjunctival progenitors can be cultured, expanded, and differentiated in media containing EGF that does not contain KGF and after 20 days in this media, goblet cells were observed that expressed MUC5AC (col. 17, lines 8 – 62; col. 19, lines 14 – 21; Figure 1E). Devi teaches the method produces a tissue system for the treatment of an injury with distorted epithelial cells and lacking goblet cells (Table 2; col. 19, lines 64 – 65). Devi teaches one month after transplanting the tissue system at the site of injury, a multilayered epithelium was formed and goblet cells reappeared (Table 2; col. 20, lines 53 – 57 and 62 – 63; col. 21, lines 6 – 15).
Devi teaches cell therapy has the potential to treat any disease that is associated with cell dysfunction or damage, including the potential for manipulating stem cells, whether embryonic stem cells or adult stem cells to repair or replace diseased or damaged tissue (col. 1, lines 54 – 60). Devi teaches adult stem cells are essential for the maintenance of a healthy ocular surface (col. 2, lines 2 – 6). Devi teaches conjunctival stem or progenitor cells continually replace conjunctival epithelium (col. 2, lines 15 – 22). Devi teaches the multiple layers of stratified conjunctival epithelium along with the interspersed goblet cells together are continuously regenerated maintaining the functional integrity of the ocular surface (col. 2, lines 32 – 37). Devi teaches the conjunctiva supports the health of the corneal epithelium and many ocular surface diseases initiate with conjunctival damage followed by corneal damage (col. 2, lines 46 – 51). Devi teaches a need for a more desirable conjunctival tissue equivalent (col. 4, lines 18 – 19).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Hayashi regarding a method of obtaining SEAM colonies with the teachings of Hayahsi-2016 regarding a method of differentiating whole SEAM colonies in a culture medium containing Y-27632 to form conjunctival cells with the teachings of Devi regarding a method of culturing conjunctival progenitors in media that contains EGF for 20 days to form conjunctival goblet cells with the teachings of Kurosawa regarding a ROCK inhibitor promotes survival of pluripotent stem cells in serum-free media and enhances differentiation to arrive at the claimed method comprising step (2) of culturing whole SEAM colonies in a differentiation medium for 3 to 5 weeks and then culturing the whole SEAM colonies in a maintenance medium for 2 to 6 weeks wherein the differentiation medium contains EGF and Y-27632 and the maintenance medium contains EGF, Y-27632, and serum replacement and neither medium contains a KGF. One would have been motivated to combine the teachings of Hayashi, Hayashi-2016, Devi, and Kurosawa in a method of generating a conjunctival tissue from pluripotent stem cells as Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development and SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise and Hayashi-2016 teaches cells in zone 3 could be developed for surgical use and Devi teaches there is a need for more desirable conjunctival tissue equivalent. One would have a reasonable expectation of success in combining the teachings as Hayashi-2016 teaches SEAM contains conjunctival cells and the whole SEAM colonies can be differentiated to produce cells within a specific zone of the SEAM and Devi teaches media containing EGF but not KGF formed conjunctival tissue that could repair ocular injury in vivo.
13. Claim(s) 21 and 24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hayashi (Hayashi, Ryuhei, et al. Nature Protocols 12.4 (2017): 683-696; previously cited), hereinafter Hayashi which is cited on the IDS filed 11/19/2020 in view of Hayashi-2016 (Hayashi, Ryuhei, et al. Nature 531.7594 (2016): 376-380; previously cited), hereinafter Hayashi-2016 which is cited on the IDS filed 11/19/2020 in view of Devi (US-8338175-B2; Filed 02/23/2007; Published 12/25/2012; previously cited), hereinafter Devi in view of Kurosawa (Kurosawa, Hiroshi. Journal of bioscience and bioengineering 114.6 (2012): 577-581; previously cited), hereinafter Kurosawa.
Hayashi in view of Hayashi-2016, Devi, and Kurosawa make obvious the limitations of claim 21 as set forth above.
Regarding claim 24, Hayashi-2016 teaches that the whole SEAM colony contains conjunctival cells in zone 3 that are PAX6+ (conjunctival progenitors) and K13+K12- (conjunctival epithelium) after 7 – 8 weeks and MUC5AC+K7+ conjunctival goblet cells after more than 12 weeks in culture (Figure 3b; page 378, left col.; Extended Data Figure 4a and 5d; page 381, left col. para. 3; Figure 3a; page 381, left col. para. 3). Hayashi-2016 teaches zone 3 cells were isolated for characterization but that these cells are present in the whole SEAM colony (page 377, left col. para. 2; page 378, left col.; Extended Data Figure 4a). Hayashi-2016 also teaches differentiation of the whole SEAM colony by a protocol which encourages retinal differentiation and RPE cells were formed in zone 2 after 7 weeks in culture (page 376, right col. para. 1; Figure 2c,d). Thus, Hayashi-2016 teaches that whole SEAM colonies can be differentiated into conjunctival epithelial, goblet, and progenitor cells where conjunctival epithelial, progenitors, and goblet cells are formed in culture media that contains Y-27632 and a serum replacement but lacks EGF.
Devi teaches conjunctival progenitors can be cultured, expanded, and differentiated in media containing EGF that does not contain KGF and after 20 days in this media, goblet cells were observed that expressed MUC5AC (col. 17, lines 8 – 62; col. 19, lines 14 – 21; Figure 1E). Devi teaches the method produces a tissue system for the treatment of an injury with distorted epithelial cells and lacking goblet cells (Table 2; col. 19, lines 64 – 65). Devi teaches one month after transplanting the tissue system at the site of injury, a multilayered epithelium was formed and goblet cells reappeared (Table 2; col. 20, lines 53 – 57 and 62 – 63; col. 21, lines 6 – 15).
Devi teaches cell therapy has the potential to treat any disease that is associated with cell dysfunction or damage, including the potential for manipulating stem cells, whether embryonic stem cells or adult stem cells to repair or replace diseased or damaged tissue (col. 1, lines 54 – 60). Devi teaches adult stem cells are essential for the maintenance of a healthy ocular surface (col. 2, lines 2 – 6). Devi teaches conjunctival stem or progenitor cells continually replace conjunctival epithelium (col. 2, lines 15 – 22). Devi teaches the multiple layers of stratified conjunctival epithelium along with the interspersed goblet cells together are continuously regenerated maintaining the functional integrity of the ocular surface (col. 2, lines 32 – 37). Devi teaches the conjunctiva supports the health of the corneal epithelium and many ocular surface diseases initiate with conjunctival damage followed by corneal damage (col. 2, lines 46 – 51). Devi teaches a need for a more desirable conjunctival tissue equivalent (col. 4, lines 18 – 19).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Hayashi regarding a method of obtaining SEAM colonies with the teachings of Hayahsi-2016 regarding a method of differentiating whole SEAM colonies in a culture medium containing Y-27632 to form conjunctival cells with the teachings of Devi regarding a method of culturing conjunctival progenitors in media that contains EGF for 20 days to form conjunctival goblet cells with the teachings of Kurosawa regarding a ROCK inhibitor promotes survival of pluripotent stem cells in serum-free media and enhances differentiation to arrive at the claimed method comprising step (2) of culturing whole SEAM colonies in a differentiation medium for 3 to 5 weeks and then culturing the whole SEAM colonies in a maintenance medium for 2 to 6 weeks wherein the differentiation medium contains EGF and Y-27632 and the maintenance medium contains EGF, Y-27632, and serum replacement and neither medium contains a KGF. One would have been motivated to combine the teachings of Hayashi, Hayashi-2016, Devi, and Kurosawa in a method of generating a conjunctival tissue from pluripotent stem cells as Hayashi teaches SEAMs generated by the protocol have potential use as in vitro cellular models for human eye development and is of substantial interest because most current information is based on mouse or avian eye development and SEAM has the potential to act as a significant research resource for human ocular cells which are difficult to obtain otherwise and Hayashi-2016 teaches cells in zone 3 could be developed for surgical use and Devi teaches there is a need for more desirable conjunctival tissue equivalent. One would have a reasonable expectation of success in combining the teachings as Hayashi-2016 teaches SEAM contains conjunctival cells and the whole SEAM colonies can be differentiated to produce cells within a specific zone of the SEAM and Devi teaches media containing EGF but not KGF formed conjunctival tissue that could repair ocular injury in vivo.
Applicant’s Arguments/ Response to Arguments
14. Applicant Argues: On page 6 para. 3 – page 7 para. 3, Applicant asserts that a person skilled in the art would reasonably understand that the colonies of SEAM cell used in step (2) of claim 1 do not contain conjunctival progenitor cells as disclosed in Devi and therefore a person skilled in the art would not expect that conjunctival tissue could be obtained as described in Devi by culturing cells that exist only during embryonic development as described in Hayashi-2016 and accordingly a person of ordinary skill in the art would not be motivated to combine Devi with Hayahsi-2016 and/or Hayashi to achieve the claimed invention with a reasonable expectation of success. Applicant asserts that the skilled person would not have had a reasonable expectation of success of arriving at the claimed invention because one would have to substantively change the principle of operation of the prior art to arrive at the claimed invention in view of the differences between the cells used in Devi and Hayashi-2016. On page 7, last para. and page 8, para. 4, Applicant asserts that Kurosawa does not disclose or suggest anything about conjunctival cells or EGF-containing medium and therefore cannot cure the defects of Devi and Hayashi-2016 and Hayashi-285 cannot cure the deficiencies of Hayashi, Hayashi-2016, Devi and/or Kurosawa as Hayashi-285 does not disclose or suggest anything about conjunctival cells or EGF-containing medium. On page 9, para. 2, Applicant asserts that the dependent claims are in condition for allowance.
Response to Arguments: This is not found persuasive because Hayashi-2016 teaches the SEAM colony contains conjunctival progenitors in Extended Data Figure 4 and Devi teaches conjunctival progenitors can be differentiated in media containing EGF where a conjunctival tissue equivalent containing conjunctival goblet cells is formed (col. 17, lines 40 – 62; col. 19, lines 11 – 21). Devi teaches that the media containing EGF was used for proliferation and differentiation of the conjunctival progenitor cells. Further, one of ordinary skill in the art would know and Devi teaches that conjunctival stem or progenitor cells continually replace conjunctival epithelium and conjunctival stem or progenitor cells reside in the conjunctiva and adult conjunctiva tissue contains conjunctival stem or progenitor cells (col. 2, lines 15 – 22; col. 16, lines 30 – 50). Therefore, a person of ordinary skill in the art would have a reasonable expectation of success that conjunctival progenitor cells in the SEAM colony of Hayashi-2016 could be cultured in media containing EGF of Devi to form a conjunctival tissue equivalent containing conjunctival progenitors and goblet cells and would be motivated to combine Hayashi, Hayashi-2016, and Devi to culture whole SEAM colonies (see rejection of new claims 22 – 24 set forth above) in media containing EGF to form conjunctival cells as Hayashi-2016 teaches cells in zone 3 could be developed for surgical use and Devi teaches there is a need for more desirable conjunctival tissue equivalent. Therefore, all previous rejections set forth in the Office action mailed 10/17/2025 are maintained and new claims 22 – 24 are rejected over the prior art.
Applicant Argues: On page 8, last two paras. And page 9, para. 1, Applicant cites MPEP 804 and Applicant submits that the claims are in condition for allowance aside from the double-patenting rejections.
Response to Arguments: This is not found persuasive as the claims (previous and new) are rejected over prior art and therefore not in condition for allowance.
Conclusion
No claims allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Z.M.B./Examiner, Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632