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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 04/07/2026 has been entered.
1. Claims 1 – 4, 6, 8, 9, 11 – 13, 15 – 28, 30, 31, 33, 48 – 50, 52, and 53 are pending. Claims 1 – 4, 6, 8, 9, 11 – 13, 15 – 28, 48 – 50, 52, and 53 are under consideration.
Withdrawn Claim Objections
2. The objections to claim 1 are withdrawn in view of Applicant’s amendment to claim 1.
Withdrawn Claim Rejections
3. The rejection of claims 1 – 4, 6, 8, 9, 11, 12, and 48 – 50 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments that Parent teaches the use of the Shh inhibitor cyclopamine.
4. The rejection of claim 47 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of the claim.
5. The rejection of claim 13 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments that Parent teaches the use of the Shh inhibitor cyclopamine.
6. The rejection of claim 51 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of the claim.
7. The rejection of claims 15 – 27 and 52 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments that Parent teaches the use of the Shh inhibitor cyclopamine.
8. The rejection of claim 28 under 35 U.S.C. 103 is withdrawn in view of Applicant’s arguments that Parent teaches the use of the Shh inhibitor cyclopamine.
Claim Objections
9. Claim 48 is objected to because of the following informalities: in line 1, “TEPS” should read “TEPs” for consistency with the abbreviation of thymic epithelial progenitors recited in claim 1. Appropriate correction is required.
10. Claim 53 is objected to because of the following informalities: in line 6, “(i)” should be deleted because there is no recitation of any addition steps. Appropriate correction is required.
11. Claim 53 is objected to because of the following informalities: in line 21, “TEPS” should read “TEPs” for consistency with the abbreviation of thymic epithelial progenitors recited in line 2. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
12. Claims 48, 49, and 53 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
13. Regarding claim 48, it is unclear how the scope of claim 48 relates to the scope of claim 1 because claim 1 recites “reconstitution in vivo” but the scope of independent claim 1 is a method of inducing differentiation of pluripotent stem cells into TECs or TEPs in vitro. Further, claim 48 does not recite any active method steps related to the scope of claim 1.
14. Regarding claim 52, it is unclear how the scope of claim 52 relates to the scope of claim 15 because claim 15 recites “reconstitution in vivo” but the scope of independent claim 15 is a method of inducing differentiation of pluripotent stem cells into TECs or TEPs. Further, claim 52 does not recite any active method steps related to the scope of claim 15.
15. Regarding claim 53, it is unclear how recitation of “wherein the TEPS or TECS support T cell reconstitution in vivo” relates to the scope of claim because of a method of inducing differentiation of pluripotent stem cells into TECs or TEPs in vitro. Further, no active steps are recited in “wherein the TEPS or TECS support T cell reconstitution in vivo”.
Claim Interpretation
16. For the purpose of applying prior art, step (b) of claim 1 is interpreted as culturing the DE with Noggin, SB431542, retinoic acid, and FGF8b.
17. For the purpose of applying prior art, step (c) of claim 1 is interpreted as continued culturing of the cells of step (b) with retinoic acid and FGF8b with addition of Shh.
18. For the purpose of applying prior art, step (d) is interpreted as culturing the cells of step (c) with Noggin.
19. For the purpose of applying prior art, step (e) is interpreted as culturing the cells of step (d) with BMP4.
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.
20. Claim(s) 1 – 4, 6, 8, 9, 11, 12, 48, and 49 is/are rejected under 35 U.S.C. 103 as being unpatentable over Green (Green, Michael D., et al. Nature biotechnology 29.3 (2011): 267-272.), hereinafter Green which is cited on the IDS filed 10/01/2021 in view of Wendling (Wendling, Olivia, et. al. Development 127.8 (2000): 1553-1562.), hereinafter Wendling which is cited on the IDS filed 10/01/2021 in view of Bain (Bain, Virginia E., et al. Development 143.21 (2016): 4027-4037.), hereinafter Bain in view of Swann (Swann, Jeremy B., et al. Scientific Reports 7.1 (2017): 8492.), hereinafter Swann which is cited on the IDS filed 10/01/2021 in view of Patel (Patel, Seema R., et al. Gene expression patterns 6.8 (2006): 794-799.), hereinafter Patel in view of Parent (Parent, Audrey V., et al. Cell stem cell 13.2 (2013): 219-229.), hereinafter Parent which is cited on the IDS filed 10/01/2021 and 01/06/2023 in view of Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795; previously cited), hereinafter Wei.
Regarding step (a) of claim 1, Green teaches a method of differentiating pluripotent stem cells into DE cells (page 2, last para.; Figure 1a and 1f; Figure 3g; Figure 4; Supplementary Figure 3b).
Regarding step (b) (i) of claim 1, Green teaches contacting DE with Noggin and SB431542 to form AFE that is competent to be pattered along dorsoventral and anteroposterior axes and expresses TBX1 (Abstract; page 3, para. 1 – 3; Figure 1f; Figure 3d, e, g; Figure 4; Supplementary Figure 3b; page 4, para. 1 – 2). Green teaches ventral AFE is formed by treatment of DE with Noggin/SB431542 (Figure 4 legend; page 5, para. 1). Green teaches the thymus and parathyroids develop from the 3rd pouch (page 2, para. 1).
Regarding step (b) (ii) of claim 1 and step (c) (i) of claim 1, Green teaches addition of FGF8b or retinoic acid individually causes a dose dependent increase in TBX1 and PAX9 where PAX9 was used as a marker of pharyngeal endoderm (Figure 1c, conditions 8 – 10 and 19 and 20; page 3, para. 1). Green does not teach addition of FGF8b and retinoic acid. However, Green teaches addition of FGF8 induced the parathyroid-specific marker GCM2 and Green teaches retinoic acid prevents expression of GCM2 (Figure 4b; page 5, last para.). Green teaches FGF8 is a marker specific for pharyngeal pouch endoderm (page 3, last para.).
Regarding step (c) (ii) of claim 1, Green teaches addition of FGF8 and SHH induces expression of the pharyngeal pouch marker GCM2 in Figure 4b and GCM2 is a parathyroid marker (page 5, last para.). Therefore, Green teaches induction of pharyngeal endoderm. Green teaches Shh is upstream of FGF8 in mouse pharyngeal pouch development because the combination of SHH and FGF8 was not additive (page 8, last para.; Figure 4b). Green teaches AFE expresses TBX1, PAX1 and PAX9 (Supplementary Figure 1b and 1c). Green does not teach addition of FGF8 and SHH immediately after Noggin/SB431542 treatment.
Regarding step (e) of claim 1, Green teaches transplanting the cells formed by treatment with Noggin/SB431542 into mice where islands of PAX9 and AIRE expressing cells were detected where AIRE is specific for medullary thymic epithelial cells (page 4, last para.). Green does not teach in vitro differentiation to TECs with BMP4.
Regarding “wherein the TEPs or TECs are FOXN1…positive” of claim 1, Green teaches the cells express PAX1, PAX9 and AIRE (page 5, para. 1; Figure 3a – 3). Green does not teach the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive. However, Green teaches Noggin/SB431542 cells expressed TBX1, PAX9, and the pharyngeal endoderm marker FOXG1 (page 4, para. 1).
Regarding claim 2, Green teaches DE formation over 1 – 5 days (Figure 1c and 1f; page 6, last para.).
Regarding claim 3, Green teaches DE formation in serum-free medium with 1 ng/mL and 0.5 ng/mL of human BMP4, 2.5 ng/mL of human bFGF, and 100 ng/mL of human activin A (page 6, last para.; Figure 1a and 1c; Figure 4).
Regarding claim 4, Green teaches addition of Noggin/SB431542 at day 5 for 2 days (Figure 4b).
Regarding claim 6, Green teaches addition of FGF8 and SHH to AFE at day 11 for about 8 days (page 5, last para.; Figure 4b).
Regarding claim 8, Green teaches culturing with 50 ng/mL FGF8b or retinoic acid at 0.1 µM in Figure 1c (conditions 9 and 20).
Regarding claim 49, Green teaches 200 ng/mL human Noggin and 10 µM SB431542 (page 6, last para.).
Green does not teach addition of FGF8b and retinoic acid of (ii) of step (b) and (i) of step (c), or incubating cells with Noggin of step (d) or in vitro differentiation to TECs with BMP4 of step (e) of claim 1 or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive of claim 1 or “wherein step (d) is performed starting at about day 11 to about day 18, for about 4 days to about 7 days” of claim 9 or “wherein step (e) is performed starting at about day 13 to about day 25 for about 5 days to about 15 days” of claim 11 or “wherein step (e) comprises culturing with about 50 ng/ml of BMP4” of claim 12 or “wherein the TEPs support T cell reconstitution in vivo of claim 48. However, Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus (Abstract). Green teaches the thymic function is severely affected by allogeneic hematopoietic stem cell transplantation leading to profound defects in T cell reconstitution (page 2, para. 1). Green teaches as the thymus involutes with age, older transplant recipients would particularly benefit from thymic replacement therapy and additionally the thymus is absent in SCID and DiGeorge syndromes (page 2, para. 1).
Regarding addition of FGF8b and retinoic acid in step (b) (ii) and step (c) (i) of claim 1, Wendling teaches treating E8 mouse embryos with a retinoic acid signaling antagonist (BMS493) resulted in undetectable expression of Fgf8 in the 3rd pouch (page 1554, left col. para. 1; page 1557, right col. para. 4; Figure 7). Wendling teaches retinoic acid is essential for the formation of 3rd pharyngeal structures during a narrow developmental window corresponding to the 7 – 10 somite stages (page 1559, left col. last para. and right col. para. 1). Wendling teaches treatment with BMS493 caused a decrease in Pax1 and Pax9 expression in the pharyngeal endoderm (page 1560, left col. last para. and right col. para. 1). Wendling teaches Pax9 is indispensable for the formation of the thymus and it is most likely that the lack of Pax9 expression in the 3rd pouch under conditions of impaired retinoic acid signaling accounts for thymus agenesis observed in retinoic acid receptor knockout fetuses (page 1560, right col. para. 1). Wendling teaches the thymus and parathyroid originate from the 3rd pouch (page 1553, left col. para. 1). One would have been motivated to combine the teachings of Green and Wendling to culture with both retinoic acid and FGF8b for differentiation towards thymus instead of parathyroid because both Green and Wendling teach the thymus and parathyroids develop from the 3rd pouch and Wendling teaches retinoic acid is essential for formation of 3rd pharyngeal structures and Green teaches retinoic acid prevents expression of the parathyroid marker GCM2 and Wendling teaches FGF8 is absent when retinoic acid signaling is inhibited. Wendling does not teach incubating cells with Noggin of step (d) or in vitro differentiation to TECs with BMP4 of step (e) of claim 1 or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive of claim 1 or “wherein step (d) is performed starting at about day 11 to about day 18, for about 4 days to about 7 days” of claim 9 or “wherein step (e) is performed starting at about day 13 to about day 25 for about 5 days to about 15 days” of claim 11 or “wherein step (e) comprises culturing with about 50 ng/ml of BMP4” of claim 12 or “wherein the TEPs support T cell reconstitution in vivo of claim 48.
Regarding step (c) (ii) of claim 1, Bain teaches activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus (page 4028, left col. para. 1). Bain teaches activation of SHH signaling showed normal Tbx1 and Fgf8 expression (page 4030, right col. para. 2). Bain teaches activation of SHH signaling delayed initial cell fate specification and reduced proliferation within the developing primordium, followed by increased thymus and reduced parathyroid fate specification (page 4031, right col. para. 1 and right col. para. 1). Bain teaches ectopic SHH signaling did not lead to an increase in the number of cells expressing Gcm2 and did not completely block Foxn1 expression (page 4031, right col. para. 2). One would have been motivated to combine the teachings of Green and Bain to culture cells with FGF8b and SHH for differentiation towards thymus instead of parathyroid as both Green and Bain teach the thymus and parathyroids develop from the 3rd pouch and Bain teaches activation of SHH signaling in the most ventral pouch does not induce Gcm2 and Bain teaches activation of SHH signaling increased thymus and reduced parathyroid fate specification and Green teaches Shh is upstream of pharyngeal pouch development and addition of SHH to ventralized AFE induces pharyngeal pouch markers. Bain does not teach incubating cells with Noggin of step (d) or in vitro differentiation to TECs with BMP4 of step (e) of claim 1 or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive of claim 1 or “wherein step (d) is performed starting at about day 11 to about day 18, for about 4 days to about 7 days” of claim 9 or “wherein step (e) is performed starting at about day 13 to about day 25 for about 5 days to about 15 days” of claim 11 or “wherein step (e) comprises culturing with about 50 ng/ml of BMP4” of claim 12 or “wherein the TEPs support T cell reconstitution in vivo of claim 48.
Regarding incubating cells with Noggin of step (d) and incubating cells with BMP4 of step (e) of claim 1, Swann teaches dampening the initial period of BMP4 signaling during early thymus development with Noggin where BMP4 in the mesenchyme induces expression of Noggin creating a time-delayed feedback inhibition of BMP signaling (page 3, para. 3; Figure 1b). Swann teaches this results in the formation of Foxn1+ and Foxn1- cells indicating that sufficient levels of BMP4 signaling are required during early development to establish stable expression of Foxn1 (page 3, para. 3; Figure 1d). Swann teaches the extent of BMP signaling and Foxn1 dosage cooperate in early embryogenesis to establish the number of epithelial cells stably expressing Foxn1 which is the hallmark of a functionally competent thymic epithelium (page 3, para. 5). Swann teaches under the condition of limiting BMP4 signaling, fewer TEPs are established (page 3, last para.). Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs (page 5, para. 3). Swann teaches the BMP-mediated thymic epithelial differentiation process is sensitive to developmental timing (page 6, last para.; page 7, para. 1). Swann teaches sufficient BMP signals are available to the thymic rudiment only during a certain developmental window, possibly associated with a particular microenvironment (page 7, para. 1). Swann teaches Foxn1 gene dosage affects the fitness of TECs (page 7, para. 3). Swann teaches that even subtle changes in BMP signaling and Foxn1 expression levels could lead to unexpectedly severe effects with respect to thymopoietic activity (page 8, para. 3). Swann teaches during embryonic development, initial expression of Foxn1 is induced in pharyngeal endoderm by mesenchyme-derived BMP4 signals (Abstract; Figure 1a; page 3, para. 2). Swann teaches thymopoiesis irreversibly fails if Foxn1 gene expression does not occur during a defining time span in mid-gestation (Abstract). Swann teaches TECs are characterized by the expression of the FOXN1 (page 3, para. 2).
Regarding incubating cells with Noggin of step (d) and incubating cells with BMP4 of step (e) of claim 1, Patel teaches Bmp4 expression is localized to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5 mouse embryos in those cells that will express Foxn1 and form the thymus (Abstract; page 794, right col. para. 2; page 795, right col. para. 2). Patel teaches noggin expression was confined to the dorsal region of the pouch at the same stage (Abstract). Patel teaches Bmp signaling is tightly regulated by noggin (page 795, left col. para. 2). Patel teaches noggin expression has been reported in the pharyngeal arch mesenchyme at E9.5 suggesting the importance of restricting Bmp signaling during pharyngeal development (page 795, para. 2). Patel teaches at E9.5, the 3rd pharyngeal pouches have just formed and neither Bmp4 nor Noggin was expressed but Noggin was expressed in the mesenchyme of the 3rd pharyngeal arch including those cells immediately adjacent to the early 3rd pharyngeal pouch (page 795, left col. para. 3; Figure 1B). Patel teaches in contrast, Bmp4 expression in the 3rd arch was restricted to a few mesenchymal cells but never in those cells closest to the endoderm (page 795, right col. para. 1; Figure 1A). Patel teaches at E10.5, mesenchymal expression of Noggin was suddenly and dramatically down regulated, unlike Bmp4 expression (page 795, right col. last para.). Patel teaches the ventral portion of the 3rd pharyngeal pouch contained no Noggin-expressing cells (page 795, right col. last para.; page 796, left col. para. 1). Patel teaches at E11.5, Bmp4 expression was confined to the ventral and posterior region of the primordium in those cells that also express Foxn1, while Noggin was restricted to cells at the dorsal/anterior region of the pouch (page 798, left col. last para.). Patel teaches Bmp4 and Noggin exhibit complementary expression patterns in the cells and tissues involved in early thymus and parathyroid organogenesis (page 798, right col. para. 3). Patel teaches the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 (page 798, right col. para. 4). Patel teaches the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling (page 798, right col. para. 4). One would have been motivated to combine the teachings of Green, Swann, and Patel to culture cells in Noggin followed by BMP4 to form TECs because Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus and both Swann and Patel teach the normal development of functional TECs depends on BMP signaling and Foxn1 dosage acting in parallel pathways and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling. Swann and Patel do not teach the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive of claim 1 or “wherein step (d) is performed starting at about day 11 to about day 18, for about 4 days to about 7 days” of claim 9 or “wherein step (e) is performed starting at about day 13 to about day 25 for about 5 days to about 15 days” of claim 11 or “wherein step (e) comprises culturing with about 50 ng/ml of BMP4” of claim 12 or “wherein the TEPs support T cell reconstitution in vivo of claim 48.
Regarding in vitro generation of TEPs and TECs of claim 1 and “wherein step (e) comprises culturing with about 50 ng/ml of BMP4 of claim 12, Parent teaches an in vitro method of differentiating pluripotent stem cells to TEPs and TECs comprising culturing ventral pharyngeal endoderm with 50 ng/ml BMP4 (claim 12) (Figure 1A, 1B, condition 7; Figure 7; page 227, left col. last para. and right col. para. 1; Figure S1B) and (Figure 1A and 1B). Parent teaches transplanting the TEPs in mice resulted in maturation characterized by upregulation of DLL4, FOXN1, K5 and K8 (page 221, left col. last para. and right col. para. 1; Figure 2A and 2C; page 227, right col. para. 1). Parent teaches HOXA3 was not induced without retinoic acid (page 226, left col.). Parent teaches Hoxa3 is essential for thymic specification in vivo (page 226, left col. 1).
Regarding “wherein the TEPs or TECs are FOXN1…positive” of claim 1, Parent teaches the cells are FOXN1, DLL4, EYA1, KRT5, and KRT8 positive (page 220, right col. para. 1; page 221, left col. para. 2 and right col. para. 1; Figure 1; Figure 2). Parent does not teach the cells are ISL1, SIX1, or IL-7 positive.
Regarding claim 48, Parent teaches the TEPs support generation of new T cells in vivo (page 221, right col. para. 3; Figure 3A; Figure 7; page 226, right col. para. 2).
Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo (page 219, right col. para. 3). Parent teaches the method comprises sequential differentiation of hESCs into DE, AFE, ventral pharyngeal endoderm, and TEPs (page 220, left col. para. 2 and right col. para. 1). Parent teaches future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified (page 226, right col. para. 1). Parent teaches Foxn1 and Hoxa3 are early and essential regulators of thymus specification and differentiation of TEPs into mature TECs (page 219, right col. last para.). Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function (page 226, right col. last para.). One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, and Parent in an in vitro method to produce TEPs because Green and Parent both teach methods for producing TEPs from pluripotent stem cells via intermediate AFE and pharyngeal endoderm formation, and both teach in vivo maturation of TEPs when transplanted in vivo and Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo and Wendling, Bain, Swann, and Patel teach the embryonic signaling events that guide thymic development in vivo. Parent does not teach the cells are ISL1, SIX1, or IL-7 positive of claim 1.
Regarding “ISL1”, “IL-7”, and “Six1” of claim 1, Wei teaches Isl1 is expressed in the ventral portion of the 3rd pp which suggested that it is expressed in TECs (page 5, left col. last para.). Wei teaches ISL1 expression was detected in all FOXN1-expressing thymic epithelial cells (page 5, right col. last para.; Figure 5). Wei teaches ISL1 and FOXG1 are expressed in most, if not all TECs (page 6, left col. para. 3; Figure 6). Wei teaches co-expression of FOXN1, FOXG1, and ISL1 in TECs (Figure 6; page 6, left col. last para. and right col. para. 1; page 7, right col. para. 2). Wei teaches IL7 is exclusively expressed in the 3rd pp and is restricted to the thymus domain of the pouch in E10.5 and E11.5 embryos (page 7, left col. para. 1). Wei teaches IL7 activation and expression is a crucial part of TEC differentiation (page 7, right col. para. 1). Wei teaches Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development and all except Tbx1 are expressed in the 3rd pp at E10.5 prior to Foxn1 expression (page 2, left col. para. 2). Wei teaches homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus (page 2, left col. para. 2).
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 Green regarding a method of differentiating pluripotent stem cells to DE and culturing DE with and Noggin/SB431542 to produce AFE cells capable of differentiating to pharyngeal pouches that express PAX1, PAX9, and AIRE with the teachings of Wendling regarding retinoic acid is essential for the formation of 3rd pharyngeal structures with the teachings of Bain regarding activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus with the teachings of Swann regarding Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs with the teachings of Patel regarding the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling with the teachings of Parent regarding an in vitro method of differentiating pluripotent stem cells to DE, AFE, ventral pharyngeal endoderm, and TEPs that express FOXN1, DLL4, EYA1, KRT5, and KRT8 with the teachings of Wei regarding co-expression of FOXN1, FOXG1, and ISL1 in TECs, and IL7 is exclusively expressed in the 3rd pp, and Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development, and homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus to arrive at the claimed method of inducing differentiation of pluripotent stem cells into thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEPs) in vitro comprising the steps of (a) differentiating the pluripotent stem cells into definitive endoderm (DE) cells; (b) differentiating the DE cells into anterior foregut endoderm (AFE) cells, wherein differentiating the DE cells into the AFE cells comprises: (i) contacting or incubating the DE cells with a Bone Morphogenic Protein (BMP) inhibitor and a TGFβ signaling inhibitor, wherein the BMP inhibitor is Noggin and the TGFβ signaling inhibitor is SB431542 and (ii) inducing expression of HOXA3 and TBXI in the DE cells, wherein inducing the expression of HOXA3 comprises contacting or incubating the DE cells with retinoic acid and inducing the expression of TBXI comprises contacting or incubating the DE cells with FGF8b, thereby generating the AFE cells; (c) differentiating the AFE cells into pharyngeal endoderm (PE) cells, wherein differentiating the AFE cells into the PE cells comprises: (i) inducing expression of HOXA3 and TBX1 in the AFE cells, wherein inducing the expression of HOXA3 and TBXI in the AFE cells comprises contacting or incubating the AFE cells with retinoic acid and FGF8b, and (ii) inducing expression of PAX1 and P AX9 in the AFE cells, wherein inducing the expression of PAX1 and PAX9 in the AFE cells comprises contacting or incubating the AFE cells with FGF8b and sonic hedgehog (Shh), thereby generating the PE cells; (d) differentiating the PE cells into distal pharyngeal pouch (PP) specification cells, wherein differentiating the PE cells into the PP specification cells comprises inducing FOXN1 expression in the PE cells, wherein inducing FOXN1 expression in the PE cells comprises: contacting or incubating the PE cells with a BMP inhibitor, wherein the BMP inhibitor is Noggin, thereby generating the PP specification cells; and (e) differentiating the PP specification cells into the TECs or TEPs, wherein differentiating the PP specification cells into the TECs or TEPs comprises inducing FOXN1 expression in the PP specification cells, wherein inducing FOXN1 expression in the PP specification cells comprises contacting or incubating the PP specification cells with BMP4, thereby generating the TECs or TEPs, wherein the TEPs or TECs are FOXNI, PAX9, PAXI, DLL4, ISLI, EYAI, SIXI, IL-7, KRT5, KRT8 and AIRE positive, thereby inducing the differentiation of pluripotent stem cells into TEPs or TECs in vitro. One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, Parent, and Wei in an in vitro method to produce TEPs or TECs as Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus and Green teaches as the thymus involutes with age, older transplant recipients would particularly benefit from thymic replacement therapy and additionally the thymus is absent in SCID and DiGeorge syndromes and Parent teaches future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified and Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function. One would have a reasonable expectation of success in combining the teachings as Green teaches Noggin/SB431542-induced cells are capable of differentiating to pharyngeal pouches and Green teaches retinoic acid inhibits expression of the parathyroid marker Gcm2 and Parent teaches the formation of ventral pharyngeal endoderm by culturing AFE with FGF8b followed by differentiation to TEPs that when transplanted in vivo support T cell reconstitution and Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo and Wendling, Bain, Swann, and Patel teach the signaling events that guide thymic development in vivo.
Regarding “starting at about day 11 to about day 18” of claim 9, Green teaches DE formation over 1 – 5 days (Figure 1c and 1f; page 6, last para.). Green teaches addition of Noggin/SB431542 at day 5 for 2 days, and addition of FGF8 and SHH for 8 days (Figure 4b). Therefore, Green makes obvious further differentiation of FGF8/SHH treated cells at about day 15.
Regarding “for about 4 days to about 7 days” of claim 9, Patel teaches at E9.5, the third pharyngeal pouches have just formed but do not express Bmp4 or Noggin (page 795, left col. last para.). Patel teaches Noggin and BMP4 are expressed in the third pharyngeal pouch endoderm by E10.5 (page 795, left col. last para. and right col. para. 2). Patel teaches at E12.5, the thymus and parathyroid rudiments are beginning to separate from each other and Noggin expression was absent (page 798, right col. para. 2). Parent teaches formation of VPE from AFE after 2 days (Figure 1A). Therefore, Patel makes obvious step (d) is performed for about 4 days (E9.5 – E12.5). Therefore, Green in view of Patel and Parent make obvious the limitations of claim 9.
Regarding “starting at about day 13 to about day 25” of claim 11, Green in view of Patel and Parent make obvious starting step (d) at about day 15 for about 4 days and therefore make obvious starting step (e) at about day 20.
Regarding “for about 5 days to about 15 days” of claim 11, Patel teaches at E9.5, the third pharyngeal pouches have just formed but do not express Bmp4 or Noggin (page 795, left col. last para.). Patel teaches Noggin and BMP4 are expressed in the third pharyngeal pouch endoderm by E10.5 (page 795, left col. last para. and right col. para. 2). Patel teaches at E12.5, the thymus and parathyroid rudiments are beginning to separate from each other and Bmp4 expression remains in the thymus while Noggin expression is absent from the thymus (page 798, right col. para. 2). Parent teaches formation of TEPs from VFE after 2 days (Figure 1A). Therefore, Patel in view of Parent make obvious step (e) is performed for about 4 days (E10.5 – E12.5 from Patel and 2 days for TEP formation from Parent). Therefore, Green in view of Patel and Parent make obvious the limitations of claim 11.
21. Claim(s) 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Green (Green, Michael D., et al. Nature biotechnology 29.3 (2011): 267-272.), hereinafter Green which is cited on the IDS filed 10/01/2021 in view of Wendling (Wendling, Olivia, et. al. Development 127.8 (2000): 1553-1562.), hereinafter Wendling which is cited on the IDS filed 10/01/2021 in view of Bain (Bain, Virginia E., et al. Development 143.21 (2016): 4027-4037.), hereinafter Bain in view of Swann (Swann, Jeremy B., et al. Scientific Reports 7.1 (2017): 8492.), hereinafter Swann which is cited on the IDS filed 10/01/2021 in view of Patel (Patel, Seema R., et al. Gene expression patterns 6.8 (2006): 794-799.), hereinafter Patel in view of Parent (Parent, Audrey V., et al. Cell stem cell 13.2 (2013): 219-229.), hereinafter Parent which is cited on the IDS filed 10/01/2021 and 01/06/2023 in view of Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795; previously cited), hereinafter Wei as applied to claims 1 – 4, 6, 8, 9, 11, 12, 48, and 49 above, and further in view of Cha (US20160002604A1; Filed 10/11/2013; Published 05/23/2017; previously cited), hereinafter Cha, which is cited on the IDS filed 10/01/2021.
Green in view of Wendling, Bain, Swann, Patel, Parent, and Wei make obvious the limitations of claim 1 as set forth above but do not teach the method further comprising contacting or incubating the TECs or TEPs with a survivin inhibitor of claim 13. It is noted that the survivin inhibitor can optionally be YM155 and the term “optionally” does not require that the survivin inhibitor be YM155. However, Green teaches undifferentiated pluripotent stem cells generated teratomas in vivo containing cells derived from all three germ layers, whereas Noggin/SB431542 treated cells produced growths lacking identifiable ectodermal or mesodermal elements (page 4, para. 3).
Cha teaches in the development of a cell therapeutic agent using pluripotent stem cells, there is a potential danger of formation of teratoma from undifferentiated pluripotent stem cells (page 1, 0005). Cha teaches there is a demand for development of a technology capable of selectively removing undifferentiated cells having the potential danger of teratoma without affecting the differentiated cells (page 1, 0005). Cha teaches a method of preparing a cell sample including undifferentiated pluripotent stem cells and differentiated cells by differentiating the pluripotent stem cells and causing the selective death of the undifferentiated pluripotent stem cells by treating the resultant cell sample with YM-155 (page 1, 0013 – 0014).
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 Green regarding a method of differentiating pluripotent stem cells to DE and culturing DE with and Noggin/SB431542 to produce AFE cells capable of differentiating to pharyngeal pouches that express PAX1, PAX9, and AIRE with the teachings of Wendling regarding retinoic acid is essential for the formation of 3rd pharyngeal structures with the teachings of Bain regarding activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus with the teachings of Swann regarding Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs with the teachings of Patel regarding the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling with the teachings of Parent regarding an in vitro method of differentiating pluripotent stem cells to DE, AFE, ventral pharyngeal endoderm, and TEPs that express FOXN1, DLL4, EYA1, KRT5, and KRT8 with the teachings of Wei regarding co-expression of FOXN1, FOXG1, and ISL1 in TECs, and IL7 is exclusively expressed in the 3rd pp, and Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development, and homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus with the teachings of Cha regarding treating a cell sample with YM-155 to arrive at the claimed method further comprising (f) culturing the TECs or TEPs with a survivin inhibitor, optionally wherein the survivin inhibitor is YM155. One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, Parent, Wei, and Cha to prepare TEPs and TECs free of undifferentiated cells as Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus and Parent teaches future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified and Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function. One would have a reasonable expectation of success in combining the teachings as Cha teaches YM-155 causes the selective death of undifferentiated pluripotent stem cells.
22. Claim(s) 50 is/are rejected under 35 U.S.C. 103 as being unpatentable Green (Green, Michael D., et al. Nature biotechnology 29.3 (2011): 267-272.), hereinafter Green which is cited on the IDS filed 10/01/2021 in view of Wendling (Wendling, Olivia, et. al. Development 127.8 (2000): 1553-1562.), hereinafter Wendling which is cited on the IDS filed 10/01/2021 in view of Bain (Bain, Virginia E., et al. Development 143.21 (2016): 4027-4037.), hereinafter Bain in view of Swann (Swann, Jeremy B., et al. Scientific Reports 7.1 (2017): 8492.), hereinafter Swann which is cited on the IDS filed 10/01/2021 in view of Patel (Patel, Seema R., et al. Gene expression patterns 6.8 (2006): 794-799.), hereinafter Patel in view of Parent (Parent, Audrey V., et al. Cell stem cell 13.2 (2013): 219-229.), hereinafter Parent which is cited on the IDS filed 10/01/2021 and 01/06/2023 in view of Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795; previously cited), hereinafter Wei as applied to claims 1 – 4, 6, 8, 9, 11, 12, 48, and 49 above, and further in view of Wertheimer (Wertheimer T, et. al. Sci Immunol. 2018 Jan 12;3(19):eaal2736), hereinafter Wertheimer.
Green in view of Wendling, Bain, Swann, Patel, Parent, and Wei make obvious the limitations of claim 1 as set forth above but do not teach the concentration of Noggin is about 100 ng/mL in step (d) of claim 50.
Wertheimer teaches Foxn1 expression can be induced in TECs in vitro by BMP4 (30 ng/mL) and this could be abrogated by addition of 100 ng/mL Noggin (page 6, last para.; Figure 6E; page 11, para. 1; page 2, para. 1). Wertheimer teaches the thymus is extremely sensitive to damage and there is a clear clinical need for therapeutic strategies to mediate rapid regeneration of thymic function following acute immune damage (page 2, para. 3). Wertheimer teaches BMP4 is a crucial mediator in the induction of TEC-like cells from pluripotent stem cells and BMP4 stimulation of TEPs may drive thymic regeneration (page 7, para. 2; page 8, para. 2).
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 Green regarding a method of differentiating pluripotent stem cells to DE and culturing DE with and Noggin/SB431542 to produce AFE cells capable of differentiating to pharyngeal pouches that express PAX1, PAX9, and AIRE with the teachings of Wendling regarding retinoic acid is essential for the formation of 3rd pharyngeal structures with the teachings of Bain regarding activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus with the teachings of Swann regarding Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs with the teachings of Patel regarding the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling with the teachings of Parent regarding an in vitro method of differentiating pluripotent stem cells to DE, AFE, ventral pharyngeal endoderm, and TEPs that express FOXN1, DLL4, EYA1, KRT5, and KRT8 with the teachings of Wei regarding co-expression of FOXN1, FOXG1, and ISL1 in TECs, and IL7 is exclusively expressed in the 3rd pp, and Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development, and homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus with the teachings of Wertheimer regarding 100 ng/mL Noggin can suppress Bmp4 signaling in TECs to arrive at the claimed method wherein step (d) comprises culturing the PE cells in about 100 ng/ml of Noggin. One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, Parent, Wei, and Wertheimer in a method of producing TEPs/TECs that recapitulates the in vivo differentiation of TECs as Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function and Wertheimer teaches the thymus is extremely sensitive to damage and there is a clear clinical need for therapeutic strategies to mediate rapid regeneration of thymic function following acute immune damage. One would have a reasonable expectation of success in combining the teachings as Wertheimer teaches 100 ng/mL of Noggin can suppress the Bmp4-mediated induction of Foxn1 expression.
23. Claim(s) 15 – 22, 24, 26, 27, and 52 is/are rejected under 35 U.S.C. 103 as being unpatentable over Green (Green, Michael D., et al. Nature biotechnology 29.3 (2011): 267-272.), hereinafter Green which is cited on the IDS filed 10/01/2021 in view of Wendling (Wendling, Olivia, et. al. Development 127.8 (2000): 1553-1562.), hereinafter Wendling which is cited on the IDS filed 10/01/2021 in view of Bain (Bain, Virginia E., et al. Development 143.21 (2016): 4027-4037.), hereinafter Bain in view of Swann (Swann, Jeremy B., et al. Scientific Reports 7.1 (2017): 8492.), hereinafter Swann which is cited on the IDS filed 10/01/2021 in view of Patel (Patel, Seema R., et al. Gene expression patterns 6.8 (2006): 794-799.), hereinafter Patel in view of Parent (Parent, Audrey V., et al. Cell stem cell 13.2 (2013): 219-229.), hereinafter Parent which is cited on the IDS filed 10/01/2021 and 01/06/2023 in view of Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795; previously cited), hereinafter Wei.
Regarding step (a) of claim 15, Green teaches a method of differentiating pluripotent stem cells into DE cells in serum-free media containing human BMP, human bFGF, and human Activin A (page 2, last para.; Figure 1a and 1f; Figure 3g; Figure 4; Supplementary Figure 3b; page 6, last para.).
Regarding step (b) (i) of claim 15, Green teaches contacting DE with Noggin and SB431542 to form AFE that is competent to be pattered along dorsoventral and anteroposterior axes and expresses TBX1 (Abstract; page 3, para. 1 – 3; Figure 1f; Figure 3d, e, g; Figure 4; Supplementary Figure 3b; page 4, para. 1 – 2). Green teaches ventral AFE is formed by treatment of DE with Noggin/SB431542 (Figure 4 legend; page 5, para. 1). Green teaches the thymus and parathyroids develop from the 3rd pouch (page 2, para. 1).
Regarding step (b) (ii) of claim 15 and step (c) (i) of claim 15, Green teaches addition of FGF8b or retinoic acid individually causes a dose dependent increase in TBX1 and PAX9 where PAX9 was used as a marker of pharyngeal endoderm (Figure 1c, conditions 8 – 10 and 19 and 20; page 3, para. 1). Green does not teach addition of FGF8b and retinoic acid. However, Green teaches addition of FGF8 induced the parathyroid-specific marker GCM2 and Green teaches retinoic acid prevents expression of GCM2 (Figure 4b; page 5, last para.). Green teaches FGF8 is a marker specific for pharyngeal pouch endoderm (page 3, last para.).
Regarding step (c) (ii) of claim 15, Green teaches addition of FGF8 and SHH to ventralized AFE induces expression of the pharyngeal pouch marker GCM2 in Figure 4b and GCM2 is a parathyroid marker (page 5, last para.). Therefore, Green teaches induction of pharyngeal endoderm. Green teaches Shh is upstream of FGF8 in mouse pharyngeal pouch development because the combination of SHH and FGF8 was not additive (page 8, last para.; Figure 4b). Green teaches AFE expresses TBX1, PAX1 and PAX9 (Supplementary Figure 1b and 1c).
Regarding step (e) of claim 15, Green teaches transplanting the cells formed by treatment with Noggin/SB431542 into mice where islands of PAX9 and AIRE expressing cells were detected where AIRE is specific for medullary thymic epithelial cells (page 4, last para.). Green does not teach in vitro differentiation to TECs with BMP4.
Regarding “wherein the TEPs or TECs are FOXN1…positive” of claim 15, Green teaches the cells express PAX1, PAX9 and AIRE (page 5, para. 1; Figure 3a – 3). Green does not teach the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive. However, Green teaches Noggin/SB431542 cells expressed TBX1, PAX9, and the pharyngeal endoderm marker FOXG1 (page 4, para. 1).
Regarding claim 16, Green teaches embryonic stem cells (HES2) and induced pluripotent stem cells (HDF2 and HDF9) (page 6, para. 3 – 4).
Regarding claim 17, Green teaches DE formation over 1 – 5 days (Figure 1c and 1f; page 6, last para.).
Regarding claim 18, Green teaches DE formation in serum-free medium with 1 ng/mL and 0.5 ng/mL of human BMP4, 2.5 ng/mL of human bFGF, and 100 ng/mL of human activin A (page 6, last para.; Figure 1a and 1c; Figure 4).
Regarding claim 19, Green teaches addition of Noggin/SB431542 at day 5 for 2 days (Figure 4b).
Regarding claim 20, Green teaches 200 ng/mL human Noggin and 10 µM SB431542 (page 6, last para.).
Regarding claim 21, Green teaches addition of FGF8 and SHH to AFE at day 11 for about 8 days (page 5, last para.; Figure 4b).
Regarding claim 22, Green teaches culturing with 50 ng/mL FGF8b or retinoic acid at 0.1 µM in Figure 1c (conditions 9 and 20).
Green does not teach addition of FGF8b and retinoic acid of (ii) of step (b) and (i) of step (c), or incubating cells with Noggin of step (d) or in vitro differentiation to TECs with BMP4 of step (e) of claim 15 or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive of claim 15 or “wherein step (d) is performed starting at about day 11 to about day 18, for about 4 days to about 7 days” of claim 24 or “wherein step (e) is performed starting at about day 13 to about day 25 for about 5 days to about 15 days” of claim 26 or “wherein step (e) comprises culturing with about 50 ng/ml of BMP4” of claim 27 or “wherein the TEPs support T cell reconstitution in vivo of claim 52. However, Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus (Abstract). Green teaches the thymic function is severely affected by allogeneic hematopoietic stem cell transplantation leading to profound defects in T cell reconstitution (page 2, para. 1). Green teaches as the thymus involutes with age, older transplant recipients would particularly benefit from thymic replacement therapy and additionally the thymus is absent in SCID and DiGeorge syndromes (page 2, para. 1).
Regarding addition of FGF8b and retinoic acid in step (b) (ii) and step (c) (i) of claim 15, Wendling teaches treating E8 mouse embryos with a retinoic acid signaling antagonist (BMS493) resulted in undetectable expression of Fgf8 in the 3rd pouch (page 1554, left col. para. 1; page 1557, right col. para. 4; Figure 7). Wendling teaches retinoic acid is essential for the formation of 3rd pharyngeal structures during a narrow developmental window corresponding to the 7 – 10 somite stages (page 1559, left col. last para. and right col. para. 1). Wendling teaches treatment with BMS493 caused a decrease in Pax1 and Pax9 expression in the pharyngeal endoderm (page 1560, left col. last para. and right col. para. 1). Wendling teaches Pax9 is indispensable for the formation of the thymus and it is most likely that the lack of Pax9 expression in the 3rd pouch under conditions of impaired retinoic acid signaling accounts for thymus agenesis observed in retinoic acid receptor knockout fetuses (page 1560, right col. para. 1). Wendling teaches the thymus and parathyroid originate from the 3rd pouch (page 1553, left col. para. 1). One would have been motivated to combine the teachings of Green and Wendling to culture with both retinoic acid and FGF8b for differentiation towards thymus instead of parathyroid because both Green and Wendling teach the thymus and parathyroids develop from the 3rd pouch and Wendling teaches retinoic acid is essential for formation of 3rd pharyngeal structures and Green teaches retinoic acid prevents expression of the parathyroid marker GCM2 and Wendling teaches FGF8 is absent when retinoic acid signaling is inhibited. Wendling does not teach incubating cells with Noggin of step (d) or in vitro differentiation to TECs with BMP4 of step (e) of claim 15 or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive of claim 15 or “wherein step (d) is performed starting at about day 11 to about day 18, for about 4 days to about 7 days” of claim 24 or “wherein step (e) is performed starting at about day 13 to about day 25 for about 5 days to about 15 days” of claim 26 or “wherein step (e) comprises culturing with about 50 ng/ml of BMP4” of claim 27 or “wherein the TEPs support T cell reconstitution in vivo of claim 52.
Regarding step (c) (ii) of claim 15, Bain teaches activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus (page 4028, left col. para. 1). Bain teaches activation of SHH signaling showed normal Tbx1 and Fgf8 expression (page 4030, right col. para. 2). Bain teaches activation of SHH signaling delayed initial cell fate specification and reduced proliferation within the developing primordium, followed by increased thymus and reduced parathyroid fate specification (page 4031, right col. para. 1 and right col. para. 1). Bain teaches ectopic SHH signaling did not lead to an increase in the number of cells expressing Gcm2 and did not completely block Foxn1 expression (page 4031, right col. para. 2). One would have been motivated to combine the teachings of Green and Bain to culture cells with FGF8b and SHH for differentiation towards thymus instead of parathyroid as both Green and Bain teach the thymus and parathyroids develop from the 3rd pouch and Bain teaches activation of SHH signaling in the most ventral pouch does not induce Gcm2 and Bain teaches activation of SHH signaling increased thymus and reduced parathyroid fate specification and Green teaches Shh is upstream of pharyngeal pouch development and addition of SHH to ventralized AFE induces pharyngeal pouch markers. Bain does not teach incubating cells with Noggin of step (d) or in vitro differentiation to TECs with BMP4 of step (e) of claim 15 or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive of claim 15 or “wherein step (d) is performed starting at about day 11 to about day 18, for about 4 days to about 7 days” of claim 24 or “wherein step (e) is performed starting at about day 13 to about day 25 for about 5 days to about 15 days” of claim 26 or “wherein step (e) comprises culturing with about 50 ng/ml of BMP4” of claim 27 or “wherein the TEPs support T cell reconstitution in vivo of claim 52.
Regarding incubating cells with Noggin of step (d) and incubating cells with BMP4 of step (e) of claim 15, Swann teaches dampening the initial period of BMP4 signaling during early thymus development with Noggin where BMP4 in the mesenchyme induces expression of Noggin creating a time-delayed feedback inhibition of BMP signaling (page 3, para. 3; Figure 1b). Swann teaches this results in the formation of Foxn1+ and Foxn1- cells indicating that sufficient levels of BMP4 signaling are required during early development to establish stable expression of Foxn1 (page 3, para. 3; Figure 1d). Swann teaches the extent of BMP signaling and Foxn1 dosage cooperate in early embryogenesis to establish the number of epithelial cells stably expressing Foxn1 which is the hallmark of a functionally competent thymic epithelium (page 3, para. 5). Swann teaches under the condition of limiting BMP4 signaling, fewer TEPs are established (page 3, last para.). Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs (page 5, para. 3). Swann teaches the BMP-mediated thymic epithelial differentiation process is sensitive to developmental timing (page 6, last para.; page 7, para. 1). Swann teaches sufficient BMP signals are available to the thymic rudiment only during a certain developmental window, possibly associated with a particular microenvironment (page 7, para. 1). Swann teaches Foxn1 gene dosage affects the fitness of TECs (page 7, para. 3). Swann teaches that even subtle changes in BMP signaling and Foxn1 expression levels could lead to unexpectedly severe effects with respect to thymopoietic activity (page 8, para. 3). Swann teaches during embryonic development, initial expression of Foxn1 is induced in pharyngeal endoderm by mesenchyme-derived BMP4 signals (Abstract; Figure 1a; page 3, para. 2). Swann teaches thymopoiesis irreversibly fails if Foxn1 gene expression does not occur during a defining time span in mid-gestation (Abstract). Swann teaches TECs are characterized by the expression of the FOXN1 (page 3, para. 2).
Regarding incubating cells with Noggin of step (d) and incubating cells with BMP4 of step (e) of claim 15, Patel teaches Bmp4 expression is localized to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5 mouse embryos in those cells that will express Foxn1 and form the thymus (Abstract; page 794, right col. para. 2; page 795, right col. para. 2). Patel teaches noggin expression was confined to the dorsal region of the pouch at the same stage (Abstract). Patel teaches Bmp signaling is tightly regulated by noggin (page 795, left col. para. 2). Patel teaches noggin expression has been reported in the pharyngeal arch mesenchyme at E9.5 suggesting the importance of restricting Bmp signaling during pharyngeal development (page 795, para. 2). Patel teaches at E9.5, the 3rd pharyngeal pouches have just formed and neither Bmp4 nor Noggin was expressed but Noggin was expressed in the mesenchyme of the 3rd pharyngeal arch including those cells immediately adjacent to the early 3rd pharyngeal pouch (page 795, left col. para. 3; Figure 1B). Patel teaches in contrast, Bmp4 expression in the 3rd arch was restricted to a few mesenchymal cells but never in those cells closest to the endoderm (page 795, right col. para. 1; Figure 1A). Patel teaches at E10.5, mesenchymal expression of Noggin was suddenly and dramatically down regulated, unlike Bmp4 expression (page 795, right col. last para.). Patel teaches the ventral portion of the 3rd pharyngeal pouch contained no Noggin-expressing cells (page 795, right col. last para.; page 796, left col. para. 1). Patel teaches at E11.5, Bmp4 expression was confined to the ventral and posterior region of the primordium in those cells that also express Foxn1, while Noggin was restricted to cells at the dorsal/anterior region of the pouch (page 798, left col. last para.). Patel teaches Bmp4 and Noggin exhibit complementary expression patterns in the cells and tissues involved in early thymus and parathyroid organogenesis (page 798, right col. para. 3). Patel teaches the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 (page 798, right col. para. 4). Patel teaches the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling (page 798, right col. para. 4). One would have been motivated to combine the teachings of Green, Swann, and Patel to culture cells in Noggin followed by BMP4 to form TECs because Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus and both Swann and Patel teach the normal development of functional TECs depends on BMP signaling and Foxn1 dosage acting in parallel pathways and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling. Swann and Patel do not teach in vitro generation of TEPs or TECs. Swann and Patel do not teach the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive of claim 15 or “wherein step (d) is performed starting at about day 11 to about day 18, for about 4 days to about 7 days” of claim 24 or “wherein step (e) is performed starting at about day 13 to about day 25 for about 5 days to about 15 days” of claim 26 or “wherein step (e) comprises culturing with about 50 ng/ml of BMP4” of claim 27 or “wherein the TEPs support T cell reconstitution in vivo of claim 52.
Regarding in vitro generation of TEPs and TECs of claim 15 and “wherein step (e) comprises culturing with about 50 ng/ml of BMP4 of claim 27, Parent teaches an in vitro method of differentiating pluripotent stem cells to TEPs and TECs comprising culturing ventral pharyngeal endoderm with 50 ng/ml BMP4 (claim 27) (Figure 1A, 1B, condition 7; Figure 7; page 227, left col. last para. and right col. para. 1; Figure S1B) and (Figure 1A and 1B). Parent teaches transplanting the TEPs in mice resulted in maturation characterized by upregulation of DLL4, FOXN1, K5 and K8 (page 221, left col. last para. and right col. para. 1; Figure 2A and 2C; page 227, right col. para. 1). Parent teaches HOXA3 was not induced without retinoic acid (page 226, left col.). Parent teaches Hoxa3 is essential for thymic specification in vivo (page 226, left col. 1).
Regarding “wherein the TEPs or TECs are FOXN1…positive” of claim 15, Parent teaches the cells are FOXN1, DLL4, EYA1, KRT5, and KRT8 positive (page 220, right col. para. 1; page 221, left col. para. 2 and right col. para. 1; Figure 1; Figure 2). Parent does not teach the cells are ISL1, SIX1, or IL-7 positive.
Regarding claim 52, Parent teaches the TEPs support generation of new T cells in vivo (page 221, right col. para. 3; Figure 3A; Figure 7; page 226, right col. para. 2).
Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo (page 219, right col. para. 3). Parent teaches the method comprises sequential differentiation of hESCs into DE, AFE, ventral pharyngeal endoderm, and TEPs (page 220, left col. para. 2 and right col. para. 1). Parent teaches future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified (page 226, right col. para. 1). Parent teaches Foxn1 and Hoxa3 are early and essential regulators of thymus specification and differentiation of TEPs into mature TECs (page 219, right col. last para.). Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function (page 226, right col. last para.). One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, and Parent in an in vitro method to produce TEPs because Green and Parent both teach methods for producing TEPs from pluripotent stem cells via intermediate AFE and pharyngeal endoderm formation, and both teach in vivo maturation of TEPs when transplanted in vivo and Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo and Wendling, Bain, Swann, and Patel teach the embryonic signaling events that guide thymic development in vivo. Parent does not teach the cells are ISL1, SIX1, or IL-7 positive of claim 15.
Regarding “ISL1”, “IL-7”, and “Six1” of claim 15, Wei teaches Isl1 is expressed in the ventral portion of the 3rd pp which suggested that it is expressed in TECs (page 5, left col. last para.). Wei teaches ISL1 expression was detected in all FOXN1-expressing thymic epithelial cells (page 5, right col. last para.; Figure 5). Wei teaches ISL1 and FOXG1 are expressed in most, if not all TECs (page 6, left col. para. 3; Figure 6). Wei teaches co-expression of FOXN1, FOXG1, and ISL1 in TECs (Figure 6; page 6, left col. last para. and right col. para. 1; page 7, right col. para. 2). Wei teaches IL7 is exclusively expressed in the 3rd pp and is restricted to the thymus domain of the pouch in E10.5 and E11.5 embryos (page 7, left col. para. 1). Wei teaches IL7 activation and expression is a crucial part of TEC differentiation (page 7, right col. para. 1). Wei teaches Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development and all except Tbx1 are expressed in the 3rd pp at E10.5 prior to Foxn1 expression (page 2, left col. para. 2). Wei teaches homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus (page 2, left col. para. 2).
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 Green regarding a method of differentiating pluripotent stem cells to DE and culturing DE with and Noggin/SB431542 to produce AFE cells capable of differentiating to pharyngeal pouches that express PAX1, PAX9, and AIRE with the teachings of Wendling regarding retinoic acid is essential for the formation of 3rd pharyngeal structures with the teachings of Bain regarding activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus with the teachings of Swann regarding Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs with the teachings of Patel regarding the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling with the teachings of Parent regarding an in vitro method of differentiating pluripotent stem cells to DE, AFE, ventral pharyngeal endoderm, and TEPs that express FOXN1, DLL4, EYA1, KRT5, and KRT8 with the teachings of Wei regarding co-expression of FOXN1, FOXG1, and ISL1 in TECs, and IL7 is exclusively expressed in the 3rd pp, and Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development, and homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus to arrive at the claimed method of inducing differentiation of pluripotent stem cells into thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEPs) in vitro comprising the steps of (a) differentiating the pluripotent stem cells into definitive endoderm (DE) cells; (b) differentiating the DE cells into anterior foregut endoderm (AFE) cells, wherein differentiating the DE cells into the AFE cells comprises: (i) contacting or incubating the DE cells with a Bone Morphogenic Protein (BMP) inhibitor and a TGFβ signaling inhibitor, wherein the BMP inhibitor is Noggin and the TGFβ signaling inhibitor is SB431542 and (ii) inducing expression of HOXA3 and TBXI in the DE cells, wherein inducing the expression of HOXA3 comprises contacting or incubating the DE cells with retinoic acid and inducing the expression of TBXI comprises contacting or incubating the DE cells with FGF8b, thereby generating the AFE cells; (c) differentiating the AFE cells into pharyngeal endoderm (PE) cells, wherein differentiating the AFE cells into the PE cells comprises: (i) inducing expression of HOXA3 and TBX1 in the AFE cells, wherein inducing the expression of HOXA3 and TBXI in the AFE cells comprises contacting or incubating the AFE cells with retinoic acid and FGF8b, and (ii) inducing expression of PAX1 and P AX9 in the AFE cells, wherein inducing the expression of PAX1 and PAX9 in the AFE cells comprises contacting or incubating the AFE cells with FGF8b and sonic hedgehog (Shh), thereby generating the PE cells; (d) differentiating the PE cells into distal pharyngeal pouch (PP) specification cells, wherein differentiating the PE cells into the PP specification cells comprises inducing FOXN1 expression in the PE cells, wherein inducing FOXN1 expression in the PE cells comprises: contacting or incubating the PE cells with a BMP inhibitor, wherein the BMP inhibitor is Noggin, thereby generating the PP specification cells; and (e) differentiating the PP specification cells into the TECs or TEPs, wherein differentiating the PP specification cells into the TECs or TEPs comprises inducing FOXN1 expression in the PP specification cells, wherein inducing FOXN1 expression in the PP specification cells comprises contacting or incubating the PP specification cells with BMP4, thereby generating the TECs or TEPs, wherein the TEPs or TECs are FOXNI, PAX9, PAXI, DLL4, ISLI, EYAI, SIXI, IL-7, KRT5, KRT8 and AIRE positive, thereby inducing the differentiation of pluripotent stem cells into TEPs or TECs in vitro. One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, Parent, and Wei in an in vitro method to produce TEPs or TECs as Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus and Green teaches as the thymus involutes with age, older transplant recipients would particularly benefit from thymic replacement therapy and additionally the thymus is absent in SCID and DiGeorge syndromes and Parent teaches future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified and Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function. One would have a reasonable expectation of success in combining the teachings as Green teaches Noggin/SB431542-induced cells are capable of differentiating to pharyngeal pouches and Green teaches retinoic acid inhibits expression of the parathyroid marker Gcm2 and Parent teaches the formation of ventral pharyngeal endoderm by culturing AFE with FGF8b followed by differentiation to TEPs that when transplanted in vivo support T cell reconstitution and Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo and Wendling, Bain, Swann, and Patel teach the signaling events that guide thymic development in vivo.
Regarding “starting at about day 11 to about day 18” of claim 24, Green teaches DE formation over 1 – 5 days (Figure 1c and 1f; page 6, last para.). Green teaches addition of Noggin/SB431542 at day 5 for 2 days, and addition of FGF8 and SHH for 8 days (Figure 4b). Therefore, Green makes obvious further differentiation of FGF8/SHH treated cells at about day 15.
Regarding “for about 4 days to about 7 days” of claim 24, Patel teaches at E9.5, the third pharyngeal pouches have just formed but do not express Bmp4 or Noggin (page 795, left col. last para.). Patel teaches Noggin and BMP4 are expressed in the third pharyngeal pouch endoderm by E10.5 (page 795, left col. last para. and right col. para. 2). Patel teaches at E12.5, the thymus and parathyroid rudiments are beginning to separate from each other and Noggin expression was absent (page 798, right col. para. 2). Parent teaches formation of VPE from AFE after 2 days (Figure 1A). Therefore, Patel makes obvious step (d) is performed for about 4 days (E9.5 – E12.5). Therefore, Green in view of Patel and Parent make obvious the limitations of claim 24.
Regarding “starting at about day 13 to about day 25” of claim 26, Green in view of Patel and Parent make obvious starting step (d) at about day 15 for about 4 days and therefore make obvious starting step (e) at about day 20.
Regarding “for about 5 days to about 15 days” of claim 26, Patel teaches at E9.5, the third pharyngeal pouches have just formed but do not express Bmp4 or Noggin (page 795, left col. last para.). Patel teaches Noggin and BMP4 are expressed in the third pharyngeal pouch endoderm by E10.5 (page 795, left col. last para. and right col. para. 2). Patel teaches at E12.5, the thymus and parathyroid rudiments are beginning to separate from each other and Bmp4 expression remains in the thymus while Noggin expression is absent from the thymus (page 798, right col. para. 2). Parent teaches formation of TEPs from VFE after 2 days (Figure 1A). Therefore, Patel in view of Parent make obvious step (e) is performed for about 4 days (E10.5 – E12.5 from Patel and 2 days for TEP formation from Parent). Therefore, Green in view of Patel and Parent make obvious the limitations of claim 26.
24. Claim(s) 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Green (Green, Michael D., et al. Nature biotechnology 29.3 (2011): 267-272.), hereinafter Green which is cited on the IDS filed 10/01/2021 in view of Wendling (Wendling, Olivia, et. al. Development 127.8 (2000): 1553-1562.), hereinafter Wendling which is cited on the IDS filed 10/01/2021 in view of Bain (Bain, Virginia E., et al. Development 143.21 (2016): 4027-4037.), hereinafter Bain in view of Swann (Swann, Jeremy B., et al. Scientific Reports 7.1 (2017): 8492.), hereinafter Swann which is cited on the IDS filed 10/01/2021 in view of Patel (Patel, Seema R., et al. Gene expression patterns 6.8 (2006): 794-799.), hereinafter Patel in view of Parent (Parent, Audrey V., et al. Cell stem cell 13.2 (2013): 219-229.), hereinafter Parent which is cited on the IDS filed 10/01/2021 and 01/06/2023 in view of Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795; previously cited), hereinafter Wei as applied to claims 15 – 22, 24, 26, 27, and 52 above, and further in view of Saldana (Saldaña JI, et. al. J Autoimmun. 2016 Apr;68:86-97), hereinafter Saldana which is cited on the IDS filed 10/01/2021.
Green in view of Wendling, Bain, Swann, Patel, Parent, and Wei make obvious the limitations of claim 15 as set forth above.
Green teaches culturing with 50 ng/mL of FGF8b (Figure 1c, condition 20) but does not teach 100 ng/mL of Shh.
Saldana teaches culture of human thymus explants in serum-free media with Shh at 125 ng/mL and Shh treatment regulates differentiation of thymocytes that mirrors the influence of the hedgehog pathway in the mouse thymus (page 90, left col. last para.; page 94, right col. last para.; Figure 6; page 95, left col. para. 1). Saldana teaches the Shh-/- thymus contains fewer thymocytes than wild type (page 92, right col. para. 2). Saldana teaches in mice with TEC-specific loss of Shh expression, the thymus contained fewer cells overall with reductions in TECs (page 94, left col. para. 4). Saldana teaches Shh is required for normal TEC differentiation and TECs are an essential component of the thymic stroma that are required to support T cell development (Abstract; page 86, left col.; page 90, left col. para. 3). Saldana teaches hedgehog signaling is active in developing TECs in the fetal thymus, suggesting a role for the hedgehog pathway in their development (page 92, left col. last para. and right col. para. 1). Saldana teaches the hedgehog pathway has been implicated in autoimmunity and allergy and it will be interesting to assess the specific contribution of hedgehog signaling in TEC differentiation and function to the induction and severity of human autoimmune disease (page 96, left col. para. 2).
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 Green regarding a method of differentiating pluripotent stem cells to DE and culturing DE with and Noggin/SB431542 to produce AFE cells capable of differentiating to pharyngeal pouches that express PAX1, PAX9, and AIRE with the teachings of Wendling regarding retinoic acid is essential for the formation of 3rd pharyngeal structures with the teachings of Bain regarding activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus with the teachings of Swann regarding BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs with the teachings of Patel regarding the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling with the teachings of Parent regarding an in vitro method of differentiating pluripotent stem cells to DE, AFE, ventral pharyngeal endoderm, and TEPs that express FOXN1, DLL4, EYA1, KRT5, and KRT8 with the teachings of Wei regarding co-expression of FOXN1, FOXG1, and ISL1 in TECs, and IL7 is exclusively expressed in the 3rd pp, and Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development, and homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus with the teachings of Saldana regarding culturing human thymus explants with 100 ng/mL Shh and the hedgehog signaling is active in developing TECs in the fetal thymus, suggesting a role for the hedgehog pathway in their development to arrive at the claimed method wherein step (c) (ii) comprises culturing the AFE cells in FGF8b in an amount of about 50 ng/mL and Shh in an amount of about 100 ng/ml. One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, Parent, Wei, and Saldana in a method of producing TEPs/TECs that recapitulates the in vivo differentiation of TECs in persons with autoimmunity disorders as Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function and Saldana teaches the hedgehog pathway has been implicated in autoimmunity and allergy and it will be interesting to assess the specific contribution of hedgehog signaling in TEC differentiation and function to the induction and severity of human autoimmune disease. One would have a reasonable expectation of success in combining the teachings as Saldana teaches treatment of thymus explants with 125 ng/mL of Shh mirrors the influence of the hedgehog pathway in the mouse thymus.
25. Claim(s) 25 is/are rejected under 35 U.S.C. 103 as being unpatentable over Green (Green, Michael D., et al. Nature biotechnology 29.3 (2011): 267-272.), hereinafter Green which is cited on the IDS filed 10/01/2021 in view of Wendling (Wendling, Olivia, et. al. Development 127.8 (2000): 1553-1562.), hereinafter Wendling which is cited on the IDS filed 10/01/2021 in view of Bain (Bain, Virginia E., et al. Development 143.21 (2016): 4027-4037.), hereinafter Bain in view of Swann (Swann, Jeremy B., et al. Scientific Reports 7.1 (2017): 8492.), hereinafter Swann which is cited on the IDS filed 10/01/2021 in view of Patel (Patel, Seema R., et al. Gene expression patterns 6.8 (2006): 794-799.), hereinafter Patel in view of Parent (Parent, Audrey V., et al. Cell stem cell 13.2 (2013): 219-229.), hereinafter Parent which is cited on the IDS filed 10/01/2021 and 01/06/2023 in view of Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795; previously cited), hereinafter Wei as applied to claims 15 – 22, 24, 26, 27, and 52 above, and further in view of Wertheimer (Wertheimer T, et. al. Sci Immunol. 2018 Jan 12;3(19):eaal2736), hereinafter Wertheimer.
Green in view of Wendling, Bain, Swann, Patel, Parent, and Wei make obvious the limitations of claim 15 as set forth above but do not teach the concentration of Noggin is about 100 ng/mL in step (d) of claim 25.
Wertheimer teaches Foxn1 expression can be induced in TECs in vitro by BMP4 (30 ng/mL) and this could be abrogated by addition of 100 ng/mL Noggin (page 6, last para.; Figure 6E; page 11, para. 1; page 2, para. 1). Wertheimer teaches the thymus is extremely sensitive to damage and there is a clear clinical need for therapeutic strategies to mediate rapid regeneration of thymic function following acute immune damage (page 2, para. 3). Wertheimer teaches BMP4 is a crucial mediator in the induction of TEC-like cells from pluripotent stem cells and BMP4 stimulation of TEPs may drive thymic regeneration (page 7, para. 2; page 8, para. 2).
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 Green regarding a method of differentiating pluripotent stem cells to DE and culturing DE with and Noggin/SB431542 to produce AFE cells capable of differentiating to pharyngeal pouches that express PAX1, PAX9, and AIRE with the teachings of Wendling regarding retinoic acid is essential for the formation of 3rd pharyngeal structures with the teachings of Bain regarding activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus with the teachings of Swann regarding Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs with the teachings of Patel regarding the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling with the teachings of Parent regarding an in vitro method of differentiating pluripotent stem cells to DE, AFE, ventral pharyngeal endoderm, and TEPs that express FOXN1, DLL4, EYA1, KRT5, and KRT8 with the teachings of Wei regarding co-expression of FOXN1, FOXG1, and ISL1 in TECs, and IL7 is exclusively expressed in the 3rd pp, and Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development, and homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus with the teachings of Wertheimer regarding 100 ng/mL Noggin can suppress Bmp4 signaling in TECs to arrive at the claimed method wherein step (d) comprises culturing the PE cells in about 100 ng/ml of Noggin. One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, Parent, Wei, and Wertheimer in a method of producing TEPs/TECs that recapitulates the in vivo differentiation of TECs as Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function and Wertheimer teaches the thymus is extremely sensitive to damage and there is a clear clinical need for therapeutic strategies to mediate rapid regeneration of thymic function following acute immune damage. One would have a reasonable expectation of success in combining the teachings as Wertheimer teaches 100 ng/mL of Noggin can suppress the Bmp4-mediated induction of Foxn1 expression.
26. Claim(s) 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Green (Green, Michael D., et al. Nature biotechnology 29.3 (2011): 267-272.), hereinafter Green which is cited on the IDS filed 10/01/2021 in view of Wendling (Wendling, Olivia, et. al. Development 127.8 (2000): 1553-1562.), hereinafter Wendling which is cited on the IDS filed 10/01/2021 in view of Bain (Bain, Virginia E., et al. Development 143.21 (2016): 4027-4037.), hereinafter Bain in view of Swann (Swann, Jeremy B., et al. Scientific Reports 7.1 (2017): 8492.), hereinafter Swann which is cited on the IDS filed 10/01/2021 in view of Patel (Patel, Seema R., et al. Gene expression patterns 6.8 (2006): 794-799.), hereinafter Patel in view of Parent (Parent, Audrey V., et al. Cell stem cell 13.2 (2013): 219-229.), hereinafter Parent which is cited on the IDS filed 10/01/2021 and 01/06/2023 in view of Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795; previously cited), hereinafter Wei as applied to claims 15 – 22, 24, 26, 27, and 52 above, and further in view of Cha (US20160002604A1; Filed 10/11/2013; Published 05/23/2017; previously cited), hereinafter Cha, which is cited on the IDS filed 10/01/2021.
Green in view of Wendling, Bain, Swann, Patel, Parent, and Wei make obvious the limitations of claim 15 as set forth above but do not teach the method further comprising contacting or incubating the TECs or TEPs with a survivin inhibitor of claim 28. It is noted that the survivin inhibitor can optionally be YM155 and the term “optionally” does not require that the survivin inhibitor be YM155. However, Green teaches undifferentiated pluripotent stem cells generated teratomas in vivo containing cells derived from all three germ layers, whereas Noggin/SB431542 treated cells produced growths lacking identifiable ectodermal or mesodermal elements (page 4, para. 3).
Cha teaches in the development of a cell therapeutic agent using pluripotent stem cells, there is a potential danger of formation of teratoma from undifferentiated pluripotent stem cells (page 1, 0005). Cha teaches there is a demand for development of a technology capable of selectively removing undifferentiated cells having the potential danger of teratoma without affecting the differentiated cells (page 1, 0005). Cha teaches a method of preparing a cell sample including undifferentiated pluripotent stem cells and differentiated cells by differentiating the pluripotent stem cells and causing the selective death of the undifferentiated pluripotent stem cells by treating the resultant cell sample with YM-155 (page 1, 0013 – 0014).
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 Green regarding a method of differentiating pluripotent stem cells to DE and culturing DE with and Noggin/SB431542 to produce AFE cells capable of differentiating to pharyngeal pouches that express PAX1, PAX9, and AIRE with the teachings of Wendling regarding retinoic acid is essential for the formation of 3rd pharyngeal structures with the teachings of Bain regarding activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus with the teachings of Swann regarding Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs with the teachings of Patel regarding the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling with the teachings of Parent regarding an in vitro method of differentiating pluripotent stem cells to DE, AFE, ventral pharyngeal endoderm, and TEPs that express FOXN1, DLL4, EYA1, KRT5, and KRT8 with the teachings of Wei regarding co-expression of FOXN1, FOXG1, and ISL1 in TECs, and IL7 is exclusively expressed in the 3rd pp, and Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development, and homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus with the teachings of Cha regarding treating a cell sample with YM-155 to arrive at the claimed method further comprising (f) culturing the TECs or TEPs with a survivin inhibitor, optionally wherein the survivin inhibitor is YM155. One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, Parent, Wei, and Cha to prepare TEPs and TECs free of undifferentiated cells as Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus and Parent teaches future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified and Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function. One would have a reasonable expectation of success in combining the teachings as Cha teaches YM-155 causes the selective death of undifferentiated pluripotent stem cells.
27. Claim(s) 52 is/are rejected under 35 U.S.C. 103 as being unpatentable over Green (Green, Michael D., et al. Nature biotechnology 29.3 (2011): 267-272.), hereinafter Green which is cited on the IDS filed 10/01/2021 in view of Wendling (Wendling, Olivia, et. al. Development 127.8 (2000): 1553-1562.), hereinafter Wendling which is cited on the IDS filed 10/01/2021 in view of Bain (Bain, Virginia E., et al. Development 143.21 (2016): 4027-4037.), hereinafter Bain in view of Swann (Swann, Jeremy B., et al. Scientific Reports 7.1 (2017): 8492.), hereinafter Swann which is cited on the IDS filed 10/01/2021 in view of Patel (Patel, Seema R., et al. Gene expression patterns 6.8 (2006): 794-799.), hereinafter Patel in view of Parent (Parent, Audrey V., et al. Cell stem cell 13.2 (2013): 219-229.), hereinafter Parent which is cited on the IDS filed 10/01/2021 and 01/06/2023 in view of Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795; previously cited), hereinafter Wei.
Regarding step (a), Green teaches a method of differentiating pluripotent stem cells into DE cells (page 2, last para.; Figure 1a and 1f; Figure 3g; Figure 4; Supplementary Figure 3b).
Regarding “Noggin, SB431542” of step (b), Green teaches contacting DE with Noggin and SB431542 for 4 days to form AFE that is competent to be pattered along dorsoventral and anteroposterior axes and expresses TBX1 (Abstract; page 3, para. 1 – 3; Figure 1c; Figure 3d, e, g; Figure 4; Supplementary Figure 3b; page 4, para. 1 – 2). Green teaches ventral AFE is formed by treatment of DE with Noggin/SB431542 (Figure 4 legend; page 5, para. 1). Green teaches the thymus and parathyroids develop from the 3rd pouch (page 2, para. 1).
Regarding “retinoic acid and FGF8b” of step (b) and step (c) (i), Green teaches addition of FGF8b or retinoic acid individually for 4 days causes a dose dependent increase in TBX1 and PAX9 where PAX9 was used as a marker of pharyngeal endoderm (Figure 1c, conditions 8 – 10 and 19 and 20; page 3, para. 1). Green does not teach addition of FGF8b and retinoic acid. However, Green teaches addition of FGF8 induced the parathyroid-specific marker GCM2 and Green teaches retinoic acid prevents expression of GCM2 (Figure 4b; page 5, last para.). Green teaches FGF8 is a marker specific for pharyngeal pouch endoderm (page 3, last para.).
Regarding “FGF8b and sonic hedgehog (Shh)” of step (c) (ii), Green teaches addition of FGF8 and SHH for 8 days induces expression of the pharyngeal pouch marker GCM2 in Figure 4b and GCM2 is a parathyroid marker (page 5, last para.). Therefore, Green teaches induction of pharyngeal endoderm. Green teaches Shh is upstream of FGF8 in mouse pharyngeal pouch development because the combination of SHH and FGF8 was not additive (page 8, last para.; Figure 4b). Green teaches AFE expresses TBX1, PAX1 and PAX9 (Supplementary Figure 1b and 1c). Green does not teach addition of FGF8 and SHH immediately after Noggin/SB431542 treatment.
Regarding step (e), Green teaches transplanting the cells formed by treatment with Noggin/SB431542 into mice where islands of PAX9 and AIRE expressing cells were detected where AIRE is specific for medullary thymic epithelial cells (page 4, last para.). Green does not teach in vitro differentiation to TECs with BMP4 for about 5 to 15 days.
Regarding “wherein the TEPs or TECs are FOXN1…positive”, Green teaches the cells express PAX1, PAX9 and AIRE (page 5, para. 1; Figure 3a – 3). Green does not teach the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive. However, Green teaches Noggin/SB431542 cells expressed TBX1, PAX9, and the pharyngeal endoderm marker FOXG1 (page 4, para. 1).
Green does not teach addition of FGF8b and retinoic acid of step (b) and (i) of step (c), of “Shh” of (ii) of step (c) or incubating cells with Noggin for about 4 to 7 days of step (d) or in vitro differentiation to TECs with BMP4 for about 5 to 15 days of step (e) or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive or “wherein the TEPs support T cell reconstitution in vivo. However, Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus (Abstract). Green teaches the thymic function is severely affected by allogeneic hematopoietic stem cell transplantation leading to profound defects in T cell reconstitution (page 2, para. 1). Green teaches as the thymus involutes with age, older transplant recipients would particularly benefit from thymic replacement therapy and additionally the thymus is absent in SCID and DiGeorge syndromes (page 2, para. 1).
Regarding addition of FGF8b and retinoic acid in step (b) and step (c) (i), Wendling teaches treating E8 mouse embryos with a retinoic acid signaling antagonist (BMS493) resulted in undetectable expression of Fgf8 in the 3rd pouch (page 1554, left col. para. 1; page 1557, right col. para. 4; Figure 7). Wendling teaches retinoic acid is essential for the formation of 3rd pharyngeal structures during a narrow developmental window corresponding to the 7 – 10 somite stages (page 1559, left col. last para. and right col. para. 1). Wendling teaches treatment with BMS493 caused a decrease in Pax1 and Pax9 expression in the pharyngeal endoderm (page 1560, left col. last para. and right col. para. 1). Wendling teaches Pax9 is indispensable for the formation of the thymus and it is most likely that the lack of Pax9 expression in the 3rd pouch under conditions of impaired retinoic acid signaling accounts for thymus agenesis observed in retinoic acid receptor knockout fetuses (page 1560, right col. para. 1). Wendling teaches the thymus and parathyroid originate from the 3rd pouch (page 1553, left col. para. 1). One would have been motivated to combine the teachings of Green and Wendling to culture with both retinoic acid and FGF8b for differentiation towards thymus instead of parathyroid because both Green and Wendling teach the thymus and parathyroids develop from the 3rd pouch and Wendling teaches retinoic acid is essential for formation of 3rd pharyngeal structures and Green teaches retinoic acid prevents expression of the parathyroid marker GCM2 and Wendling teaches FGF8 is absent when retinoic acid signaling is inhibited. Wendling does not teach incubating cells with Noggin of step (d) or in vitro differentiation to TECs with BMP4 of step (e) or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive or “wherein the TEPs support T cell reconstitution in vivo.
Regarding step (c) (ii), Bain teaches activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus (page 4028, left col. para. 1). Bain teaches activation of SHH signaling showed normal Tbx1 and Fgf8 expression (page 4030, right col. para. 2). Bain teaches activation of SHH signaling delayed initial cell fate specification and reduced proliferation within the developing primordium, followed by increased thymus and reduced parathyroid fate specification (page 4031, right col. para. 1 and right col. para. 1). Bain teaches ectopic SHH signaling did not lead to an increase in the number of cells expressing Gcm2 and did not completely block Foxn1 expression (page 4031, right col. para. 2). One would have been motivated to combine the teachings of Green and Bain to culture cells with FGF8b and SHH for differentiation towards thymus instead of parathyroid as both Green and Bain teach the thymus and parathyroids develop from the 3rd pouch and Bain teaches activation of SHH signaling in the most ventral pouch does not induce Gcm2 and Bain teaches activation of SHH signaling increased thymus and reduced parathyroid fate specification and Green teaches Shh is upstream of pharyngeal pouch development and addition of SHH to ventralized AFE induces pharyngeal pouch markers. Bain does not teach incubating cells with Noggin of step (d) or in vitro differentiation to TECs with BMP4 of step (e) or the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive or “wherein the TEPs support T cell reconstitution in vivo of claim.
Regarding incubating cells with Noggin of step (d) and incubating cells with BMP4 of step (e), Swann teaches dampening the initial period of BMP4 signaling during early thymus development with Noggin where BMP4 in the mesenchyme induces expression of Noggin creating a time-delayed feedback inhibition of BMP signaling (page 3, para. 3; Figure 1b). Swann teaches this results in the formation of Foxn1+ and Foxn1- cells indicating that sufficient levels of BMP4 signaling are required during early development to establish stable expression of Foxn1 (page 3, para. 3; Figure 1d). Swann teaches the extent of BMP signaling and Foxn1 dosage cooperate in early embryogenesis to establish the number of epithelial cells stably expressing Foxn1 which is the hallmark of a functionally competent thymic epithelium (page 3, para. 5). Swann teaches under the condition of limiting BMP4 signaling, fewer TEPs are established (page 3, last para.). Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs (page 5, para. 3). Swann teaches the BMP-mediated thymic epithelial differentiation process is sensitive to developmental timing (page 6, last para.; page 7, para. 1). Swann teaches sufficient BMP signals are available to the thymic rudiment only during a certain developmental window, possibly associated with a particular microenvironment (page 7, para. 1). Swann teaches Foxn1 gene dosage affects the fitness of TECs (page 7, para. 3). Swann teaches that even subtle changes in BMP signaling and Foxn1 expression levels could lead to unexpectedly severe effects with respect to thymopoietic activity (page 8, para. 3). Swann teaches during embryonic development, initial expression of Foxn1 is induced in pharyngeal endoderm by mesenchyme-derived BMP4 signals (Abstract; Figure 1a; page 3, para. 2). Swann teaches thymopoiesis irreversibly fails if Foxn1 gene expression does not occur during a defining time span in mid-gestation (Abstract). Swann teaches TECs are characterized by the expression of the FOXN1 (page 3, para. 2).
Regarding incubating cells with Noggin of step (d) and incubating cells with BMP4 of step (e), Patel teaches Bmp4 expression is localized to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5 mouse embryos in those cells that will express Foxn1 and form the thymus (Abstract; page 794, right col. para. 2; page 795, right col. para. 2). Patel teaches noggin expression was confined to the dorsal region of the pouch at the same stage (Abstract). Patel teaches Bmp signaling is tightly regulated by noggin (page 795, left col. para. 2). Patel teaches noggin expression has been reported in the pharyngeal arch mesenchyme at E9.5 suggesting the importance of restricting Bmp signaling during pharyngeal development (page 795, para. 2). Patel teaches at E9.5, the 3rd pharyngeal pouches have just formed and neither Bmp4 nor Noggin was expressed but Noggin was expressed in the mesenchyme of the 3rd pharyngeal arch including those cells immediately adjacent to the early 3rd pharyngeal pouch (page 795, left col. para. 3; Figure 1B). Patel teaches in contrast, Bmp4 expression in the 3rd arch was restricted to a few mesenchymal cells but never in those cells closest to the endoderm (page 795, right col. para. 1; Figure 1A). Patel teaches at E10.5, mesenchymal expression of Noggin was suddenly and dramatically down regulated, unlike Bmp4 expression (page 795, right col. last para.). Patel teaches the ventral portion of the 3rd pharyngeal pouch contained no Noggin-expressing cells (page 795, right col. last para.; page 796, left col. para. 1). Patel teaches at E11.5, Bmp4 expression was confined to the ventral and posterior region of the primordium in those cells that also express Foxn1, while Noggin was restricted to cells at the dorsal/anterior region of the pouch (page 798, left col. last para.). Patel teaches Bmp4 and Noggin exhibit complementary expression patterns in the cells and tissues involved in early thymus and parathyroid organogenesis (page 798, right col. para. 3). Patel teaches the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 (page 798, right col. para. 4). Patel teaches the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling (page 798, right col. para. 4). One would have been motivated to combine the teachings of Green, Swann, and Patel to culture cells in Noggin followed by BMP4 to form TECs because Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus and both Swann and Patel teach the normal development of functional TECs depends on BMP signaling and Foxn1 dosage acting in parallel pathways and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling. Swann and Patel do not teach the cells are FOXN1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, or KRT8 positive or “wherein the TEPs support T cell reconstitution in vivo.
Regarding in vitro generation of TEPs and TECs, Parent teaches an in vitro method of differentiating pluripotent stem cells to TEPs and TECs comprising culturing ventral pharyngeal endoderm with BMP4 for 2 days (Figure 1A, 1B, condition 7; Figure 7; page 227, left col. last para. and right col. para. 1; Figure S1B) and (Figure 1A and 1B). Parent teaches transplanting the TEPs in mice resulted in maturation characterized by upregulation of DLL4, FOXN1, K5 and K8 (page 221, left col. last para. and right col. para. 1; Figure 2A and 2C; page 227, right col. para. 1). Parent teaches HOXA3 was not induced without retinoic acid (page 226, left col.). Parent teaches Hoxa3 is essential for thymic specification in vivo (page 226, left col. 1).
Regarding “wherein the TEPs or TECs are FOXN1…positive”, Parent teaches the cells are FOXN1, DLL4, EYA1, KRT5, and KRT8 positive (page 220, right col. para. 1; page 221, left col. para. 2 and right col. para. 1; Figure 1; Figure 2). Parent does not teach the cells are ISL1, SIX1, or IL-7 positive.
Regarding “wherein the TEPS or TECs support T cell reconstitution in vivo”, Parent teaches the TEPs support generation of new T cells in vivo (page 221, right col. para. 3; Figure 3A; Figure 7; page 226, right col. para. 2).
Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo (page 219, right col. para. 3). Parent teaches the method comprises sequential differentiation of hESCs into DE, AFE, ventral pharyngeal endoderm, and TEPs (page 220, left col. para. 2 and right col. para. 1). Parent teaches future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified (page 226, right col. para. 1). Parent teaches Foxn1 and Hoxa3 are early and essential regulators of thymus specification and differentiation of TEPs into mature TECs (page 219, right col. last para.). Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function (page 226, right col. last para.). One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, and Parent in an in vitro method to produce TEPs because Green and Parent both teach methods for producing TEPs from pluripotent stem cells via intermediate AFE and pharyngeal endoderm formation, and both teach in vivo maturation of TEPs when transplanted in vivo and Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo and Wendling, Bain, Swann, and Patel teach the embryonic signaling events that guide thymic development in vivo. Parent does not teach the cells are ISL1, SIX1, or IL-7 positive.
Regarding “ISL1”, “IL-7”, and “Six1”, Wei teaches Isl1 is expressed in the ventral portion of the 3rd pp which suggested that it is expressed in TECs (page 5, left col. last para.). Wei teaches ISL1 expression was detected in all FOXN1-expressing thymic epithelial cells (page 5, right col. last para.; Figure 5). Wei teaches ISL1 and FOXG1 are expressed in most, if not all TECs (page 6, left col. para. 3; Figure 6). Wei teaches co-expression of FOXN1, FOXG1, and ISL1 in TECs (Figure 6; page 6, left col. last para. and right col. para. 1; page 7, right col. para. 2). Wei teaches IL7 is exclusively expressed in the 3rd pp and is restricted to the thymus domain of the pouch in E10.5 and E11.5 embryos (page 7, left col. para. 1). Wei teaches IL7 activation and expression is a crucial part of TEC differentiation (page 7, right col. para. 1). Wei teaches Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development and all except Tbx1 are expressed in the 3rd pp at E10.5 prior to Foxn1 expression (page 2, left col. para. 2). Wei teaches homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus (page 2, left col. para. 2).
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 Green regarding a method of differentiating pluripotent stem cells to DE and culturing DE with and Noggin/SB431542 to produce AFE cells capable of differentiating to pharyngeal pouches that express PAX1, PAX9, and AIRE with the teachings of Wendling regarding retinoic acid is essential for the formation of 3rd pharyngeal structures with the teachings of Bain regarding activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus with the teachings of Swann regarding Swann teaches BMP signaling and Foxn1 dosage predominantly act in parallel pathways converging on the number of functional TECs with the teachings of Patel regarding the presence of BMP4 in the posterior-ventral endoderm cells of the 3rd pharyngeal pouch at E10.5 occurs prior to the onset of high level Foxn1 expression in the same domain at E11.25 and is consistent with a role for Bmp4 in the induction of Foxn1 and the timing of Noggin expression within the pouch corresponds to that of Bmp4, consistent with its primary role in opposing Bmp signaling with the teachings of Parent regarding an in vitro method of differentiating pluripotent stem cells to DE, AFE, ventral pharyngeal endoderm, and TEPs that express FOXN1, DLL4, EYA1, KRT5, and KRT8 with the teachings of Wei regarding co-expression of FOXN1, FOXG1, and ISL1 in TECs, and IL7 is exclusively expressed in the 3rd pp, and Hoxa3, Tbx1, Pax1, Pax9, Six1, and Eya are necessary for 3rd pp development, and homozygous mutants of Hoxa3, Eya1, Six1, Pax9, and Tbx1 either fail to form the 3rd pp or exhibit defects in the formation of the thymus to arrive at the claimed method of inducing differentiation of pluripotent stem cells into thymic epithelial cells (TECs) or thymic epithelial cell progenitors (TEPs) in vitro comprising the steps of (a) differentiating the pluripotent stem cells into definitive endoderm (DE) cells; (b) differentiating the DE cells into anterior foregut endoderm (AFE) cells, wherein differentiating the DE cells into the AFE cells comprises: (i) contacting or incubating the DE cells with a Bone Morphogenic Protein (BMP) inhibitor and a TGFβ signaling inhibitor, wherein the BMP inhibitor is Noggin and the TGFβ signaling inhibitor is SB431542 and (ii) inducing expression of HOXA3 and TBXI in the DE cells, wherein inducing the expression of HOXA3 comprises contacting or incubating the DE cells with retinoic acid and inducing the expression of TBXI comprises contacting or incubating the DE cells with FGF8b, thereby generating the AFE cells; (c) differentiating the AFE cells into pharyngeal endoderm (PE) cells, wherein differentiating the AFE cells into the PE cells comprises: (i) inducing expression of HOXA3 and TBX1 in the AFE cells, wherein inducing the expression of HOXA3 and TBXI in the AFE cells comprises contacting or incubating the AFE cells with retinoic acid and FGF8b, and (ii) inducing expression of PAX1 and P AX9 in the AFE cells, wherein inducing the expression of PAX1 and PAX9 in the AFE cells comprises contacting or incubating the AFE cells with FGF8b and sonic hedgehog (Shh), thereby generating the PE cells; (d) differentiating the PE cells into distal pharyngeal pouch (PP) specification cells, wherein differentiating the PE cells into the PP specification cells comprises inducing FOXN1 expression in the PE cells, wherein inducing FOXN1 expression in the PE cells comprises: contacting or incubating the PE cells with a BMP inhibitor, wherein the BMP inhibitor is Noggin, thereby generating the PP specification cells; and (e) differentiating the PP specification cells into the TECs or TEPs, wherein differentiating the PP specification cells into the TECs or TEPs comprises inducing FOXN1 expression in the PP specification cells, wherein inducing FOXN1 expression in the PP specification cells comprises contacting or incubating the PP specification cells with BMP4, thereby generating the TECs or TEPs, wherein the TEPs or TECs are FOXNI, PAX9, PAXI, DLL4, ISLI, EYAI, SIXI, IL-7, KRT5, KRT8 and AIRE positive, thereby inducing the differentiation of pluripotent stem cells into TEPs or TECs in vitro. One would have been motivated to combine the teachings of Green, Wendling, Bain, Swann, Patel, Parent, and Wei in an in vitro method to produce TEPs or TECs as Green teaches the ability to differentiate pluripotent cells into AFE derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as thymus and Green teaches as the thymus involutes with age, older transplant recipients would particularly benefit from thymic replacement therapy and additionally the thymus is absent in SCID and DiGeorge syndromes and Parent teaches future studies will focus on defining conditions that enhance efficiency of differentiation to the thymic lineage as well as improving the purity of the cells by isolating them using cell surface markers yet to be identified and Parent teaches the ability to generate functional thymic epithelium from human pluripotent stem cells will have numerous applications including enabling modeling of human immune diseases using patient-specific iPSCs and will make possible the use of stem cells as a potential source of TECs to enhance or restore thymic function. One would have a reasonable expectation of success in combining the teachings as Green teaches Noggin/SB431542-induced cells are capable of differentiating to pharyngeal pouches and Green teaches retinoic acid inhibits expression of the parathyroid marker Gcm2 and Parent teaches the formation of ventral pharyngeal endoderm by culturing AFE with FGF8b followed by differentiation to TEPs that when transplanted in vivo support T cell reconstitution and Parent teaches the method recapitulates the embryonic signaling events that guide thymic development in vivo and Wendling, Bain, Swann, and Patel teach the signaling events that guide thymic development in vivo.
Regarding “for about 4 to 7 days” of step (d), Green teaches DE formation over 1 – 5 days (Figure 1c and 1f; page 6, last para.). Green teaches addition of Noggin/SB431542 at day 5 for 2 days, and addition of FGF8 and SHH for 8 days (Figure 4b). Therefore, Green makes obvious further differentiation of FGF8/SHH treated cells at about day 15. Patel teaches at E9.5, the third pharyngeal pouches have just formed but do not express Bmp4 or Noggin (page 795, left col. last para.). Patel teaches Noggin and BMP4 are expressed in the third pharyngeal pouch endoderm by E10.5 (page 795, left col. last para. and right col. para. 2). Patel teaches at E12.5, the thymus and parathyroid rudiments are beginning to separate from each other and Noggin expression was absent (page 798, right col. para. 2). Parent teaches formation of VPE from AFE after 2 days (Figure 1A). Therefore, Patel makes obvious step (d) is performed for about 4 days (E9.5 – E12.5).
Regarding “for about 5 days to about 15 days” of step (e), Green in view of Patel and Parent make obvious starting step (d) at about day 15 for about 4 days and therefore make obvious starting step (e) at about day 20. Patel teaches at E9.5, the third pharyngeal pouches have just formed but do not express Bmp4 or Noggin (page 795, left col. last para.). Patel teaches Noggin and BMP4 are expressed in the third pharyngeal pouch endoderm by E10.5 (page 795, left col. last para. and right col. para. 2). Patel teaches at E12.5, the thymus and parathyroid rudiments are beginning to separate from each other and Bmp4 expression remains in the thymus while Noggin expression is absent from the thymus (page 798, right col. para. 2). Parent teaches formation of TEPs from VFE after 2 days (Figure 1A). Therefore, Patel in view of Parent make obvious step (e) is performed for about 4 days (E10.5 – E12.5 from Patel and 2 days for TEP formation from Parent).
Applicant’s Arguments/ Response to Arguments
28. Applicant Argues: Applicant asserts that Snoeck is silent about the use of FGF8b and RA for inducing HOXA3.
Response to Arguments: This is not found persuasive because in the new rejection set forth above, Parent teaches in Figure 1A that the VPE produced by the method expresses HOXA3 when cultured with RA and FGF8b and that the method recapitulates thymus organogenesis by using Foxn1 and Hoxa3 expression as readouts for thymic specification.
Applicant Argues: Applicant asserts that Snoeck describes the induction of the parathyroid fate with SHH and FGFs.
Response to Arguments: This is not found persuasive because in the new rejection set forth above, similar to Snoeck, Green teaches that retinoic acid reduces expression of the parathyroid marker Gcm2.
Applicant Argues: Applicant asserts that Parent teaches culturing with the Shh inhibitor cyclopamine.
Response to Arguments: This is found persuasive and the previous rejection has been withdrawn. In the new rejection set forth above, Bain teaches activation of SHH signaling in the most ventral pouch does not induce Gcm2 and does not suppress Foxn1 and this may be due to high levels of Bmp4 expression that allows ventral pouch cells to differentiate as thymus (page 4028, left col. para. 1). Therefore, Bain teaches SHH treatment of ventral pharyngeal endoderm does not induce parathyroid fate.
Applicant Argues: Applicant asserts that none of Snoeck or Parent disclose a differentiation protocol that yields TEPs or TECs that are positive for the recited markers.
Response to Arguments: This is not found persuasive because Parent teaches the method recapitulates thymus organogenesis by using Foxn1 and Hoxa3 expression as readouts for thymic specification and combined with the teachings of Green, Wendling, Bain, Swann, Patel, and Wei, one of ordinary skill in the art would expect a method that recapitulates thymus organogenesis would result in TEPs or TECs that are positive for the recited markers.
Applicant Argues: Applicant asserts that references that are generally directed at the analysis of embryonic/fetal development do not provide any guarantee that exposing pluripotent stem cells in vitro to a series of differentiation factors would yield the terminally differentiated TECs and/or TEPs.
Response to Arguments: This is not found persuasive because Green teaches methods of differentiating pluripotent stem cells to AFE cells that are capable of differentiating into pharyngeal pouches and Parent teaches differentiation of ventral pharyngeal endoderm to TEPs and TECs. Therefore, when combined with the teachings of Wendling, Bain, Swann, and Patel, one of ordinary skill in the art would expect mirroring thymus development in vivo would yield TECs and/or TEPs that are positive for the recited markers.
Applicant Argues: Applicant asserts that the claimed method is an improved method that yields cells resembling naturally occurring TEPs and TECs more closely than described in any of the cited references.
Response to Arguments: Should Applicant provide arguments regarding how the claimed method provides improvements over the combined references cited in the new rejections set forth above, Applicant may overcome the rejections over the prior art upon further consideration.
Conclusion
No claims allowed.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632
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