Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Amendment to the claims filed 19 Jun 2026 are acknowledged. Claims 2-4, 6-9, and 11 are canceled. Claim 1 is amended. Claims 1, 5, and 10 are pending and under consideration.
Priority
Claims 1, 5, and 10 contain subject matter, the Wnt inhibitor IWR-1-endo, which was disclosed in the Provisional Application 63/666,348. Therefore, the earliest effective filing date of the pending claims is the filing date of the present application, 01 Jun 2025.
Specification
The use of the terms APEL™ 2, StemPro™ Serum Replacement, , Essential 8™ medium, KnockOut™ Serum Replacement, and B-27® supplement, which are trade names or marks used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology, be capitalized wherever it appears, and, where appropriate, include a proper symbol indicating use in commerce such as ™, SM, or ® following the term. Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
The chemical "IWR-endo-1" should be changed to the common name "IWR-1-endo" to avoid confusion and clarify that it does not refer to a different Wnt inhibitor.
Claim Objections
Claims 1 and 10 are objected to because of the following informalities:
Regarding claim 1 (a)(vi), the chemical "IWR-endo-1" should be corrected to the common name "IWR-1-endo".
Regarding claim 1, the first instance of any abbreviation recited in the claims should be accompanied by the full, written out term, then the abbreviation may be used alone thereafter, such as "BMP" recited in part (a)(iii), "FSH" recited in part (b)(i)(2), and "MEF" recited in part (b)(ii).
Regarding claim 1, lines 89-90 on p. 5 should be corrected to "...formation of round spermatids is evidenced by the appearance..." to remove the extra "b".
Regarding claim 1, the units "ug" and "uM" should be corrected to standard international units "μg" and "μM", respectively.
Regarding claim 10, the term "rate" should be corrected to "ratio".
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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.
Claims 1, 5, and 10 are rejected under 35 U.S.C. § 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claims 1, 5, and 10 are rejected under 35 U.S.C. § 112(b) on the basis the use of parentheticals in the present case renders the claims indefinite. It is unclear whether the limitations within the parentheticals are part of the claimed invention or merely exemplary of the preceding limitation. Claim 1 (b)(i)(6) recites the limitation "c-KIT receptor ligand (KITLG)". KITLG is not the only ligand for the c-KIT receptor; therefore it is unclear whether the scope of the claim is limited to only KITLG or to any c-KIT ligand. See MPEP § 2173.05(d). Claims 5 and 10 are included in this rejection as they depend from claim 1 and do not resolve the issue.
Claim 1 (c)(v) contains the recitation "conditioned media comprising MEF-conditioned third culture medium" and claim 10 recites "wherein the third culture medium comprises MEF-conditioned third cultured medium at a rate of one part MEF-conditioned third cell culture medium to three parts third culture medium". It is unclear how a composition can comprise a portion of itself. If making third culture medium requires a step of conditioning with MEF cells following by diluting with more third culture medium, then it is unclear whether or not the conditioned portion is diluted since the third culture medium is already MEF-conditioned.
Claim 1 contains the trademark/trade name αMEM (alpha Minimum Essential Medium) and KSR (KnockOut™ Serum Replacement), . Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. § 112(b). See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a basal cell culture medium and a serum substitute, respectively, and, accordingly, the identification/description is indefinite.
Conclusion
No claim is allowed.
The following references are cited but not relied upon by the examiner:
Zhang, X., Gunewardena, S. & Wang, N. Nutrient restriction synergizes with retinoic acid to induce mammalian meiotic initiation in vitro. Nat Commun 12, 1758 (2021)
Zhou, Q. et al. Complete Meiosis from Embryonic Stem Cell-Derived Germ Cells In Vitro. Cell Stem Cell 18, 330–340 (2016)
Stanners, C. P., Eliceiri, G. L. & Green, H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol 230, 52–54 (1971)
Yuan, F. et al. Pluripotency factor Tex10 finetunes Wnt signaling for spermatogenesis and primordial germ cell development. Nat Commun 16, 1900 (2025)
Clevers, H. & Nusse, R. Wnt/β-Catenin Signaling and Disease. Cell 149, 1192–1205 (2012)
Van Maaren, J., Alves, L. F., Van Wely, M., Van Pelt, A. M. M. & Mulder, C. L. Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis. Front. Cell Dev. Biol. 11, 1330830 (2024)
Tesarik, J., Guido, M., Mendoza, C. & Greco, E. Human Spermatogenesis in Vitro: Respective Effects of Follicle-Stimulating Hormone and Testosterone on Meiosis, Spermiogenesis, and Sertoli Cell Apoptosis. J Clin Endocrinol Metab 83, 4467–4473 (1998)
Sousa, M., Cremades, N., Alves, C., Silva, J. & Barros, A. Developmental potential of human spermatogenic cells co-cultured with Sertoli cells. Hum Reprod 17, 161–172 (2002)
Funahashi, H. Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro. Reproduction 130, 889–898 (2005)
Dumont, L. et al. Vitamin A prevents round spermatid nuclear damage and promotes the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue. Mol Hum Reprod 22, 819–832 (2016)
Dong, W.-Z., Hua, J.-L., Shen, W.-Z. & Dou, Z.-Y. In vitro production of haploid sperm cells from male germ cells of foetal cattle. Animal Reprod Sci 118, 103–109 (2010)
The claims contain subject matter that is free of the prior art. Zhang (X. Zhang, et al., Nat Commun, 2021) and Zhou (Q. Zhou, et al. Cell Stem Cell, 2016) teach a two-step method of generating round spermatids from spermatogonial stem cells (SSC) or embryonic stem cell (ESC)-derived primordial germ cell-like cells (PGCLC) differentiated through an SSC stage by culturing in diluted minimal essential medium alpha (αMEM) (nutrient restriction) supplemented with a serum replacement, retinoic acid, bone morphogenic proteins 2, 4, and 7 (BMP2, BMP4, and BMP7, respectively), activin A, and insulin in the first culture step and culturing in αMEM supplemented with a serum replacement, follicle-stimulating hormone (FSH), testosterone, bovine pituitary extract, and L-glutamine on a mouse embryonic fibroblast (MEF) feeder cell layer in the second step. Yuan (F. Yuan, et al., Nat Commun, Feb 2025) and Clevers (H. Clevers and R. Nusse, Cell, 2012) teach addition of the Wnt inhibitor IWR-1-endo to promote induction of meiosis in SSC (first culture step). Van Maaren teaches that 32 °C is the physiological temperature for spermatogonial cells and that temperatures below the standard culture temperature of 37 °C favor spermatogonial cell survival in vitro. Each of the characterization methods in the last paragraph of claim 1 are also taught in the cited references and are obvious to apply to any spermatogonial differentiation protocol. Thus, each of the elements of claim 1 (a) and (b) are taught by the art. Zhang and Zhou further teach that the two-step method is sufficient to produce post-meiotic round spermatids, but not elongated spermatids or spermatozoa.
However, claim 1 step (c) contains limitations not taught in the art. Tesarik (J. Tesarik, et al., J Clin Endocrinol Metab, 1998) and Sousa (M. Sousa, et al., Hum Reprod, 2002) teach differentiation of round spermatids by culturing in a nutrient-rich medium with a serum substitute, testosterone, lactate, and conditioned medium from a feeder cell layer. Funahashi (H. Funahashi, Reproduction, 2005) teaches addition of 2-mercaptoethanol to culture media to reduce oxidative stress in spermatozoa. However, the art only teaches conditioned medium from Vero cells (an African green monkey kidney cell line), a materially different cell line than MEF, or Sertoli cells, a testicular epithelial cell. The art does not teach or suggest use of MEF or conditioned medium from MEF to differentiate round spermatids. Furthermore, the art does not teach or suggest addition of about retinoic acid to medium for differentiation of round spermatids. Dumont (L. Dumont, et al., Mol Hum Reprod, 2016) teaches that vitamin A (which is metabolized to retinoic acid in vivo) deficiency inhibits spermatogenesis in vivo and supplementation restores spermatogenesis in a non-analogous in vitro culture condition, but does not provide sufficient teaching to motivate addition of retinoic acid at 4-6 μM in a different culture medium. Dong (W.Z. Dong, et al., Animal Reprod Sci, 2010) teaches addition of retinoic acid to medium for differentiation of SSC prior to maturation of round spermatid, but teaches away from concentrations greater than 0.1 μM. Therefore, the art does not teach or suggest addition of about 4 to about 6 μM retinoic to medium for the differentiation of round spermatids into spermatozoa.
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Eric B Wright, PhD
Examiner
Art Unit 1632
/Eric B Wright/Examiner, Art Unit 1632 /VALARIE E BERTOGLIO/Primary Examiner, Art Unit 1632
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