DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 10/20/2025 has been entered.
Claims 25, 30, 51, 53, 55, 58-61, 66, and 69-76, of record 10/20/2025, are pending and subject to prosecution. Claim 25 is amended.
Information Disclosure Statement
In the IDS filed 10/20/2025, several references are lined through as not having been provided by the applicant.
Status of Prior Rejections/Response to Arguments
RE: Rejection of claims 25, 30, 32, 51, 53, 55, 58-61, and 69-75 under 35 U.S.C. 103 over Furue et al. (US 20190127707 A1) in view of Steiner et al. (Nature Biotechnology, 2010), further in view of Giulitti et al. (Nature Cell Biology, 2019) and Watanabe et al. (Nature Biotechnology, 2007):
RE: Rejection of claims 25, 30, 32, 51, 53, 55, 58-61, 66, and 69-75 under 35 U.S.C. 103 over Furue et al. (US 20190127707 A1) in view of Steiner et al. (Nature Biotechnology, 2010), Giulitti et al. (Nature Cell Biology, 2019), and Watanabe et al. (Nature Biotechnology, 2007), further in view of Amit et al. (Nature Protocols, 2011):
RE: Rejection of claims 25, 30, 32, 51, 53, 55, 58-61, and 69-76 under 35 U.S.C. 103 over Furue et al. (US 20190127707 A1) in view of Steiner et al. (Nature Biotechnology, 2010), Giulitti et al. (Nature Cell Biology, 2019), and Watanabe et al. (Nature Biotechnology, 2007), further in view of Ito et al. (WO 2020022511 A1):
The applicant asserts that the prior art references do not teach or suggest removal of a ROCK inhibitor or a GSK inhibitor from the culture medium after one day, as now required in the amended claim (Applicant Remarks, page 6). The applicant also asserts unexpected results in the form of optimal proliferation from culture conditions wherein the ROCK inhibitor Y-27632 and the GSK inhibitor CHIR99021 were paired only at the time of passaging and omitted from the culture medium thereafter (Applicant Remarks, page 6-7).
In light of the amendment to independent claim 25 to require culture medium comprising a ROCK inhibitor and a GSK inhibitor only for the first day after passaging, the rejections are withdrawn.
Even though the rejection has been withdrawn in light of the amendments to the claim, the applicant’s argument regarding unexpected results is not found persuasive. Inclusion of a ROCK inhibitor in stem cell culture medium for only the first day post-passaging was known to improve cell survival (See Gao et al., page 7, col. 2, ¶1), and GSK inhibitors had been established as promoting pluripotency in stem cells (See Naujok et al., page 1, col. 1, ¶1). The cytotoxicity of GSK inhibitors was also known, as Naujok et al. showed the dose-dependent cytotoxicity of several GSK3 inhibitors in ESCs (See fig. 1) and Laco et al. demonstrated that CHI99021 is cytotoxic to hPSC monolayer and EB cultures, with a decrease in EB cell number and monolayer cell protein mass occurring after 24 h exposure (See page 1855, col. 1, ¶1-2 and fig. 1). A finding that optimal growth occurs with exposure of pluripotent stem cells to a ROCK inhibitor and a GSK inhibitor only for the first day after passaging would therefore not be surprising in light of the art.
New Rejections
Claim Interpretation
Claim 25 recites the limitation “wherein the second composition does not include the GSK3 inhibitor or the ROCK inhibitor, or salts thereof, or esters thereof”. This limitation is interpreted as requiring that both the GSK3 inhibitor and the ROCK inhibitor (or their salts or esters) are excluded from the second composition.
If the applicant wishes to exclude only one of the GSK3 inhibitor and the ROCK inhibitor from the second composition, the following language is suggested: “… wherein the second composition does not include the GSK3 inhibitor, or salts thereof, or esters thereof, or does not include the ROCK inhibitor, or salts thereof, or esters thereof…”.
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.
Claim 73 is 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.
Claim 73 recites “[t]he method of claim 32”. Because parent claim 32 has been cancelled, the scope of claim 73 cannot be determined.
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.
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.
Claims 25, 30, 51, 53, 55, 58-61, and 69-75 are rejected under 35 U.S.C. 103 as being unpatentable over Steiner et al. (Nature Biotechnology, 2010), of record, in view of Gao et al. (Stem Cell Research, 2019), Naujok et al. (BMC Research Notes, 2014), and Laco et al. (Stem Cell Reports, 2018), and evidenced by Watanabe et al. (Nature Biotechnology, 2007), Garcia-Gonzalo et al., (PLoS One, 2008), and Life Technologies (Product data sheet, 2014).
This is a new rejection necessitated by the applicant’s amendments to the claims.
Regarding claims 25, 30, 51, 55, 58-59, 61, 69-70, and 73-75: Steiner et al. teach the propagation of hESCs (which read on “pluripotent stem cells”) as aggregates in suspension culture in 12-well tissue culture dishes (which read on “culture vessel” and “multi-well plate”) (See Online Methods, page 1, col. 1, ¶2). The aggregates were cultured in Neurobasal medium (which reads on “cell culture basal medium”) with Knockout serum replacement (which reads on “serum-free”), FGF2 (which reads on “one or more mitogenic growth factors” and “bFGF”), BDNF (which reads on “at least two mitogenic growth factors”) and fibronectin and laminin (which read on “one or more extracellular matrix proteins”) (See Online Methods, page 1, col. 1, ¶2). A ROCK inhibitor (which reads on “inhibits ROCK activity and/or ROCK2 activity”)was present in the culture medium for 48 h following passaging (which reads on “contacting with the first medium is at a seeding step or a passaging step”) to reduce cell death (See page 363, col. 2, full ¶1). Steiner et al. do not teach removal of the ROCK inhibitor 24 h after seeding or the use of a GSK inhibitor.
Gao et al. teach that treatment of dissociated hESCs with the ROCK inhibitor Y-27632 is usually limited to 24 h in order to optimize its pro-survival effects (See page 7, col. 2, ¶1).
Naujok et al. teach that small molecule GSK3 inhibitors can be used to maintain pluripotency in ESCs (See Page 1, col. 1, ¶1). GSK3 inhibitors induced dose-dependent cytotoxicity in mouse ESC lines, with CHIR99021 able to maintain the best balance between Wnt activation/pluripotency marker expression and cell viability (See fig. 1-3).
Laco et al. teach that CHIR99021 has a cytotoxic effect on hPSC monolayer cultures and embryoid bodies, with a gradual loss of cell number in the embryoid bodies and in monolayer cell protein mass after 24 h exposure (See page 1855, col. 1, ¶1-2 and fig. 1).
It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method of Steiner et al. to comprise limiting ROCK inhibitor treatment of the hESCs to 24 h. One would be motivated to make this modification because Gao et al. teach that 24 h exposure to a ROCK inhibitor is commonly performed for optimizing its effects (See page 7, col. 2, ¶1). There would be a reasonable expectation of success in making this modification because the post-passaging culture medium in the method of Steiner et al. could be readily replaced after 24 h with a medium lacking a ROCK inhibitor, which would read on “contacting the PSCs in the suspension culture with a second composition the day after the contacting with the first composition”.
It also would have been obvious to modify the method of Steiner et al. to comprise the short-term addition of a GSK3 inhibitor such as CHIR99021 to the culture medium. One would be motivated to make this modification because Naujok et al. teach that GSK3 inhibitors promote pluripotency (See page 1, col. 1, ¶1), though the results of Naujok et al. and Laco et al. suggest that exposure time be limited in order to reduce cytotoxicity. There would be a reasonable expectation of success in making this modification because a GSK3 inhibitor such as CHIR99021 could be readily added to the culture medium upon passaging and omitted upon changing of the medium at 24 h.
Regarding claim 53: Following the discussion of claims 25, 30, 51, 55, 58-59, 61, 69-70, and 73-75, Steiner et al. do not expressly teach the ROCK inhibitor used. However, Steiner et al. cite (See Online Methods, page 1, col. 1, ¶2) the work of Watanabe et al., who focus on the effects of the ROCK inhibitor Y-27632 on hESCs (See Abstract), which indicates Y-27632 was the inhibitor used by Steiner et al.
Regarding claim 60: Following the discussion of claims 25, 30, 51, 55, 58-59, 61, 69-70, and 73-75, Steiner et al. do not expressly teach the inclusion of albumin in the medium. Garcia-Gonzalo et al. teach that Knockout serum replacement comprises the lipid-rich albumin AlbuMAX (See table 1), which is bovine serum albumin (See Life Technologies, product data sheet).
Regarding claims 71-72: Following the discussion of claims 25, 30, 51, 55, 58-59, 61, 69-70, and 73-75, Steiner et al. teach that medium changes were performed by replacing 80% of the medium (which reads on “exchanging a portion of the first composition with the second composition” and “exchanging at least 25% of the suspension culture medium with an equivalent amount of fresh second composition”) (See Online Methods, page 1, col. 1, ¶2).
Claims 25, 30, 51, 53, 55, 58-61, 66, and 69-76 are rejected under 35 U.S.C. 103 as being unpatentable over Steiner et al. (Nature Biotechnology, 2010), of record, in view of Gao et al. (Stem Cell Research, 2019), Naujok et al. (BMC Research Notes, 2014), and Laco et al. (Stem Cell Reports, 2018), and evidenced by Watanabe et al. (Nature Biotechnology, 2007), Garcia-Gonzalo et al., (PLoS One, 2008), and Life Technologies (Product data sheet, 2014), further in view of Ito et al. (WO 2020022511 A1, machine translation), of record.
The teachings of Steiner et al., Gao et al., Naujok et al., Laco et al., Watanabe et al., Garcia-Gonzalo et al., and Life Technologies are set forth in the rejection above and are incorporated herein in their entirety.
Regarding claim 66: Following the discussion of claims 25, 30, 51, 53, 55, 58-61, and 69-75, Steiner et al., modified by Gao et al., Naujok et al., and Laco et al., and evidenced by Watanabe et al., Garcia-Gonzalo et al., and Life Technologies, render obvious a method for growing PSCs in suspension but do not teach the agitation of the suspension culture.
Ito et al. teach that stirring or shaking (which read on “agitating”) can be used to efficiently obtain iPSC aggregates having a controlled size (See ¶0034, 0038, 0041, and 0047).
It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to modify the method of Steiner et al., modified by Gao et al., Naujok et al., and Laco et al., and evidenced by Watanabe et al., Garcia-Gonzalo et al., and Life Technologies, to comprise stirring or shaking of the suspension culture, as taught by Ito et al. One would be motivated to make this modification because Ito et al. teach that the size of the stem cell aggregates can be controlled by agitation (See ¶0034 and 0041). There would be a reasonable expectation of success in doing so because the cultures of Steiner et al., modified by Gao et al., Naujok et al., and Laco et al., and evidenced by Watanabe et al., Garcia-Gonzalo et al., and Life Technologies, could be readily shaken or stirred.
Regarding claim 76: Following the discussion of claims 25, 30, 51, 53, 55, 58-61, and 69-75, Steiner et al., modified by Gao et al., Naujok et al., and Laco et al., and evidenced by Watanabe et al., Garcia-Gonzalo et al., and Life Technologies, render obvious a method for growing PSCs in suspension but do not teach the inclusion of a surfactant in the culture media.
Ito et al. teach the use of water-soluble polymers having surface activity (which read on “surfactants”) for preventing precipitation of serum-free medium components in the suspension culture of cells such as pluripotent stem cells (See ¶0004, 0006, 0022, 0034, and 0041). The one or more polymers can be nonionic surfactants (See ¶0011-0013).
It would have been obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to modify the culture medium in the method of Steiner et al., modified by Gao et al., Naujok et al., and Laco et al., and evidenced by Watanabe et al., Garcia-Gonzalo et al., and Life Technologies, to comprise at least one surfactant, such as those taught by Ito et al. One would be motivated to make this modification because Ito et al. teach that a surfactant can prevent precipitation of serum-free media components, such as insulin, thereby increasing the efficiency of suspension culture (See ¶0034 and 0041). There would be a reasonable expectation of success in doing so because a surfactant could be readily added to the serum-free medium of Steiner et al., modified by Gao et al., Naujok et al., and Laco et al., and evidenced by Watanabe et al., Garcia-Gonzalo et al., and Life Technologies.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JENNIFER S SPENCE, whose telephone number is 571-272-8590. The examiner can normally be reached M-F 8:30-5:30.
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/J.S.S./Examiner, Art Unit 1633
/CHRISTOPHER M BABIC/Supervisory Patent Examiner, Art Unit 1633