DETAILED ACTION
Applicant’s amendment and Arguments/Remarks received on 03 September 2025 have been entered. Claims 1-16 were previously pending in the application. Claim 3 has been cancelled by Applicant. Claims 1-2 and 4-16 are currently pending in the application. Claims 1, 10, 12, and 16 are independent claims. The election of Groups I and II (rejoined), drawn to a method for generating a population of cardiac fibroblast cells and a population of cardiac fibroblast cells, remains in effect in the instant application.
Claims 12-16 remain withdrawn from consideration as being directed to a nonelected invention. Claims 1-2 and 4-11 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
37 CFR 1.121(c)
The claim amendments filed 03 September 2025 is objected to under 37 CFR 1.121(c) because Applicant’s claim listing is not in compliance with 37 CFR 1.121(c) which states that the claim listing must provide the status of all claims, that all claims being currently amended in an amendment paper shall be presented in the claim listing, indicate a status of "currently amended," and be submitted with markings to indicate the changes that have been made relative to the immediate prior version of the claims, and that the text of all pending claims not being currently amended shall be presented in the claim listing in clean version, i.e., without any markings in the presentation of text. The presentation of a clean version of any claim having the status of "original," "withdrawn" or "previously presented" will constitute an assertion that it has not been changed relative to the immediate prior version, except to omit markings that may have been present in the immediate prior version of the claims of the status of "withdrawn" or "previously presented."
Claim 1 is marked “Currently Amended” and contains markings to indicate inserted and deleted text. However, not all of the inserted text was presented in an underlined format. Specifically, “cell population comprising cardiac fibroblast cells” on line 5 was inserted without an underline.
In the interests of compact prosecution, the claim listing has been entered. However, future claim listings must include the correct status of all claims, including appropriate mark-ups for changes to the claims, in order for the claim listing to meet the requirements for entry under 37 CFR 1.121(c) or a Notice of Non-Compliant Amendment will be mailed to applicant.
Priority
The present application, filed 09 June 2022, claims priority to U.S. Provisional application No. 63/209,429, filed 11 June 2021.
Thus, the earliest possible priority for the instant application is 11 June 2021.
Information Disclosure Statement
The information disclosure statement filed 03 September 2025 has been considered by the Examiner. Examiner notes the filing of IDS Size Fee assertions for the IDS filed 03 September 2025, as required under 37 CFR 1.98, indicating that no IDS size fee is required under 37 CFR 1.17(v) at this time
Claim Objections
The objection to amended claim 8 for reciting “vimentin, and/or GATA4” in a list of two species, is withdrawn in view of the amendment to claim 8 deleting the extra “,”.
Claim Rejections - 35 USC § 112(b)
The rejection of amended claim 8 under 35 U.S.C. 112(b) as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for reciting “wherein the cardiac fibroblast cells stain positive for a TE-7 antibody”, is withdrawn in view of Applicant’s amendments to claim 8 such that claim 8 now recites, “wherein the cardiac fibroblast cells stain positively with a TE-7 antibody”.
Claim Rejections - 35 USC § 112(a)
The rejection of amended, previously presented, original, and cancelled claims 1-11 under 35 U.S.C. 112(a) for while being enabling for”
generating a population of cardiac fibroblast cells by culturing epicardial progenitor cells derived from human induced pluripotent stem cells in culture media comprising 5 ng/mL basic fibroblast growth factor (bFGF) such that the cardiac fibroblast cells are capable of undergoing expansion for up to 10 days and/or up to 5 cell passages in medium equivalent to FibroGRO growth medium (EMD Millipore) with manufacturer’s supplements, supplemental glutamine equivalent to 1X GlutaMAX® (ThermoFisher), and 2% fetal bovine serum (FBS) (Specification paragraphs 0053, 0083, 0099, and 00106),
does not reasonably provide enablement for:
generating a population of cardiac fibroblast cells by incubating epicardial fibroblast cells with any amount of bFGF and any additional cell culture conditions which are capable of undergoing expansion for about 60 days (amended claim 1), wherein the cardiac fibroblast cells stain positive with a TE-7 antibody and maintain expression of vimentin, and/or GATA4 for the about 60 days (amended claim 8), and/or wherein the cardiac fibroblast cells are capable of undergoing at least 15 cell passages (original claim 9),
is withdrawn over cancelled claim 3 and maintained over claims 1-2 and 4-11. Applicant’s amendments to the claims and arguments have been fully considered but have not been found persuasive in overcoming the rejection for reasons of record as discussed in detail below.
Applicant amended independent claim 1 to recite a duration of culturing epicardial progenitor cells (EpiCs) (i.e., about 9-15 days) and to limit the culture medium for the differentiation to be a chemically defined, xenogeny-free, and serum-free culture medium. Applicant additionally amended independent claim 1 to recite that the cardiac fibroblast cells obtained are capable of undergoing expansion for 60 days in a cell culture media supplemented with 2% fetal bovine serum. Claim1 was also amended to recite that “the cardiac fibroblast cells do not express MYH6 and MYH7, cTnt, or calponin”, such that as now written, the cardiac fibroblast cells are not expressing MYH6 and MYH7 (but could express one of the two), are not expressing cTnt, and are not expressing calponin.
Applicant’s amendments to claim 51 have narrowed the scope of the claim to be more closely aligned with the scope identified by the office in the prior action. However, the scope of amended claim 1 is still not commensurate with the enabled scope previously identified. As described in the prior action, Applicant’s specification has demonstrated maintenance of cardiac fibroblast cells capable of undergoing expansion for up to 10 days and/or up to 5 cell passages in medium equivalent to FibroGRO growth medium (EMD Millipore) with manufacturer’s supplements, supplemental glutamine equivalent to 1X GlutaMAX® (ThermoFisher), and 2% fetal bovine serum (FBS) (Specification paragraphs 0053, 0083, 0099, and 00106). However, Applicant’s disclosure does not enable any person skilled in the art to which it pertains to generate cardiac fibroblast cells by the claimed method which are capable of undergoing expansion for about 60 days in any cell culture supplemented with 2% fetal bovine serum. As such, the specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
As discussed in the prior action, the specification does not provide an enabling disclosure for culturing the differentiated cardiac fibroblast cells beyond 10 days or 5 cell passages using Applicant’s claimed methods. The methods presented in the specification disclose that following differentiation in 5 ng/ml bFGF, the EpiC-FB cells were cryopreserved or passaged at 7,000 cells/cm2 or approximately 1:3-1:6 split with ACCUTASE® into FIBROGRO® media on a cell culture treated plate, including the FIBROGRO® basal media with manufacturer’s supplements, GLUTAMAX® supplemented for glutamine, and 2% FBS, with media changes every 2 days until fibroblasts reached approximately 80-90% confluency, at which point they were passaged again with ACCUTASE® (specification paragraph 0053). The specification further discloses that all experiments were performed between P1 and P5 unless otherwise noted, but no indication of experiments being performed at passages higher than P5 are provided in the specification (specification paragraph 0053). These methods do not specify the exact supplements included nor the concentrations of components contained within or added to the media that could promote maintenance of the cardiac fibroblast cells in extended culture without activation or transdifferentiation into myofibroblasts. The culturing conditions described simply and broadly disclose culturing cardiac fibroblast cells in FIBROGRO® media with manufacturer’s supplements, GLUTAMAX®, and 2% FBS for up to 5 cell passages beyond differentiation.
Therefore, merely reciting general conditions that the EpiCs are differentiated into cardiac fibroblasts (EpiC-FBs) in a chemically defined, xenogeny-free, and serum-free culture media and that the EpiC-FBs obtained thereby are cultured in any culture media supplemented with 2% FBS does not align the claim with the enabled scope. As such, Applicant’s amendments do not overcome this scope of enablement rejection under 35 USC 112(a).
Applicant argues that:
No reference cited in the Action establishes that long-term culture of iPSC/hESC-derived cardiac fibroblasts under low-serum, non-activating conditions leads to spontaneous or unpredictable transdifferentiation into myofibroblasts;
2% serum is significantly below the threshold typically required to induce robust myofibroblast differentiation, such that the use of 2% serum in the present application is added to support cell viability while preventing activation, wherein the absence of α-SMA expression under these conditions serves as a functional indicator that the cells retain their fibroblast identity;
The protocol described in Floy et al. 2022 explicitly addresses long-term passaging, in that Floy teaches that passaged EpiC-FBs are expected to exhibit morphology similar to that in Figure 6, which depicts freshly differentiated fibroblasts, which combined with the teaching of Hillsley et al. 2022 that cell size and shape are reliable predictors of fibroblast activation, the morphological consistency strongly supports the conclusion that long-term passaged cardiac fibroblasts remain unactivated;
Floy additionally teaches that maintenance of EpiC-FBs in medium containing higher percentages of serum increased cell size, a marker associated with FB stress, reinforces the notion that 2% FBS does not lead to myofibroblast conversion, whereas higher serum concentrations do; and
Instant Figure 2X shows the growth rate of EpiC-FB over 60 days and maintenance of TE7 expression by flow cytometry for two cell lines in three independent differentiations, wherein the inventors observe senescence after approximately 60 days (15 passages), confirming that the compositions and methods provided herein are capable of expanding a self-renewing population of CFBs for at least 60 days.
However, this is not agreed.
Regarding Applicant’s argument 1), Zhang was cited for teaching that transdifferentiation of iPSC-derived cardiac fibroblasts into myofibroblasts occurs within the first 5 passages following differentiation into cardiac fibroblast cells when cultured in a fibroblast growth medium (Zhang et al. 2019, Circ. Res., Vol. 125(5), pages 560-561, Figure 5, IDS). Zhang et al. were able to reduce transdifferentiation with the addition of a TFGβ inhibitor to the media, and yet transdifferentiation was still observable in >5% of cells by passage 5 (pages 560-561 and Figure 5). However, Zhang is silent regarding the presence of serum in the iPSC-derived cardiac fibroblast culture post-differentiation beyond implying a higher than 0.5% FBS level by teaching that after confluent monolayers of CFs were formed, cells were synchronized in a low serum media (0.5% FBS) for wound healing assays, [supplemental methods page 2 ¶ 1, page 5 ¶ 2, page 7 ¶ 4].
Additionally, Landry was cited for teaching that primary cardiac fibroblast cells are “notoriously difficult to maintain for extended periods of time in cell culture, due to the plasticity of their phenotype and sensitivity to mechanical input” (Landry et al. 2019, Sci. Rep., Vol. 9, article 12889, pages 1-2.) Landry et al. also teaches improved cell culture conditions allowing for maintenance of cardiac fibroblasts in culture for at least 3 days (pages 1-3 and Figure 1). Baum & Duffy were cited for teaching that isolated fibroblasts maintain fibroblast markers only in early passage cultures and transdifferentiate into myofibroblasts, with myofibroblast markers expressing as early as the second passage (Baum & Duffy 2011, J. Cardiovasc. Pharmacol., Vol. 57(4), 376-379).
Landry also teaches supplementing primary cardiac fibroblast culture media with 2% FBS reduces α-SMA expression, but is insufficient as a single variable to prevent activation and transdifferentiation of the cardiac fibroblasts by 72 hours of culture, and that any improvement from reduced (2% compared to 10% serum levels) were also dependent on the substrate used [page 2 ¶ 6- page 3 ¶ 1, Figures 1, 3]. Additionally, even in the best conditions (e.g., 2% FBS and 5 kPa substrate), significant a-SMA expression was observed after only 72 hours of culture [page 2 ¶ 6- page 3 ¶ 1, Figure 1, 2, 3].
Although the cited art do not explicitly recite iPSC-derived cardiac fibroblasts cultured in 2% FBS spontaneously or unpredictable transdifferentiation into myofibroblasts during long-term culture, the art establishes a lack of predictability in preventing transdifferentiation into myofibroblasts over long term culturing.
Regarding Applicant’s argument 2), that 2% serum is significantly below the threshold typically required to induce robust myofibroblast differentiation, such that the use of 2% serum in the present application is added to support cell viability while preventing activation, wherein the absence of α-SMA expression under these conditions serves as a functional indicator that the cells retain their fibroblast identity, note first that Applicant has not shown a lack of α-SMA expression past 10 days or 5 passages.
Additionally, as discussed above, Landry teaches that while 2% FBS reduces the amount of transdifferentiation in a cardiac fibroblast cell culture, it is insufficient to prevent transdifferentiation, as demonstrated by α-SMA expression after 72 hours of culture [page 2 ¶ 6- page 3 ¶ 1, Figure 1, 2, 3].
Additionally, Applicant has not provided any evidence that cardiac fibroblasts generated and cultured according to their claimed method are capable of being expanded for about 60 days or 15 passages without transdifferentiating.
Regarding Applicant’s argument 3), that the protocol described in Floy et al. 2022 explicitly addresses long-term passaging, in that Floy teaches that passaged EpiC-FBs are expected to exhibit morphology similar to that in Figure 6, which depicts freshly differentiated fibroblasts, which combined with the teaching of Hillsley et al. 2022 that cell size and shape are reliable predictors of fibroblast activation, indicates that the morphological consistency strongly supports the conclusion that long-term passaged cardiac fibroblasts remain unactivated, note that a teaching that the authors expect that the cells will maintain the morphology of freshly differentiated cells during long-term culture is not evidence that the cells actually do maintain that morphology.
Floy does not provide any phenotyping data for the cells beyond day 10 or passage 5 other than that senescence was observed after about 60 days (15 passages), and as such does not support that the cells are propagated for the about 60 days or 15 passages without activation or transdifferentiation [page 24¶ 1-2, page 25 ¶ 5, page 27 ¶ 19, page 33 ¶ 2, 9, 10, page 34 ¶ 3, Figure 6, 7, 11].
Applicant likewise has not provided any evidence that cardiac fibroblasts generated and cultured according to their claimed method are capable of being expanded for about 60 days without transdifferentiating.
Regarding Applicant’s argument 4), that Floy additionally teaches maintenance of EpiC-FBs in medium containing higher percentages of serum increased cell size, a marker associated with FB stress, reinforces the notion that 2% FBS does not lead to myofibroblast conversion, whereas higher serum concentrations do; note that Floy teaches that medium containing higher percentages of serum increased cell size, as shown in Figure 7, which is a marker associated with FB stress [page 25 ¶ 5]. Floy does not teach that the FB stress is associated with myofibroblast conversion. Additionally, Floy teaches that even cells cultured in the 2% FBS condition expressed α-SMA in as many as 10% of the cells at the time of flow cytometric analysis presented in Figure 11, which was performed over four days using day 10 EpiC-FB cells.
As such, Applicant has not provided any evidence that cardiac fibroblasts generated and cultured according to their claimed method are capable of being expanded for about 60 days or 15 passages without transdifferentiating.
Regarding Applicant’s argument 5), as discussed in the prior action, Figure 2C of the drawings discloses the growth rate of epicardial cell-derived cardiac fibroblasts (EpiC-FB) over the course of 60 days (Specification paragraph 0014 and Figure 2C). However, no supporting methods are provided disclosing any cell culturing conditions different from the general culturing conditions provided in paragraph 0053 of the specification which allowed these cells to continue expanding for the claimed 60 days beyond differentiation into cardiac fibroblast cells. Further, no evidence is provided to indicate that the cells used in this assay have not activated or transdifferentiated into myofibroblasts.
Figure 2C also shows TE-7 expression for 60 days. However, Goodpaster was cited in the prior action for teaching that Goodpaster et al. teaches that TE-7 and vimentin antibodies stain both fibroblast and myofibroblast cells [pages 347, 348, 349, 354, 356, and Figures 7], and as such, neither cell expansion nor TE-7 expression indicate a lack of transdifferentiation of the cardiac fibroblast cells. Therefore, the cells expressing TE-7 antigen in Figure 2C of the present application, if initially cardiac fibroblasts, lacking any evidence to the contrary, could have transdifferentiated into myofibroblasts over the course of the 60 days and not maintained their identity as cardiac fibroblasts for the duration of the experiment.
Applicant has not provided any evidence that their methods as disclosed actually do allow maintenance of the cardiac fibroblast identity over the extended time and/or passage numbers claimed. Additionally, Applicant did not provide any evidence that they actually assessed the cardiac markers indicated to determine that the cells maintained their cardiac fibroblast identities over the extended time and/or passage numbers claimed in excess of 10 days or 5 passages. As such, Applicant’s arguments do not overcome the rejection of record and the scope of enablement rejection under 35 USC 112(a) is maintained.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Dr. KATIE L PENNINGTON whose telephone number is (703)756-4622. The examiner can normally be reached M-Th 8:30 am - 5:30 pm, Friday 8:30 am - 12:30 pm CT.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634