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 .
Status of the Claims
Claims 13-15, 24-25, 28-33, and 36-40 are pending (claim set as filed on 07/23/2026).
Applicant’s election without traverse of Group I directed to the product(s) is again acknowledged. Claims 24-25 stand withdrawn as being directed to the non-elected method claims.
Therefore, only product claims 13-15, 28-33, and 36-40 are presented for examination.
Priority
This application is a DIV of application no.: 15/048,789 (now U.S. Patent no. 11,674,123), which has a PRO 62/118,553 filed on 02/20/2015.
Withdrawal of Rejections
The response and amendments filed on 07/23/2026 are acknowledged. Any previously applied minor objections and/or minor rejections (i.e., formal matters), not explicitly restated herein for brevity, have been withdrawn necessitated by Applicant’s formality corrections and/or amendments. For the purposes of clarity of the record, the reasons for the Examiner’s withdrawal, and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner’s response to arguments section.
Briefly, the previously applied claim rejection under §101 subject matter eligibility has been withdrawn necessitated by amendments such that the claimed invention, as a whole, is now drawn to a cell culture instead of a product of nature (isolated substantially pure stem cells).
Briefly, the previous obviousness combination over McCloskey in view of Lian has been updated with new secondary references to address the cell culture component limitations. Moreover, the amended claims no longer recite product-by-process phrases (e.g., obtained from).
The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
New Grounds of Rejection Necessitated by Amendments
Claim Rejections - 35 USC §103, Obviousness
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 13-15, 28-33, and 37-40 are rejected under 35 U.S.C. 103 as being unpatentable over McCloskey (US 2012/0064040 A1 - cited in the IDS filed on 05/01/2023) and in view of Lian (Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Progenitors via Small-Molecule Activation of WNT Signaling, published on 05/01/2023) and Esmeraldo (WO 2014/097192 A2 – cited in the IDS filed on 05/01/2023) and Klein (US Patent No. 6,441,053 B1 – cited in the IDS filed on 10/21/2023) as evidenced by ATCC (Formulation for DMEM).
McCloskey’s general disclosure relates to the field of derivation of endothelial cells from embryonic stem cells using a serum free chemically defined medium (see abstract & ¶ [0003]).
Regarding the base claims, McCloskey teaches methods that are particularly useful for preparing a population of endothelial cells (e.g., vascular or arterial endothelial cells) wherein the method comprises differentiating embryonic stem cells by culturing said stem cells in a chemically defined medium that is serum-free wherein the medium comprises vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), amino acids, antibiotics, and admixed in a basal medium of DMEM (see ¶ [0090]-[0091], [0104]-[0106], [0013]). McCloskey discloses that the cells are cultured under conditions (temperature, growth, or culture medium) and for an appropriate amount of time to attain exponential proliferation and further discloses that induction times varies between cell lines; cells were allowed to grow up for at least 7 days (see ¶ [0099], [0103], [0105]).
Regarding the claims’ limitations pertaining to the arterial endothelial cell expression markers and percentage population, McCloskey teaches “an arterial endothelial cell is a specialized endothelial cell population that lines the lumen of arteries. Both arterial and arterial progenitors will express varying levels of the aforementioned markers and will also express of the following arterial EC markers: Alkl, Bmx, CD44, Connexin37, Connexin40, CXCR4, Delta-like 4, Depp, ephrinB2, GFBP-5P, Jagged1, Neuropilin1, Notch1, Notch4, and Unc5b” (see ¶ [0050], [0112]) and venous endothelial cells have the Eph4 expression marker (see [0051]). McCloskey further teaches a range of obtaining a substantially homogeneous cell population which describes a population of cells in which more than about 80% of the cells are of the same or similar phenotype (see ¶ [0041]).
Regarding claims’ limitations pertaining to the mesodermal cells, McCloskey teaches an induction stage of culturing murine embryonic stem cells in the chemically defined medium comprising FGF, VEGF, bone morphogenetic protein 4 (BMP) for 2-5 days wherein BMP-4 and Activin A succeed in promoting mesodermal specific gene expression in serum free differentiation conditions (see ¶ [0116]-[0119], [0104]-[0106]). Activin A is known to enhance mesoderm induction (see ¶ [0058]). McCloskey teaches a mesodermal progenitor cell is a cell that is committed to the mesodermal lineage, as opposed to ectoderm or endoderm. Mesodermal tissues include muscle, cardiovascular tissue, and hematopoietic cells. Markers for identification of a mesodermal progenitors include but are not limited to: Flk-1, Brachyury, CD31 (PECAM1), CD325 (M-cadherin), CD34, (Mucosialin), NF-YA and Sca-1 (Ly6A/E) (see ¶ [0053]).
Regarding claims’ limitations pertaining to the type of pluripotent cells, McCloskey teaches differentiating an isolated embryonic stem cell, an embryoid body, a parthenogenetic stem cell or an induced pluripotent stem (iPS) cell to an endothelial cell progenitor or an endothelial cell or a population of endothelial progenitors or endothelial cells. The isolated stem cell is of animal origin, e.g., a mammalian cell, e.g., a human cell, a simian cell, a bovine cell or a murine cell (see ¶ [0017], [0033], [0047], [0073]).
McCloskey teaches this invention also provides a cell culture system or “kit” comprising of the cell culture mediums as described above alone or in combination with each other; a cell culture container and instructions for culturing and differentiating the cells (see ¶ [0087]-[0089]). ATCC (evidence document) discloses that the formulation of DMEM is albumin-free and insulin-free (see page 1).
However, McCloskey does not teach wherein the medium comprises: (a) a Notch agonist; (b) TGF-β inhibitor of SB431542 (claim 37); or (c) an inhibitor of inositol mono-phosphatase (IMPase) of L-690,330 (claim 38); or an activator of Wnt/β-catenin signaling (claim 29(a)’s limitation).
Lian’s general disclosure relates to a method to efficiently produce endothelial cells from human pluripotent stem cells (hPSC) via GSK3 inhibition and culture in defined media to direct hPSC differentiation to endothelial progenitors (see abstract). Lian teaches endothelial progenitors are derived from a subpopulation of mesoderm that co-expresses brachyury and teaches hPSCs cultured as embryoid bodies were exposed to a growth factor cocktail containing Activin A, BMP4, FGF2, and VEGF to induce differentiation to endothelial progenitors and teaches the TGF-β signaling inhibitor SB431542 promoted endothelial cell generation and expansion (see pages 804-805, adjoining ¶). Lian further teaches “to assess whether these GSK3 inhibitor-induced brachyury+ mesendoderm progenitors also have the potential to differentiate to endothelial progenitors, we treated induced pluripotent stem cells (iPSCs) with the GSK3 inhibitor CHIR99021 and subsequently cultured the cells in StemPro-34 medium supplemented with VEGF, both of which have been reported to stimulate vascular cell differentiation of hPSCs. After optimizing the concentrations and timing of application of various components, the differentiation protocol consisted of two phases. Phase I generated brachyury+ cells by treatment of undifferentiated hPSCs with CHIR990211 for 2 days, whereas phase II directed the brachyury+ progenitors to CD34+CD31+ cells by culture in StemPro-34 supplemented with VEGF for 3 days. We found that without CHIR99021 treatment, very few CD34+CD31+ cells were generated from human iPSCs, even in the presence of VEGF. However, treatment of the human iPSCs with CHIR99021 generated more than 20% CD34+CD31+ cells” (see page 805, right col.). Lian also tested other GSK3 inhibitors of BIO-acetoxime and CHIR98014 (other species of GSK3 inhibitors in claim 9) and found that both also effectively induced endothelial progenitor differentiation (see page 805, right col. last ¶). Lian discloses GSK3 inhibition can affect multiple signaling pathways, including the desired activation of canonical WNT signaling by stabilizing β-catenin protein (see page 806, left col.). Lian discloses that “GSK3 inhibition has been shown to be a potent inducer of mesendoderm lineage commitment in hPSCs. The WNT pathway exhibits crosstalk with a variety of other developmental signaling networks, including TGF-β superfamily, FGF, Notch, Hippo, and retinoic acid signaling … canonical WNT signaling is not the only pathway required for endothelial progenitor specification, but it may act as a master regulator to orchestrate signaling that leads to endothelial progenitor differentiation” (see page 811, adjoining ¶ of left & right col.).
Esmeraldo teaches that human pluripotent stem cells can differentiate in arterial endothelial cells characterized at protein level by the expression of EphB2 wherein the activation of Shh and Notch signaling pathways enhance arterial specification and increases significantly the percentage of cells already committed into an arterial sub-phenotype (see page 12, 2nd full ¶). Esmeraldo teaches the use of the Notch activator of DLL4 (see pages 6-7, adjoining ¶).
Klein discloses the administration of lithium to an animal, or of another inhibitor of GSK-3, can be used to activate Wnt/signaling, enhance glycogen synthesis, alter cell differentiation, and increase transcription enhancement mediated by c-Jun (see col. 28, lines 6-24 & col. 2, lines 33-49). Klein discloses the Wnt pathway is a highly conserved signaling pathway that regulates cell fate decisions (see col. 4, lines 1-4). Klein teaches “The bisphosphonate compound L-690,330 is an approximately thousand-fold more potent inhibitor of IMPase (inositol mono-phosphatase) activity than is lithium” and has mild toxicity on embryos at high doses (see col. 16, lines 1-40).
It would have been first obvious to one of ordinary skill in the art to employ the TGF-β inhibitor and the GSK3 inhibitor (i.e., an activator of Wnt/β-catenin signaling) such as taught by Lian in the method of McCloskey for producing arterial endothelial cells. The ordinary artisan would have been motivated to do so is because Lian teaches that: (a) a TGF-β inhibitor promoted endothelial cell generation and expansion; and further teaches that (b) fewer endothelial cells were generated without the GSK3 inhibitor of CHIR99021, and suggests the role of GSK3 inhibition on Wnt/β-catenin signaling pathway which may act as a master regulator to orchestrate signaling that leads to endothelial progenitor differentiation. Thus, the ordinary artisan would have reasonably recognized that a TGF-β inhibitor and a GSK3 inhibitor are advantageous additives for McCloskey’s purpose for inducing/directing the differentiation of pluripotent stem cells into endothelial cells as each of their function in influencing to endothelial cell lineage commitment are already known in the art. The ordinary artisan would have had a reasonable expectation of success because the references of McCloskey and Lian are both directed to culture media agents to produce endothelial cells from pluripotent stem cells.
Furthermore, it would have been secondly obvious to one of ordinary skill in the art to add a Notch agonist such as taught by Esmeraldo and an inhibitor of IMPase such as taught by Klein in the method of modified-McCloskey-Lian’s objective for producing arterial endothelial cells from pluripotent stem cells using a chemically defined medium. As individually discussed above, the motivation to employ a Notch agonist was suggested by Esmeraldo because it enhances arterial specification and increases significantly the percentage of cells already committed into an arterial sub-phenotype and the motivation to employ an inhibitor of inositol mono-phosphatase (IMPase) is to activate the WNT/β-catenin signaling pathway to promote endothelial progenitor differentiation as disclosed by Klein. Since the composition components are known, it is considered prima facie obvious to combine them into a single composition useful for the very same purpose (e.g., such a composition is typically referred to in the stem cell arts as a cocktail composition which promotes/induces stem cell commitment to a specific lineage and subsequently to a specialized cell type population, in this case, an arterial endothelial phenotype). Moreover, the ordinary artisan would initially recognize from Lian’s disclosure the role of GSK3 inhibition in activating the WNT/β-catenin signaling pathway which may act as a master regulator to orchestrate signaling that leads to endothelial progenitor differentiation (see Lian’s page 811, adjoining ¶). Therefore, the motivation to add L-690,330 is because Klein teaches that L-690,330 is considered a very potent inhibitor IMPase (inositol mono-phosphatase) with mild toxicity on cells and the nexus is that L-690,330 can activate the WNT/signaling pathway to promote endothelial progenitor differentiation as Klein favors its use over lithium. The ordinary artisan would have had a reasonable expectation of success is because both Lian and Klein are directed to agents that activate the WNT/β-catenin signaling pathway.
Claim 36 is rejected under 35 U.S.C. 103 as being unpatentable over McCloskey in view of Lian, Esmeraldo, and Klein as applied to the claims above, and in further view of Zhang (US 2014/0248694 A1 – cited in the IDS filed on 05/01/2023).
The combined teachings of McCloskey, Lian, Esmeraldo, and Klein, herein referred to as modified-McCloskey, is taught above as it pertains to a method of obtaining arterial endothelial cells comprising culturing embryonic stem cells in a chemically defined medium that is serum-free, albumin-free, insulin-free and wherein the medium comprises VEGF, FGF, a Notch agonist, a TGF-β inhibitor, BMP, Activin A, and an activator of Wnt/β-catenin (GSK3/IMPase inhibitor).
However, modified-McCloskey does not teach: wherein the Notch agonist is selected from valproic acid or suberoyl bishdroxamic acid (claim 36).
Zhang teaches the Notch signaling pathway agonist can be a histone deacetylase (HDAC) inhibitor selected from the group consisting of valproic acid (VPA), suberoyl bishydroxamic acid (SBHA), and sodium butyrate (see ¶ [0011]).
As discussed above, it was obvious to activate both the Shh and Notch pathway such as taught by Esmeraldo for method modified-McCloskey’s objective of producing arterial endothelial cells because Esmeraldo teaches that activating both pathway enhances the arterial specification and increases significantly the percentage of cells already committed into an arterial sub-phenotype (i.e. promoting the differentiation towards the specialized cell population of arterial endothelial cell lineage). The addition of a Shh and Notch agonist/activator would have been reasonably advantageous to modified-McCloskey’s purpose of producing a substantially homogeneous population of arterial endothelial phenotypes. Furthermore, it would have been secondly or further obvious to employ the specific Notch agonist of valproic acid (VPA) or suberoyl bishydroxamic acid (SBHA) such as taught from the small genus of Zhang because VPA and SBHA are already known in the art as Notch signaling pathway agonist. Hence, the substitution is no more than the predictable use of prior art elements according to their established functions resulting in the simple substitution of one known element for another for a predictable result. The ordinary artisan would have had a reasonable expectation of success is because the references of modified-McCloskey-Lian, Esmeraldo, and Zhang are all directed to culture medium agents that promote the differentiation of stem cells to the desired terminal cell phenotype.
Conclusion
No claims were 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 extension fee 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 date of this final action.
Correspondence Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NGHI V NGUYEN whose telephone number is (571)270-3055. The examiner can normally be reached Mon-Fri: 9 - 3 pm (EST).
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/NGHI V NGUYEN/Primary Examiner, Art Unit 1653
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