DETAILED ACTION
Notice of Pre-AIA or AIA Status
The text of those sections of Title 35, U.S. Code not included in this action can
be found in a prior Office action.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This action is in response to the papers filed on 04/02/2026. Claims 1 and 9-22 are currently pending as per claims filed on 04/02/2026. Claims 2-8 and 23-30 are cancelled and Claims 1, 10, 12-15, 17, 18 and 20 and have been amended by Applicants’ amendment filed on 04/02/2026. No new claims have been added.
Applicant’s election without traverse of Group I, claims 1-4 and 9-22 in the reply filed on 03/04/2026 was previously acknowledged.
Therefore, claims 1 and 9-22 are under examination to which the
following grounds of rejection are applicable. Claim 1 is an independent claim.
Priority
The instant application claims domestic benefit to US provisional patent application number 63/390,458 filed on 07/19/2022. Thus, the earliest possible priority for the instant application is 07/19/2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 07/01/2026 was filed before the mailing date of the current office action. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Withdrawn objections/rejection in response to Applicants’ arguments or
Amendment
Claim Rejections - 35 USC § 112 (b)
In view of Applicants’ amendment of claim 1, 2, 3, 10-15, 17, 18, and 20 to specify “a first culture medium”, “a second culture medium”, and “a third culture media”, and respectively, “the first culture medium”, “the second culture medium”, and “the third culture media”, claim rejections have been withdrawn.
In view of Applicants’ amendment of claim 1 to specify “a first cell culture”, “a second cell culture”, and “a third cell culture”, claim rejections have been withdrawn.
Maintained Claim Rejections in response to Applicants’ arguments
Claim Rejections - 35 USC § 103
Claim(s) 1-4 and 9-22 are rejected under 35 U.S.C. 103 as being unpatentable over Orlova et al (Nature Protocols, 2014, pages 1514-1531; as cited in IDS), and further in view of Watanabe et al (Nature Biotechnology, 2007, pages 681-686; as cited in Office Action filed 04/02/2026).
Regarding claim 1, Orlova teaches a method of differentiating human pluripotent stem cells (hPSCs) into pericytes (i.e. a species of stromal cells) comprising:
Culturing hPSCs to induce mesoderm specification by the addition of bone morphogenetic protein 4 (BMP4), activin A, small-molecule inhibitor of glycogen synthase kinase-3β (CHIR), and vascular endothelial growth factor (VEGF) (page 1516, left col, para 1) to a base culture medium (B(P)EL medium (page 1521, Mesoderm induction medium; page 1523, step 13 – Monolayer differentiation of hPSCs));
Removal of mesoderm-inductive factors on day 3 of differentiation and replace with vascular specification medium (base culture medium (B(P)EL medium supplemented with VEGF and the transforming growth factor-β (TGF-β) pathway small-molecule inhibitor SB431542) (page 1521, vascular specification medium; page 1516, left col, para 1);
Cell sorting to isolate endothelial cells (ECs) fractions from pericytes fraction by Dynabead isolation; and
Passage of pericyte cell fraction into DMEM–10% (vol/vol) FBS (base culture medium) with TGF-β3 and PDGF-BB and incubate for 3 days to expand pericytes (page 1524, Expansion of pericytes: step (v))
rendering obvious a method for differentiating pluripotent stem cells into stromal cells, comprising:
culturing pluripotent stem cells in a first culture medium comprising a base culture medium, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), a glycogen synthase kinase 3 (GSK3) inhibitor, activin A to form a first culture cell culture.
culturing the first cell culture of (i) in a second culture medium comprising a base culture medium, VEGF and a transforming growth factor β (TGFβ) inhibitor to form a second cell culture;
culturing the second cell culture of (ii) in a third culture medium comprising a base culture medium, TGFβ3 and a platelet- derived growth factor (PDGF) to form stromal cells.
Orlova does not teach Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor in step (i).
However, Watanabe teaches the use of the ROCK inhibitor Y-27632 enables “human embryonic stem cells to grow and differentiate under unfavorable culture conditions” (page 684, right col, para 3; page 685, left col, para 1).
It would have been prima facie obvious to one of ordinary skill, in the art at the time of the effective filing date, to modify the teachings of a method of differentiating pluripotent stem cells into pericytes (i.e. a species of stromal cells) from Orlova in the culture step (I) to further add the ROCK inhibitor from Watanabe to increase the survival of the pluripotent cells undergoing differentiation as the process of differentiation is stressful to cells and the addition of ROCK inhibitor would mitigate cell loss during culture. One would be motivated to do so to maximize cell survival and, therefore, maximize the number of differentiating cells in the culture. As use of ROCK inhibitor to increase cell viability during cell culture is known in the art, one would have a reasonable expectation of success.
Regarding claim 9, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Watanabe teaches using the ROCK inhibitor Y-27632 (page 684, right col, para 3), rendering obvious wherein the ROCK inhibitor is Y-27632.
Regarding claim 10, the teachings of Orlova and Watanabe render obvious claim 1 and 9. Moreover, Watanabe teaches that 10 μm is a commonly used concentration for Y-27632 in cell culture (page 681, right col, para 2), rendering obvious wherein Y-27632 is present in the first culture medium at a concentration of about 10 μM.
Regarding claim 11, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches using the small-molecule inhibitor of glycogen synthase kinase-3β (CHIR) (page 1516, left col, para 1), rendering obvious wherein the GSK3 inhibitor is CHIR99021.
Regarding claim 12, the teachings of Orlova and Watanabe render obvious claim 1 and 11. Moreover, Orlova teaches that the mesoderm induction media contains 1.5 μm CHIR (page 1521, mesoderm induction media), rendering obvious wherein CHIR99021 is present in the first culture medium at a concentration of about 1.5μM.
Regarding claim 13, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches that the mesoderm induction media contains 30 ng/ml BMP4 (page 1521, mesoderm induction media), rendering obvious wherein BMP4 is present in the first culture medium at a concentration of about 30 ng/mL.
Regarding claim 14, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches that the mesoderm induction media contains 50 ng/ml VEGF (page 1521, mesoderm induction media), rendering obvious wherein VEGF is present in the first culture medium at a concentration of about 50 ng/mL.
Regarding claim 15, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches that the mesoderm induction media contains 25 ng/ml Activin A (page 1521, mesoderm induction media), rendering obvious wherein activin A is present in the first culture medium at a concentration of about 25 ng/mL.
Regarding claim 16, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches the transforming growth factor-β (TGF-β) pathway small-molecule inhibitor is SB431542 (page 1516, left col, para 1; page 1521, vascular specification media), rendering obvious wherein the TGF3 inhibitor is SB431542.
Regarding claim 17, the teachings of Orlova and Watanabe render obvious claim 1 and 16. Moreover, Orlova teaches the transforming growth factor-β (TGF-β) pathway small-molecule inhibitor SB431542 has a concentration of 10 μM (page 1521, vascular specification media), rendering obvious wherein SB431542 is present in the second culture medium at a concentration of about 10 μM.
Regarding claim 18, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches the cells are passaged and incubated with media containing 2 ng/mL of TGF-β3 (page 1524, Expanding pericytes step (v)), rendering obvious wherein TGFβ3 is present in the third culture medium at a concentration of about 2 ng/mL.
Regarding claim 19, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches the cells are passaged and incubated with media containing PDGF-BB (page 1524, Expanding pericytes step (v)), rendering obvious wherein the PDGF is PDGF-BB.
Regarding claim 20, the teachings of Orlova and Watanabe render obvious claim 1 and 19. Moreover, Orlova teaches the cells are passaged and incubated with media containing 4 ng/mL of PDGF-BB (page 1524, Expanding pericytes step (v)), rendering obvious wherein PDGF-BB is present in the third culture medium at a concentration of about 4 ng/mL.
Regarding claim 21, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches a protocol for the generation of pericytes from human pluripotent stem cells (abstract, page 1), rendering obvious wherein the stromal cells are pericytes.
Regarding claim 22, the teachings of Orlova and Watanabe render obvious claim 1. Moreover, Orlova teaches not fully confluent cells at Day 0 (Figure 2D shows Day 0 cells that are not fully confluent), rendering obvious wherein the pluripotent stem cells are not fully confluent at the start of the culturing of (i).
Response to Arguments as they apply to rejection of claims 1, 9-22 under 35 USC § 103 as they applied to Orlova and Watanabe
Applicant's arguments filed 07/01/2026 have been fully considered but have not
been found persuasive in overcoming the rejection for reasons of record as discussed in
detail below.
Applicant acknowledges that Orlova discloses a method of differentiating endothelial cells and pericytes from human pluripotent stem cells, but does not teach or suggest using a ROCK inhibitor in the culture media (page 3 of Applicant remarks). Applicant asserts that Watanabe is directed to a fundamentally different objective than Orlova and it discloses that the ROCK inhibitor Y-27632 enhances survival and proliferation of undifferentiated hESCs, maintain cells in an undifferentiated state, and does not teach or suggest using a ROCK inhibitor to promote differentiation into a specific cell type. Hence, applicant asserts there is no motivation to combine Orlova and Watanabe to arrive at the method of claim 1 (page 4 of Applicants’ remarks).
While Watanabe teaches the use of ROCK inhibitor for hESC pluripotency maintenance, Watanabe also teaches that ROCK inhibitor elicits beneficial effects to pluripotent stem cells as shown by Y-27632-treated hESCs displaying increased proliferation and survival and ability to retain competence to differentiate into not only neuronal cells, as noted by Applicants’ remarks, but also mesoderm cells (page 681, right col, para 3) which is the germ layer origin of stromal cells and pericytes. Indeed, Watanabe teaches that hESCs undergoing differentiation were exposed to Y-27632 and factors including BMP (page 684, left col, para 1-2) which is a factor recited in the instantly claimed method. Collectively, Watanabe teaches that the ROCK inhibitor has protective ability to permit the survival and proliferation of hESCs, even when undergoing differentiation, therefore it would have been obvious to include Y-27632 in the media to enhance cell viability and therefore increase yield of resulting stromal cells upon following the steps of the claimed method. It is also noted claim 1 recites the ROCK inhibitor as a component of the media for the first(i) step in which the cells could be in an undifferentiated state since the claims, as recited, do not require cells to be differentiated.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. 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.
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/JULIANA IRENE CANDELARIA/Examiner, Art Unit 1634
/MARIA MARVICH/Primary Examiner, Art Unit 1634
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