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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 11/25/2025 has been entered.
Claim Status
1. The amendment filed 11/25/2025 has been entered. Claims 1, 3, 6, 7, 10 – 13, 15, 16, 23, 24, 26, 28 – 34, 36 – 38, 41, 44, 46, 47, 52, 92, 123, and 124 remain pending.
2. Claims 1, 3, 6, 7, 10 – 13, 15, 16, 23, 24, 26, 28 – 34, 36 – 38, 41, 44, 46, 47, 123, and 124 are under consideration.
Election/Restrictions
3. Applicant’s election without traverse of Group I (claims 1 – 7, 10 – 13, 15 – 16, 23 – 24, 26, 28 – 34, 36 – 38, 41, 44, 46 – 47, and 123) in the reply filed on 10/25/2024 is acknowledged.
4. Claims 52 and 92 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/25/2024.
Priority
5. This application claims the benefit of United States Provisional Patent Application Nos. 62/861,640, filed June 14, 2019, 62/865,806, filed June 24, 2019, and 63/038,564, filed June 12, 2020.
Information Disclosure Statement
6. The information disclosure statement (IDS) submitted on 10/20/2025 and 09/25/2025 are acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
7. The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Withdrawn Claim Objection
8. The objection to claim 7 is withdrawn in view of Applicant’s amendment to the claim.
Withdrawn Specification Objection
9. The objection to the specification for improper trade name or marker usage is withdrawn in view of Applicant’s amendment to the specification.
Withdrawn Claim Rejections
10. The rejection of claims 1, 3, 10, 12, 13, 15, 16, 23, 36, 37, 38, 44, and 47 under 35 U.S.C. 102(a)(1) is withdrawn in view of Applicant’s amendment to the claims.
11. The rejection of claims 1, 3, 12, 13, 15, 23, 26, and 28 under 35 U.S.C. 102(a)(1) is withdrawn in view of Applicant’s amendment to the claims.
12. The rejection of claims 1, 6, 7, 11, and 123 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claims.
13. The rejection of claims 1, 23, and 24 under 35 U.S.C. 103 are withdrawn in view of Applicant’s amendment to the claims.
14. The rejection of claims 1, 28 – 31 and 46 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claims.
15. The rejection of claims 1, 28, 32 and 34 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claims.
16. The rejection of claims 1, 36, and 41 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claims.
17. The rejection of claim 33 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claims.
18. The rejection of claim 124 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claims.
Claim Interpretation
19. For the purpose of applying prior art, “deposited by plasma polymerization” of claim 3 is interpreted as a product-by-process limitation (see MPEP 2113) and not an active method step.
New Claim Objections
20. Claim 31 is objected to because of the following informalities: in line 1 “the differentiated cells” should read “the MSCs” because claim 31 depends from claim 30 and claim 30 requires the MSCs are positive for CD73. Appropriate correction is required.
Claim Rejections Necessitated by Amendment - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
21. Claim 33 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. 37 CFR 1.118 (a) states that “No amendment shall introduce new matter into the disclosure of an application after the filing date of the application”.
Claim 33 has been amended to recite “The method of claim 32, wherein the MSCs are cultured in the presence of pericyte medium comprising FGFβ, EGF, and IGF-1 in the absence of
extracellular proteins and endothelial cells”.
The specification provides no implicit or explicit support for the context of culturing MSCs in the absence of endothelial cells. The specification has only provided support for culturing in the absence of extracellular proteins. Applicants are reminded that it is their burden to show where the specification supports any amendments to the claims. See 37 CFR 1.121 (b)(2)(iii), the MPEP 714.02, 3rd paragraph, last sentence and also the MPEP 2163.07, last sentence.
MPEP 2163.06 notes “If new matter is added to the claims, the examiner should reject the claims under 35 U.S.C. 112, first paragraph - written description requirement. In re Rasmussen, 650 F.2d 1212, 211 USPQ 323 (CCPA 1981).” MPEP 2163.02 teaches that “Whenever the issue arises, the fundamental factual inquiry is whether a claim defines an invention that is clearly conveyed to those skilled in the art at the time the application was filed...If a claim is amended to include subject matter, limitations, or terminology not present in the application as filed, involving a departure from, addition to, or deletion from the disclosure of the application as filed, the examiner should conclude that the claimed subject matter is not described in that application. MPEP 2163.06 further notes “When an amendment is filed in reply to an objection or rejection based on 35 U.S.C. 112, first paragraph, a study of the entire application is often necessary to determine whether or not “new matter” is involved. Applicant should therefore specifically point out the support for any amendments made to the disclosure [or point to case law supporting incorporation of such a limitation as in the instant case]” (emphasis added).
New 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.
22. Claim 24 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.
23. Regarding claim 24, it is unclear if “an amine surface to generate HPCs” of step (a) is the same positively charged amine surface recited in step (a) of claim 1 or a different amine surface because claim 24 recites “an amine surface” and not “the positively charged amine surface”.
Claim Interpretation
24. For the purpose of applying prior art, “an amine surface to generate HPCs” of step (a) of claim 24 is interpreted as the “positively charged amine surface” recited in step (a) of claim 1.
25. For the purpose of applying prior art, “neutrally charged surface” of claim 37 is interpreted to include the Corning Primaria surface based on Applicant’s specification at para. 00175.
Claim Rejections Necessitated by Amendment - 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
26. Claim(s) 1, 6, 7, 10, 11, 12, 13, 15, 16, 23, 36, 37, 38, 41, 44, 47, 123, and 124 are rejected under 35 U.S.C. 103 as being unpatentable over Salvagiotto (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829.), hereinafter Salvagiotto in view of Abud (Abud, Edsel M., et al. Neuron 94.2 (2017): 278-293.), hereinafter Abud as evidenced by ThermoFisher (ThermoFisher: (September 26, 2025). “Cell Culture Vessels and Plastics”. https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/culture-plastics.html, Accessed 02/06/2026), hereinafter ThermoFisher in view of Burton (WO-2018067826-A1; previously cited), hereinafter Burton which is cited on the IDS filed 10/09/2023 in view of Muffat (Muffat J, et. al. Nat Med. 2016 Nov;22(11):1358-1367), hereinafter Muffat.
Regarding claim 1, Salvagiotto teaches an in vitro method for differentiating iPSCs to HPCs (step (a)) and differentiating the HPCs to endothelial cells (step (b) and “endothelial cells”) comprising culturing the iPSCs on murine collagen IV or human fibronectin (page 2, left col. last para.; Figure 1A; page 3, left col. last para. and right col. para. 1; page 5, right col. para. 2 – 3; page 6; Figure 4; page 7). Salvagiotto does not teach the surface is charged or the absence of extracellular matrix proteins.
Regarding claim 12, Salvagiotto teaches culturing the iPSCs in defined, serum-free media (page 2, left col. para. 2; page 5, right col. para. 2 – 3; page 6).
Regarding claim 13, Salvagiotto teaches culturing the iPSCs in the presence of a ROCK inhibitor (page 5, right col. last para.).
Regarding claim 23, Salvagiotto teaches differentiating the HPCs to endothelial cells (page 5, left col. last para. and right col. last para.; page 2, left col. para. 2 – 4; page 3, left col. para. 1 and right col.).
Regarding claim 47, Salvagiotto teaches the method is performed under hypoxic conditions (page 7, left col. para. 1; page 2, left col. para. 3 – 4; Figure 1C).
Regarding claim 123, Salvagiotto teaches the iPSCs are cultured in as a two-dimensional culture (Abstract; page 1, right col. last para.; Figure 1A).
Regarding claim 124, Salvagiotto teaches embryoid bodies are not formed (Abstract; Figure 1A).
Salvagiotto does not teach culturing iPSCs on either a positively charged amine surface or negatively charged surface in the absence of extracellular matrix proteins of step (a) of claim 1 or “the negatively charged surface is a carboxyl surface” of claim 6, “the negatively charged surface comprises oxygen-containing functional groups” of claim 7, “the surface is a polymeric surface” of claim 10, “the polymeric surface is a polystyrene surface” of claim 11 or culturing is carried out in the absence of the proteins recited in claim 15 or “comprising engineering the iPSCs to have disrupted expression of MeCP2 prior to step (a)” of claim 16 or “microglia” of claim 36 or “culturing the HPCs on a neutrally charged surface of ultralow attachment surface in the presence of microglia differentiation media” of claim 37 or “the microglia differentiation media comprises IL34, TGF, and/or MCSF” of claim 38 or “the microglia are positive for CD45, CD11b, and CD33” of claim 41, or “wherein step (b) of the method does not comprise purification of the cells” of claim 44. However, Salvagiotto teaches murine collagen IV and human fibronectin used in the method allowed for cell attachment (page 2, left col. last para.). Salvagiotto teaches the method is a 2D, feeder-free, serum-free, defined system (page 3, right col. last para.). Salvagiotto teaches for the potential use of hiPSCs in pre- and clinical settings the major challenge is to define culture conditions to differentiate progenitor cells into a selected lineage with high efficiency and purity (page 5, left col. last para.). Salvagiotto teaches their method can be easily converted to xenogenic-free conditions for potential clinical applications as the only reagents of non-human origin used in the method was bFGF (page 3, right col. last para.). Salvagiotto teaches iPSCs are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes for drug discovery, and in clinical cell therapy applications (Abstract). Salvagiotto teaches a robust differentiation method together with the accessibility of patient-specific pluripotent cell lines provide a novel approach to study blood disorders and the generation of patient-specific HPCs could eventually be used in cellular therapy (page 1, left col.). Salvagiotto teaches current methods for hematopoietic differentiation of pluripotent stem cells rely on the use of serum or co-culture and the poorly defined factors present in bovine serum prompted the development of a new defined animal product-free differentiation system to generate clinical grade hematopoietic progenitors (page 1, right col.).
Regarding “negatively charged surface” and “in the absence of extracellular matrix proteins” of step (a) of claim 1, “the negatively charged surface is a carboxyl surface” of claim 6, “the negatively charged surface comprises oxygen-containing functional groups” of claim 7, “the surface is a polymeric surface” of claim 10, “the polymeric surface is a polystyrene surface” of claim 11, Abud teaches a method of differentiating iPSCs to HPCs by culturing iPSCs on ThermoFisher tissue-culture treated plates under hypoxic conditions (page e4 of Star Methods, para. 2 and 7 – 8; page e5 of Star Methods, para. 1 – 5). ThermoFisher tissue culture treated plates have a polymeric surface (claim 10) of polystyrene (claim 11) that has a negative charge (“negatively charged surface” of step (a) of claim 1) that has oxygen-containing functional groups (claim 7) including carboxyl groups (claim 6) as evidenced by ThermoFisher (page 1, para. 1 – 3).
Regarding claim 15, Abud teaches culturing the iPSCS without the proteins recited in claim 15 (page e4 of Star Methods, para. 2 and 7 – 8; page e5 of Star Methods, para. 1 – 5).
Regarding claim 36, Abud teaches differentiating the HPCs to microglia (page e5 of Star Methods, para. 6 – 10; page 281, left col. para. 3 – 4).
Regarding claim 37, Abud teaches differentiating HPCs to microglia on Matrigel coated plates in the presence of microglia differentiation media (page e5 of Star Methods, para. 7) but does not teach “a neutrally charged surface or ultralow attachment surface”.
Regarding claim 38, Abud teaches the microglia differentiation media comprises IL34, TGFb1, and MCSF (page e5 of Star Methods, para. 7).
Regarding claim 41, Abud teaches the microglia are positive for CD45, CD11b, and CD33 (page 281, left col. para. 4 and right col. para. 1; Figure 1D – F; Figure 3B; page 286, left col. para. 1; Figure 4C).
Regarding claim 44, Abud teaches the microglia differentiated from HPCs were transplanted into rat brains without purification (e7 of Star Methods, para. 4).
Abud does not teach “engineering the iPSCs to have disrupted expression of MeCP2 prior to step (a)” of claim 16 or “a neutrally charged surface or ultralow attachment surface” of claim 37. However, Abud teaches the microglia differentiated from iPSCs can be used to study their function in neurological diseases like Alzheimer’s disease (Summary; page 291, left col. para. 3). Abud teaches a critical prerequisite of the two-step defined protocol is the robust differentiation of iPSCs to HPCs because this recapitulates microglia ontogeny as HPCs represent early primitive hematopoietic cells derived from the yolk sac that give rise to microglia during development (page 281, left col. para. 3). Abud teaches the method produces HPCs with a >90% purity resembling a commercial source (page 281, left col. para. 3). One would have been motivated to substitute the fibronectin or collagen surface of Salvagiotto with the tissue culture treated surface of Abud to reduce the cost of the method and further define the culture conditions because Abud teaches the method using tissue culture treated plates produces HPCs with a >90% purity resembling a commercial source.
Regarding “engineering the iPSCs to have disrupted expression of MeCP2 prior to step (a)” of claim 16, Burton teaches a method of culturing iPSCs to produce HPCs where iPSCs with MeCP disruption are generated through engineering and the MeCP knockout (MeCP2KO) iPSCs may then be differentiated into HPCs under defined feeder free, serum free conditions (page 16, para. 0080; page 65, para. 00247; Figure 1A; page 75, para. 00278). Burton teaches MeCP2KO iPSCs were adapted to hypoxia and subjected to differentiation to produce HPCs and further differentiated to microglia by the method of Abud (page 75, para. 00278). Burton teaches that knockout of MeCP2 resulted in increased purity of the microglia cell population as determined by the percent of cells positive for IBA, P2RY, and TREM-2 (page 75 – 76, para. 00280). Burton does not teach “a neutrally charged surface or ultralow attachment surface” of claim 37. One would have been motivated to combine the teachings of Salvagiotto, Abud, and Burton in a method to produce pure microglia from HPCs differentiated from iPSCs with MeCP2 knocked out Burton teaches that knockout of MeCP2 resulted in increased purity of the microglia cell population.
Regarding “a neutrally charged surface or ultralow attachment surface” of claim 37, Muffat teaches differentiating iPSCs to microglia in microglia differentiation media in ultra-low attachment six-well plates (“ultralow attachment surface”) followed by selective culturing of microglia on Primaria plates (“neutrally charged surface”) for selective culturing of microglia differentiated from hiPSCs (pMGLs) as the Primaria surface provides adherent maintenance of microglia (page 1368, right col. para. 2; page 1359, left col. para. 2; Figure 2a; Supplementary Figure 1). Muffat teaches to exclude the growth of neuro-ectodermal derivatives, Primaria plastic is used to positively select pMGLs (page 1366, left col. para. 3). Muffat teaches pMGLs faithfully recapitulated the expected ontogeny and characteristics of their in vivo counterparts and they resemble primary fetal human and mouse microglia (Abstract). Muffat teaches the availability of a robust protocol to generate and maintain microglia from patients with neurodegenerative diseases allows for study of the interaction of neurons and microglia and will facilitate the investigation of these diseases in defined culture conditions (page 1366, right col. para. 3).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Salvagiotto regarding a method of culturing iPSCs to form HPCs and differentiating HPCs to endothelial cells with the teachings of Abud regarding a method of culturing iPSCS to HPCs on a negatively charged surface in the absence of extracellular matrix proteins and a method of differentiating the HPCs to microglia with the teachings of Burton regarding a method of culturing iPSCs with MeCP2 knocked out to produce HPCs with the teachings of Muffat regarding differentiating iPSCs to microglia with ultra-low attachment plates and Primaria plates to arrive at the claimed method comprising:
(a) culturing the iPSCs on a negatively charged surface in the absence of extracellular matrix proteins to produce hematopoietic precursor cells (HPCs), and (b) differentiating the HPCs to endothelial cells, or microglia. One would have been motivated to combine the teachings of Salvagiotto, Abud, Burton, and Muffat in a defined method of producing microglia of high purity to study brain development and neurodegenerative diseases as Salvagiotto teaches for the potential use of hiPSCs in pre- and clinical settings the major challenge is to define culture conditions to differentiate progenitor cells into a selected lineage with high efficiency and purity and Muffat teaches the availability of a robust protocol to generate and maintain microglia from patients with neurodegenerative diseases allows for study of the interaction of neurons and microglia and will facilitate the investigation of these diseases in defined culture conditions and Abud teaches a critical prerequisite of the two-step defined protocol is the robust differentiation of iPSCs to HPCs because this recapitulates microglia ontogeny as HPCs represent early primitive hematopoietic cells derived from the yolk sac that give rise to microglia during development and Abud teaches the microglia differentiated from iPSCs can be used to study their function in neurological diseases like Alzheimer’s disease. One would have a reasonable expectation of success in combining the teachings as Abud teaches the method using the negatively charged surface produces HPCs with a >90% purity resembling a commercial source and Muffat teaches pMGLs faithfully recapitulated the expected ontogeny and characteristics of their in vivo counterparts and they resemble primary fetal human and mouse microglia.
27. Claim(s) 3 and 24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Salvagiotto (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829.), hereinafter Salvagiotto in view of Abud (Abud, Edsel M., et al. Neuron 94.2 (2017): 278-293.), hereinafter Abud as evidenced by ThermoFisher (ThermoFisher: (September 26, 2025). “Cell Culture Vessels and Plastics”. https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/culture-plastics.html, Accessed 02/06/2026), hereinafter ThermoFisher in view of Burton (WO-2018067826-A1; previously cited), hereinafter Burton which is cited on the IDS filed 10/09/2023 in view of Muffat (Muffat J, et. al. Nat Med. 2016 Nov;22(11):1358-1367), hereinafter Muffat as applied to claims 1, 6, 7, 10, 11, 12, 13, 15, 16, 23, 36, 37, 38, 41, 44, 47, 123, and 124 above, and further in view of Ying (US-10975350-B2; Filed 11/30/2016; Published 04/13/2021), hereinafter Ying as evidenced by Lim (Lim, Wai Feng, et al. Stem cell research & therapy 4.3 (2013): 71.), hereinafter Lim in view of Boespflug (Boespflug, Gaël, et al. Plasma Processes and Polymers 14.7 (2017): 1600139), hereinafter Boespflug.
Salvagiotto in view of Abud, Burton, and Muffat make obvious the limitations of claim 1 and 23 for culturing iPSCs on a negatively charged surface as set forth above. Salvagiotto teaches culturing iPSCs to generate HPCs and differentiation of HPCs to endothelial cells (page 2, left col. last para.; Figure 1A; page 3, left col. last para. and right col. para. 1; page 5, right col. para. 2 – 3; page 6; Figure 4; page 7). Salvagiotto teaches pluripotent cells offer a powerful system to create in vitro models of human development and disease, provide a valuable source of large quantities of mature cell types of consistent quality and purity for drug discovery and testing, and have strong potential for clinical cell replacement therapies (page 1, left col.). Salvagiotto teaches a robust differentiation method together with the accessibility of patient-specific pluripotent cell lines provide a novel approach to study blood disorders and patient-specific HPCs could eventually be used in cellular therapy (page 1, left col.). Salvagiotto teaches the method is completely defined and generates endothelial cells at very high efficiency (page 3, right col. last para.). Salvagiotto, Abud, Burton, and Muffat do not teach “a positively charged amine surface comprising amine monomers” of claim 3 or “step (a) comprises culturing on an amine surface to generate HPCs and step (b) comprises culturing on a carboxyl surface in the presence of endothelial differentiation media to produce endothelial cells” of claim 24.
Regarding “a positively charged amine surface” of step (a) of claim 1 and claim 3, Burton teaches pluripotent cells may be cultured on polylysine (page 53, para. 00212). Therefore, Burton teaches iPSCs may be cultured on a positively charged amine surface comprising amine monomers but does not teach that the positively charged amine surface does not comprise polylysine.
Regarding “a positively charged amine surface” and “in the absence of extracellular matrix proteins” and “wherein the positively charged amine surface does not comprise polylysine” of step (a) of claim 1 and claim 3 and “step (a) comprises culturing on an amine surface to generate HPCs” of claim 24, Ying teaches a positively charged amine surface (NP2) for culturing hiPSCs in defined serum-free media where having the amine functional group is essential to obtaining attachment of the cells on the cell culture substrate (col. 21, lines 30 – 35; col. 22, lines 40 and 65 – 67; col. 23, lines 28 – 29). Ying teaches it was remarkable that the percentage of cells that stained positive for the various pluripotent markers were either comparable to or higher than that achieved for extracellular matrices (col. 23, lines 52 – 55; Table 4). Ying teaches the NP2 surface was able to support the xeno-free culture of pluripotent stem cells (col. 25, lines 50 – 67; Figure 13). Ying teaches the cells cultured on the positively charged amine surface maintained their potential to differentiate into mesoderm indicating their potential to differentiate into cell types of the mesoderm (col. 25, lines 1 – 5 and 24 – 26; Figure 11a). iPSC-derived hematopoietic progenitors come from the mesoderm, which recapitulates in vivo development of the hematopoietic system as evidenced by Lim (page 1, left col. para. 2; Figure 1).
Regarding “amine monomers” of claim 3, Ying teaches the surface is a positively charged amine surface comprising amine monomers (Figure 1a; col. 15, lines 4 – 35; Table 3; col. 21, lines 30 – 35).
Ying does not teach “step (b) comprises culturing on a carboxyl surface in the presence of endothelial differentiation media to produce endothelial cells” of claim 24. However, Ying teaches when pluripotent stem cells were cultured on NP2, the differentiated cells would not attach to the substrate, leaving only the pluripotent colonies growing on the substrates which would be a very beneficial feature in practical applications (col. 25, lines 35 – 41). Ying teaches iPSCs have enormous potential for regenerative medicine for a number of diseases including spinal cord and cardiac injuries, Parkinson’s disease, brain cancer, motor neuron diseases, and multiple sclerosis, but the application of iPSCs in tissue engineering remains limited because long-term culture still requires the use of expensive recombinant extracellular matrix proteins or animal-derived matrices which are sources of variability, immunogenicity, and xenogeneic contamination (col. 1, lines 333 – 44). Ying teaches although there has been a shift towards the improvement of media formulations, most systems still involve either undefined or expensive substrates and therefore to enable clinical and industrial applications of stem cells improved, scalable, and affordable culture methods are required (col. 1, lines 47 – 53). One would have been motivated to combine the teachings of Burton and Ying because both teach that iPSCs can be cultured and differentiated on positively charged amine surfaces comprising amine monomers.
Regarding “step (b) comprises culturing on a carboxyl surface in the presence of endothelial differentiation media to produce endothelial cells” of claim 24, Boespflug teaches carboxyl surfaces for culturing endothelial cells (Figure 8; Table 2; page 7, left col. para. 3). Boespflug teaches in Figure 8 that endothelial cells adhere and show cellular activity on carboxyl surfaces (L-PPE:O) and tissue culture polystyrene and Primaria plates that contain carboxyl groups (Table 4). Boespflug teaches oxygen-containing surface functionalities are of great interest to support cell colonization as illustrated by the example of tissue culture polystyrene (TCP) (page 2, right col. para. 2). Boespflug teaches oxygen plasma-functionalized TCP containing a carboxyl surface (Table 3) is very satisfactory for HUVEC colonization and culture (page 8, left col. last para.). Boespflug teaches that carboxylic or ester groups are important in promoting endothelial colonization (page 10, left col. para. 1).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Salvagiotto regarding a method of culturing iPSCs to form HPCs and differentiating HPCs to endothelial cells with the teachings of Burton regarding iPSCs can be cultured on a positively charged amine surface with the teachings of Muffat regarding selective culturing on Primaria plates with the teachings of Ying regarding a positively charged amine surface comprising amine monomers for culturing iPSCs and differentiating the iPSCs to mesoderm with the teachings of Boespflug regarding endothelial cells adhere and can be cultured on carboxyl containing surfaces including Primaria plates to arrive at the claimed method comprising: (a) culturing the iPSCs on an amine surface to produce hematopoietic precursor cells (HPCs), and (b) culturing on a carboxyl surface in the presence of endothelial differentiation media to produce endothelial cells. One would have been motivated to combine the teachings of Salvagiotto, Burton, Muffat, Ying, and Boespflug in a defined, xeno-free method of producing endothelial cells for regenerative medicine as Salvagiotto teaches a robust differentiation method together with the accessibility of patient-specific pluripotent cell lines and patient-specific HPCs could eventually be used in cellular therapy and Ying teaches iPSCs have enormous potential for regenerative medicine for a number of diseases including cardiac injuries but the application of iPSCs in tissue engineering remains limited because long-term culture still requires the use of expensive recombinant extracellular matrix proteins or animal-derived matrices which are sources of variability, immunogenicity, and xenogeneic contamination and Ying teaches when pluripotent stem cells were cultured on NP2, the differentiated cells would not attach to the substrate, leaving only the pluripotent colonies growing on the substrates which would be a very beneficial feature in practical applications and Muffat teaches selective culturing of desired cell types on Primaria plates. One would have a reasonable expectation of success in combining the teachings as Salvagiotto teaches the method is completely defined and generates endothelial cells at very high efficiency and Ying teaches the NP2 surface was able to support the xeno-free culture of pluripotent stem cells and their potential to differentiate into mesoderm and both Salvagiotto and Ying teach the differentiated cells were floating and Boespflug teaches endothelial cells adhere and show cellular activity on carboxyl surfaces including Primaria plates that contain carboxyl groups.
28. Claim(s) 26 is/are rejected under 35 U.S.C. 103 as being unpatentable over Salvagiotto (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829.), hereinafter Salvagiotto in view of Abud (Abud, Edsel M., et al. Neuron 94.2 (2017): 278-293.), hereinafter Abud as evidenced by ThermoFisher (ThermoFisher: (September 26, 2025). “Cell Culture Vessels and Plastics”. https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/culture-plastics.html, Accessed 02/06/2026), hereinafter ThermoFisher in view of Burton (WO-2018067826-A1; previously cited), hereinafter Burton which is cited on the IDS filed 10/09/2023 in view of Muffat (Muffat J, et. al. Nat Med. 2016 Nov;22(11):1358-1367), hereinafter Muffat as applied to claims 1, 6, 7, 10, 11, 12, 13, 15, 16, 23, 36, 37, 38, 41, 44, 47, 123, and 124 above, and further in view of Minami (Minami, Haruka, et al. PLoS One 10.6 (2015): e0128890.), hereinafter Minami.
Salvagiotto in view of Abud, Burton, and Muffat make obvious the limitations of claim 1 and 23 as set forth above. Salvagiotto teaches culturing iPSCs to generate HPCs and differentiation of HPCs to endothelial cells (page 2, left col. last para.; Figure 1A; page 3, left col. last para. and right col. para. 1; page 5, right col. para. 2 – 3; page 6; Figure 4; page 7). Salvagiotto teaches pluripotent cells offer a powerful system to create in vitro models of human development and disease, provide a valuable source of large quantities of mature cell types of consistent quality and purity for drug discovery and testing, and have strong potential for clinical cell replacement therapies (page 1, left col.). Salvagiotto teaches a robust differentiation method together with the accessibility of patient-specific pluripotent cell lines provide a novel approach to study blood disorders and patient-specific HPCs could eventually be used in cellular therapy (page 1, left col.). Salvagiotto teaches the method is completely defined and generates endothelial cells at very high efficiency (page 3, right col. last para.). Abud teaches the microglia differentiated from iPSCs can be used to study their function in neurological diseases like Alzheimer’s disease (Summary; page 291, left col. para. 3). Muffat teaches the availability of a robust protocol to generate and maintain microglia from patients with neurodegenerative diseases allows for study of the interaction of neurons and microglia and will facilitate the investigation of these diseases in defined culture conditions (page 1366, right col. para. 3). Burton teaches that knockout of MeCP2 resulted in increased purity of the microglia cell population as determined by the percent of cells positive for IBA, P2RY, and TREM-2 (page 75 – 76, para. 00280). Salvagiotto, Abud, Burton, and Muffat do not teach “further comprising differentiating the endothelial cells to brain microvascular endothelial cells (BMECs) or lymphatic endothelial cells” of claim 26.
Minami teaches human PSC-derived endothelial cells are expected to be used as sources for human BMECs (page 2, para. 2). Minami teaches a method of differentiating iPSCs to endothelial cells (iPS-ECs) under serum- and feeder-free conditions followed by differentiating the iPS-ECs to BMEC cells (page 2, para. 4; Figure 1A; page 9, para. 2; page 11, para. 2; page 4, para. 1 and last para.; page 6; page 7, para. 1). Minami teaches the blood brain barrier (BBB) is formed by BMECs and tightly regulates the transport of molecules from blood to neural tissues and in vitro BBB models would be useful for research on the BBB development and function but also for drug-screening for neurological disease (Abstract; page 7, para. 2; page 8, para. 4). Minami teaches the use of primary BMECs to model the BBB has drawbacks including their limited range of sources and differences in function from batch to batch (page 2, para. 1).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Salvagiotto regarding a method of differentiating iPSCs to endothelial cells with the teachings of Abud regarding a method of generating glial cells to study their function in neurological diseases with the teachings of Muffat regarding a robust protocol to generate glial cells from patients with neurodegenerative diseases allows for the study of their role in diseases in defined culture conditions with the teachings of Burton regarding a method of increasing the purity of iPSC-derived glial cells with the teachings of Minami regarding a method of differentiating iPSCs to BMECs to arrive at the claimed method further comprising differentiating the endothelial cells to brain microvascular endothelial cells (BMECs). One would have been motivated to combine the teachings of Salvagiotto, Abud, Muffat, Burton, and Minami in a defined method of producing BMECs with high purity to study the BBB as Minami teaches the BBB is formed by BMECs and in vitro BBB models would be useful for research on the BBB development and function but also for drug-screening for neurological disease and Minami teaches human PSC-derived endothelial cells are expected to be used as sources of human BMECs. One would have a reasonable expectation of success in combining the teachings as Salvagiotto teaches the method is completely defined and generates endothelial cells at very high efficiency and Minami teaches maturation of human iPSC-derived endothelial cells to BMECs.
29. Claim(s) 28 – 31 is/are rejected under 35 U.S.C. 103 as being unpatentable over Salvagiotto (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829.), hereinafter Salvagiotto in view of Abud (Abud, Edsel M., et al. Neuron 94.2 (2017): 278-293.), hereinafter Abud as evidenced by ThermoFisher (ThermoFisher: (September 26, 2025). “Cell Culture Vessels and Plastics”. https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/culture-plastics.html, Accessed 02/06/2026), hereinafter ThermoFisher in view of Burton (WO-2018067826-A1; previously cited), hereinafter Burton which is cited on the IDS filed 10/09/2023 in view of Muffat (Muffat J, et. al. Nat Med. 2016 Nov;22(11):1358-1367), hereinafter Muffat as applied to claims 1, 6, 7, 10, 11, 12, 13, 15, 16, 23, 36, 37, 38, 41, 44, 47, 123, and 124 above, and further in view of Kopher (Kopher, Ross A., et al. Bone 47.4 (2010): 718-728.), hereinafter Kopher in view of Chua (Chua KN, et. al. Biomaterials. 2006 Dec;27(36):6043-51), hereinafter Chua.
Salvagiotto in view of Abud, Burton, and Muffat make obvious the limitations of claim 1 as set forth above. Salvagiotto, Abud, Burton, and Muffat do not teach “differentiating HPCs to MSCs” of claim 28 or “culturing the HPCs on an amine surface in the presence of MSC media and absence of extracellular matrix proteins” of claim 29 or “the MSCs are positive for CD73, CD44, and CD105” of claim 30 or “at least 90% of the differentiated cells are positive for CD73” of claim 31. However, Salvagiotto teaches the differentiation of pluripotent stem cells results in CD34+ progenitor cells that includes mesenchymal progenitor cells (page 3, left col. para. 2 and right col. para. 1). Salvagiotto teaches that under the optimized condition for hematopoietic differentiation, no consistent differences were observed between hESC and iPSC (page 3, right col. para. 1).
Regarding “differentiating the HPCs to MSCs” of claim 28 and “the MSCs are positive for CD73, CD44, and CD105” of claim 30 and “at least 90% of the differentiated cells are positive for CD73” of claim 31, Kopher teaches hESC-derived CD34+CD73- cells can serve as MSC progenitor cells and differentiate into MSCs (claim 28) (Abstract; page 720, right col. last para.; page 724, right col. last para.; page 725, left col. last para. and right col. para. 1). Kopher teaches culturing CD34-CD73+ cells in MSC media results in MSC cells that express CD73, CD44, and CD105 (claim 30) with more than 90% are positive for CD73 (claim 31) (page 720, right col. last para.; Figure 1A – C; page 721, left col.). Kopher teaches culturing CD34+CD73- and CD34+CD73+ cells in MSC media under conditions conducive to attachment and expansion where both cell populations gradually lost CD34 expression while maintaining CD73 expression and showed MSC morphology (page 723, left col.; Figure 3; page 724, left col. para. 1). Kopher teaches the MSCs expressed CD73, CD105, and CD44 and could be differentiated to osteogenic, chondrogenic, and adipogenic lineages (page 724, left col. para. 1; Figure 4). Kopher teaches culture of hESC-CD34+CD73- cells cultured in MSC media developed into 90 – 95% CD73+ population (page 724, left col. para. 2; Figure 5). Kopher teaches the specific isolation of functional MSCs from hESC-derived CD34+CD73- cells that differentiate into CD34+CD73+ and CD34-CD73+ cells provides new insight for mesodermal development and differentiation (page 724, right col. last para.). Kopher teaches they have previously shown the endothelial potential of hESC-derived CD34+ cells but now they show a mesenchymal potential of hESC-derived CD34+ cells (page 725, left col. para. 1). Kopher teaches CD34 has traditionally been used as a marker that selects against MSCs; however tow progenitor cell populations expressing CD34 have differentiated into functional MSCs (page 726, right col. last para.). Kopher teaches that because fetal- and adult-derived MSCs appear to have some limitations, hESC-derived MSCs will provide improved opportunities for regenerative medicine (page 726, right col. para. 2; Abstract; page 718, right col. para. 1). Kopher teaches MSCs are currently under study to aid in several therapies (page 718, left col.). Kopher does not teach “culturing the HPCs on an amine surface in the presence of MSC media and absence of extracellular matrix proteins” of claim 29. One would have been motivated to combine the teachings of Salvagiotto and Kopher because both teach that CD34+ progenitor cells are mesenchymal progenitor cells.
Regarding “an amine surface” and “absence of extracellular matrix proteins” of claim 29, Chua teaches expansion of CD34+ HPCs on unmodified, carboxylated and animated nanofibers where the aminated nanofibers were the most efficient in supporting the expansion of the HPCs and supported a higher degree of cell adhesion (Abstract; Figure 1D; Table 1; page 6046, right col.; Figure 2; page 6047, left col. para. 1; page 6050, left col. para. 1). Chua teaches the expansion conditions included using commercially available serum-free stem cell media (page 6050, left col. para. 1). Chua teaches the HPCs adhered better to aminated surfaces (page 6048, left col. para. 2 – 3 and right col. para. 1). Chua teaches evidence suggests the importance of surface chemistry on the rate of HPC proliferation and CD34+ cell expansion where surface choice can significantly affect the outcome of ex vivo expansion (page 6044, left col. para. 1). Chua teaches an efficient and practical ex vivo expansion strategy is necessary to produce sufficient quantity of HPCs for therapy (page 6043).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Salvagiotto regarding a method of producing iPSC-derived HPCs containing CD34+ MSC progenitor cells with the teachings of Kopher regarding a method of differentiating CD34+ MSC progenitors to MSCs by expanding these progenitors in MSC media with the teachings of Chua regarding expanding CD34+ HPCs on an amine surface to arrive at the claimed method where differentiating comprises culturing the HPCs on an amine surface in the presence of MSC media and absence of extracellular matrix proteins. One would have been motivated to combine the teachings of Salvagiotto, Kopher, and Chua in a defined method of producing MSCs for regenerative therapy as Kopher teaches that because fetal- and adult-derived MSCs appear to have some limitations, hESC-derived MSCs will provide improved opportunities for regenerative medicine and Chua teaches an efficient and practical ex vivo expansion strategy is necessary to produce sufficient quantity of HPCs for therapy. One would have a reasonable expectation of success in combining the teachings as Salvagiotto teaches CD34+ cells contain mesenchymal progenitors, Kopher teaches expansion of CD34+ mesenchymal progenitors produced MSCs and Chua teaches an amine surface showed the best performance for expansion of HPCs.
30. Claim(s) 28, 32, 33, and 34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Salvagiotto (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829.), hereinafter Salvagiotto in view of Abud (Abud, Edsel M., et al. Neuron 94.2 (2017): 278-293.), hereinafter Abud as evidenced by ThermoFisher (ThermoFisher: (September 26, 2025). “Cell Culture Vessels and Plastics”. https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/culture-plastics.html, Accessed 02/06/2026), hereinafter ThermoFisher in view of Burton (WO-2018067826-A1; previously cited), hereinafter Burton which is cited on the IDS filed 10/09/2023 in view of Muffat (Muffat J, et. al. Nat Med. 2016 Nov;22(11):1358-1367), hereinafter Muffat as applied to claims 1, 6, 7, 10, 11, 12, 13, 15, 16, 23, 36, 37, 38, 41, 44, 47, 123, and 124 above, and further in view of Kumar (Kumar, Akhilesh, et al. Cell reports 19.9 (2017): 1902-1916.), hereinafter Kumar in view of Peters (Brown Peters, Erica Cho. "A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies." (2015); previously cited), hereinafter Peters.
Salvagiotto in view of Abud, Burton, and Muffat make obvious the limitations of claim 1 as set forth above.
Regarding “differentiating HPCs to MSCs” of claim 28, Salvagiotto teaches the differentiation of pluripotent stem cells results in the formation of HPCs that contain mesenchymal progenitor cells (“MSCs”) (page 3, left col. para. 2 and right col. para. 1). Salvagiotto, Abud, Burton, and Muffat do not teach “differentiating the MSCs to pericytes” of claim 32, or “the pericytes are positive for NG2, PDGFRβ, and/or CD146” of claim 34.
Regarding “differentiating the MSCs to pericytes” of claim 32, Kumar teaches a method of differentiating mesenchymal progenitors (“MSCs”) to pericytes (page 1915, left col. para. 4; Figure 1A; page 1905, left col. para. 3 and right col. para. 1; Figure 7). Kumar teaches the pericytes are derived from MSCs that are derived from iPSCs (Figure S2; page 1905, right col. para. 1).
Regarding claim 33, Kumar teaches FGF2 (“FGFb”) in Figure 1A and Figure 7but does not teach EGF or IGF-1.
Regarding “the pericytes are positive for NG2, PDGFRβ, and/or CD146” of claim 34, Kumar teaches the pericytes expressed NG2, PDGFRβ, and CD146 (page 1905, left col. para. 3; Figure 1B – C).
Kumar does not teach “EGF, and IGF-1” of claim 33. However, Kumar teaches pericytes can be used for modeling genetic diseases associated with vascular and skeletal abnormalities by employing patient-specific iPSCs and the method is reproducible for the scalable generation of distinct populations of pericytes of mesodermal origin from hPSCs for potential applications in regenerative medicine (page 1903, right col. page 1915, left col. para. 1). Kumar teaches the pericytes showed HUVEC tube supporting capability mimicking the position of pericytes in vessels in vivo and strongly supported the formation of neovessels containing circulating blood cells in vivo (page 1911, right col. para. 1 and 3; Figure 4 and 6). One would have been motivated to combine the teachings of Salvagiotto and Kumar because both teach methods for deriving mesenchymal progenitors from iPSCs.
Regarding “EGF, and IGF-1” of claim 33, Peters teaches MSCs are able to function as mural cells (pericytes) by supporting human cord blood-derived endothelial progenitor cells (hCB-EPC) network formation only when cultured with endothelial growth media supplement comprising FGF-2, EGF, and IGF-1 (page 182; page 213, para. 1; Figure 5.10; page 216, para. 1). Peters teaches MSCs can serve as pericyte-progenitor cells revealing novel opportunities for their use in vascular therapies (page 182, last para.). Peters teaches this tissue engineered model serves as a non-invasive research tool and has shown promise for direct translation into pro-angiogenic therapies where MSCs and hCB-EPCs are promising allogenic sources for tissue engineered-microvessel structures due to their noninvasive isolation and proliferative potential (page 215, para. 1; page 216, para. 2).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Salvagiotto regarding a method of producing HPCs that also produces mesenchymal progenitor cells with the teachings of Kumar regarding a method of producing pericytes from mesenchymal progenitor cells where the pericytes support HUVEC tubes in vitro and support the formation of neovessels in vivo with the teachings of Peters regarding a hCB-EPC network comprising MSCs that function as pericytes only in the presence of FGF-2, EGF, and IGF-1 to arrive at the claimed method where MSCs are differentiated to pericytes. One would have been motivated to combine the teachings of Salvagiotto, Kumar, and Peters in a method of producing pericytes to study vascular diseases and for regenerative therapy as Kumar teaches pericytes can be used for modeling genetic diseases associated with vascular and skeletal abnormalities by employing patient-specific iPSCs and the method is reproducible for the scalable generation of distinct populations of pericytes of mesodermal origin from hPSCs for potential applications in regenerative medicine. One would have a reasonable expectation of success in combing the teachings as Kumar teaches the pericytes produced from mesenchymal progenitors support neovessel formation in vivo and Peters teaches the MSCs act as pericytes in the hCB-EPC when cultured in the defined media.
31. Claim(s) 46 is/are rejected under 35 U.S.C. 103 as being unpatentable over Salvagiotto (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829.), hereinafter Salvagiotto in view of Abud (Abud, Edsel M., et al. Neuron 94.2 (2017): 278-293.), hereinafter Abud as evidenced by ThermoFisher (ThermoFisher: (September 26, 2025). “Cell Culture Vessels and Plastics”. https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/culture-plastics.html, Accessed 02/06/2026), hereinafter ThermoFisher in view of Burton (WO-2018067826-A1; previously cited), hereinafter Burton which is cited on the IDS filed 10/09/2023 in view of Muffat (Muffat J, et. al. Nat Med. 2016 Nov;22(11):1358-1367), hereinafter Muffat as applied to claims 1, 6, 7, 10, 11, 12, 13, 15, 16, 23, 36, 37, 38, 41, 44, 47, 123, and 124 above, and further in view of Kaupisch (Kaupisch, A., et al. Journal of cardiovascular translational research 5.5 (2012): 605-617.), hereinafter Kaupisch.
Salvagiotto in view of Abud, Burton, and Muffat make obvious the limitations of claim 1 as set forth above but do not teach the method is GMP compliant. However Salvagiotto the method is a 2D, feeder-free, serum-free, defined system (page 3, right col. last para.). Salvagiotto teaches for the potential use of hiPSCs in pre- and clinical settings the major challenge is to define culture conditions to differentiate progenitor cells into a selected lineage with high efficiency and purity (page 5, left col. last para.). Salvagiotto teaches their method can be easily converted to xenogeneic-free conditions for potential clinical applications as the only reagents of non-human origin used in the method was bFGF (page 3, right col. last para.). Salvagiotto teaches iPSCs are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes for drug discovery, and in clinical cell therapy applications (Abstract). Salvagiotto teaches a robust differentiation method together with the accessibility of patient-specific pluripotent cell lines provide a novel approach to study blood disorders and the generation of patient-specific HPCs could eventually be used in cellular therapy (page 1, left col.). Salvagiotto teaches current methods for hematopoietic differentiation of pluripotent stem cells rely on the use of serum or co-culture and the poorly defined factors present in bovine serum prompted the development of a new defined animal product-free differentiation system to generate clinical grade hematopoietic progenitors (page 1, right col.). Abud teaches a method of differentiating iPSCs to HPCs by culturing iPSCs on ThermoFisher tissue-culture treated plates under hypoxic conditions (page e4 of Star Methods, para. 2 and 7 – 8; page e5 of Star Methods, para. 1 – 5).
Kaupisch teaches a GMP-compliant method for the production of endothelial cells from hESCs (page 607, left col. and right col. para. 1 – 2; page 610, right col. para. 1). Kaupisch teaches the derivation of the hESCs was conducted using materials which were accredited by suppliers as GMP compliant and in almost all instances free of animal components (page 607, left col. para. 2). Kaupisch teaches the media for culture could be custom-made to be GMP-compliant and the components for differentiation were supplied as GMP compliant or suitable for GMP processing (page 607, right col. para. 1 – 2; page 612, left col. last para. and right col. para. 1). Kaupisch teaches modifying a previous protocol to be GMP compliant and that the endothelial cells are produced at a standard suitable for clinical trial purposes (page 614, right col. para. 2; page 615, right col. para. 2). Kaupisch teaches revascularization of ischemic tissue remains an area of substantial unmet clinical need in cardiovascular disease (Abstract). Kaupisch teaches endothelial cells will have to be manufactured to the very high standards described in the appropriate directives and regulations underpinned by the principles of GMP (page 606, left col. last para. and right col. para. 1). Kaupisch teaches the GMP-compliant method provides an important proof of concept for further studies to enhance differentiation and generate stable populations of progenitor and mature endothelial cells for safe and therapeutic investigations in the context of peripheral and/or myocardial ischemia (page 615, right col. para. 2).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Salvagiotto regarding a method of culturing iPSCs to form HPCs and differentiating HPCs to endothelial cells where the method can be easily converted to xenogeneic-free conditions for potential clinical applications with the teachings of Abud regarding a method of culturing iPSCS to HPCs on a negatively charged surface in the absence of extracellular matrix proteins with the teachings of Burton regarding a method of culturing iPSCs with MeCP2 knocked out to produce HPCs with the teachings of Kaupisch regarding a GMP-compliant method of producing endothelial cells from pluripotent cells to arrive at the claimed method where the method is GMP compliant. One would have been motivated to combine the teachings of Salvagiotto, Abud, Burton, and Kaupisch in a defined method for producing large quantities of highly pure endothelial cells for clinical therapies as Salvagiotto teaches iPSCs are an attractive source of cells of high quantity and purity to be used in clinical cell therapy applications and Kaupisch teaches revascularization of ischemic tissue remains an area of substantial unmet clinical need in cardiovascular disease and Kaupisch teaches endothelial cells will have to be manufactured to the very high standards described in the appropriate directives and regulations underpinned by the principles of GMP. One would have a reasonable expectation of success in combining the teachings as Salvagiotto teaches their method can be easily converted to xenogeneic-free conditions for potential clinical applications and Abud teaches the method used commercially available tissue culture plates and Kaupisch teaches the cell culture media and differentiation reagents could be made GMP compliant by sourcing reagents from GMP compliant vendors.
Applicant’s Arguments/ Response to Arguments
32c. Applicant Argues: On page 8, last para. and page 9, para. 5, Applicant asserts that Burton does not anticipate amended claim 1 because Burton teaches culturing on polylysine and claim 1 has been amended to exclude polylysine.
Response to Arguments: The previous rejection of the claims under 35 U.S.C. 102(a)(1) is withdrawn in view of Applicant’s amendment to the claims. In the new rejection of amended claim 1 set forth above, Burton is still cited for teaching that iPSCs can be cultured on a positively charged amine surface in paragraph 00212, which is reproduced below. Burton teaches that 1 component may be used for culturing the pluripotent cells in the absence of extracellular matrix proteins.
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Applicant Argues: On page 9, paragraph 3, Applicant requests removal of the rejection using the teachings of Lewis.
Response to Arguments: The previous rejection of the claims under 35 U.S.C. 102(a)(1) is withdrawn in view of Applicant’s amendment to the claims.
Applicant Argues: On page 9, last para. – page 10, para. 1 – 2, Applicant asserts that one of skill in the art would not have expected that HPCs could be efficiently produced without the presence of ECM proteins based on the disclosure of Fryer and Ilmarinen teaches differentiation is performed in the presence of ECM proteins.
Response to Arguments: The previous rejection of the claims using the teachings of Lewis and Ilmarinen have been withdrawn in view of Applicant’s amendment to the claims.
Applicant Argues: On page 10, last para. and page 11, para. 1, Applicant asserts that the charged surfaces in the absence of matrix proteins allows for the production of HPCs and the additional cell types without needing to perform purification such as MACS and that the cited art does not teach or suggest the culture of iPSCs on an amine surface or negatively charged surface to differentiate to HPCs in the absence of ECM proteins which are then further efficiently differentiated to produce highly pure populations of the various cell types such as without the need for purification of the cells.
Response to Arguments: In the new rejection set forth above, Abud teaches a method of differentiating iPSCs to HPCs by culturing iPSCs on ThermoFisher tissue-culture treated plates under hypoxic conditions (page e4 of Star Methods, para. 2 and 7 – 8; page e5 of Star Methods, para. 1 – 5). ThermoFisher tissue culture treated plates have a polymeric surface of polystyrene that has a negative charge that has oxygen-containing functional groups including carboxyl groups as evidenced by ThermoFisher (page 1, para. 1 – 3). Abud does not teach culturing the iPSCS with any ECM proteins (page e4 of Star Methods, para. 2 and 7 – 8; page e5 of Star Methods, para. 1 – 5). Abud teaches differentiating the HPCs to microglia (page e5 of Star Methods, para. 6 – 10; page 281, left col. para. 3 – 4). Abud teaches the microglia differentiated from HPCs were transplanted into rat brains without purification (e7 of Star Methods, para. 4). Thus, Abud does not teach a need for performing purification of the microglia prior to transplantation.
Muffat teaches differentiating iPSCs to microglia in microglia differentiation media in ultra-low attachment six-well plates followed by culturing of microglia on Primaria plates for selective culturing of microglia differentiated from hiPSCs (pMGLs) as the Primaria surface provides adherent maintenance of microglia (page 1368, right col. para. 2; page 1359, left col. para. 2; Figure 2a; Supplementary Figure 1). Muffat teaches to exclude the growth of neuro-ectodermal derivatives, Primaria plastic is used to positively select pMGLs (page 1366, left col. para. 3). Thus, Muffat teaches that culturing using specific surfaces allows for purification without the need for performing purification protocols such as MACS.
Applicant Argues: On page 15, para. 3, Applicant asserts that there is no teaching or disclosure in Peters on differentiating MSCs to pericytes in the absence of ECM and endothelial cells and in fact teaches that the absence of ECM proteins may hinder the ability of MSCs to support EC network formation.
Response to Arguments: The previous rejections of claim 33 has been withdrawn and new rejections set forth above. In the new rejection, Kumar teaches a method of differentiating mesenchymal progenitors to pericytes (page 1915, left col. para. 4; Figure 1A; page 1905, left col. para. 3 and right col. para. 1; Figure 7). Kumar teaches the pericytes are derived from MSCs that are derived from iPSCs (Figure S2; page 1905, right col. para. 1). Kumar teaches FGF2 (“FGFb”) in Figure 1A and Figure 7 but does not teach EGF or IGF-1. Kumar teaches the pericytes expressed NG2, PDGFRβ, and CD146 (page 1905, left col. para. 3; Figure 1B – C). Kumar teaches pericytes can be used for modeling genetic diseases associated with vascular and skeletal abnormalities by employing patient-specific iPSCs and the method is reproducible for the scalable generation of distinct populations of pericytes of mesodermal origin from hPSCs for potential applications in regenerative medicine (page 1903, right col.page 1915, left col. para. 1). Kumar teaches the pericytes showed HUVEC tube supporting capability mimicking the position of pericytes in vessels in vivo and strongly supported the formation of neovessels containing circulating blood cells in vivo (page 1911, right col. para. 1 and 3; Figure 4 and 6).
Peters teaches MSCs are able to function as mural cells (pericytes) by supporting human cord blood-derived endothelial progenitor cells (hCB-EPC) network formation only when cultured with endothelial growth media supplement comprising FGF-2, EGF, and IGF-1 (page 182; page 213, para. 1; Figure 5.10; page 216, para. 1). Peters teaches MSCs can serve as pericyte-progenitor cells revealing novel opportunities for their use in vascular therapies (page 182, last para.). Peters teaches this tissue engineered model serves as a non-invasive research tool and has shown promise for direct translation into pro-angiogenic therapies where MSCs and hCB-EPCs are promising allogenic sources for tissue engineered-microvessel structures due to their noninvasive isolation and proliferative potential (page 215, para. 1; page 216, para. 2).
One would have been motivated to combine the teachings of Salvagiotto, Kumar, and Peters in a method of producing pericytes to study vascular diseases and for regenerative therapy as Kumar teaches pericytes can be used for modeling genetic diseases associated with vascular and skeletal abnormalities by employing patient-specific iPSCs and the method is reproducible for the scalable generation of distinct populations of pericytes of mesodermal origin from hPSCs for potential applications in regenerative medicine. One would have a reasonable expectation of success in combing the teachings as Kumar teaches the pericytes produced from mesenchymal progenitors support neovessel formation in vivo and Peters teaches the MSCs act as pericytes in the hCB-EPC when cultured in the defined media.
Applicant Argues: On page 11, para. 2 – 4 – page16, Applicant asserts that claimed invention is distinct from the cited teachings of Lewis, Oh, Lu, Ramachandran, Crook, Wang, Loibl, Hickman, Peters, Fryer, and Saha.
Response to Arguments: The previous rejections of the claims using the cited teachings of Lewis, Oh, Lu, Ramachandran, Crook, Wang, Loibl, Hickman, Fryer, and Saha have been withdrawn and therefore arguments addressing these cited teachings are moot.
Conclusion
No claims allowed.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632