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 .
Claims 1, 10 – 13, 15, 16, 23, 24, 26 – 34, 36 – 38, 41, 44, 46, 47, 52, 92, 123, 124, and new claims 125 and 126 are pending. Claims 1, 10 – 13, 15, 16, 23, 24, 26, 28 – 34, 36 – 38, 41, 44, 46, 47, and 123 – 126 are under consideration. Claims 3, 6, and 7 have been canceled.
Withdrawn Claim Objection
The objection to claim 31 is withdrawn in view of Applicant’s amendment to the claim.
Withdrawn Claim Rejections
The rejection of claim 33 under 35 U.S.C. 112(a) as failing to comply with the written description requirement is withdrawn in view of Applicant’s amendment to the claim.
The rejection of claim 24 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to the claim.
The rejection of claims 6 and 7 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancelation of these claims.
The rejection of claim 3 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancelation of the claim.
The rejection of claims 1, 6, 7, 10, 11, 12, 13, 15, 16, 23, 36, 37, 38, 41, 44, 47, 123, and 124 under 35 U.S.C. 103 in view of Applicant’s amendment to claim 1 requiring a “plasma polymerized amine surface”.
The rejection of claim 24 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
The rejection of claim 26 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1 and 26.
The rejection of claims 28 – 31 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
The rejection of claims 28, 32, 33, and 34 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1 and claim 33.
The rejection of claim 46 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
Claim Objections and Rejections Necessitated by Amendment
Claim Objections
Claim 33 is objected to because of the following informalities: in line 2, “consisting of MSCs” should read “consisting of the MSCs” for consistency with “the MSCs” recited in line 1 and in line 4, “extracellular proteins” should read “extracellular matrix proteins” based on Applicant’s specification at para. 00207. Appropriate correction is required.
Claim 125 is objected to because of the following informalities: in line 1 – 2, “wherein the method produces at least 90% CD31+CD144+CD105+ endothelial cells” should read “wherein the method produces endothelial cells that are at least 90% positive for CD31, CD144, and CD105” to clarify that 90% refers to the expression of the markers based on Figure 3. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1, 10 – 13, 15, 16, 23, 24, 26 – 34, 36 – 38, 41, 44, 46, 47, and 123 – 126 are 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.
Regarding claim 1, it is unclear if recitation of “a positively charged plasma polymerized amine surface comprising amine monomers” means that the surface comprises both polymerized amines and amine monomers, or that the surface is a polymerized amine surface produced by plasma polymerization of amine monomers. It is unclear how the polymerized amine surface would also contain amine monomers if the claim requires the surface to be polymerized. Applicant’s specification at para. 00177 describes the plasma polymerization technique where monomers are deposited onto cell culture surfaces which are then polymerized. Applicant’s specification at para. 00176 states that the cell culture surface may be coated with a plasma polymerized film; the source of the plasma polymerization is one or more monomers; and useful polymerizable monomers may include unsaturated organic compounds such as olefinic amines. The exemplary HPC differentiation method and Examples 1 and 2 in Applicant’s specification state “Amine culture” dishes at para. 00131, 00203, and 00205. Therefore, the structure of the culture surface recited as “a positively charged plasma polymerized amine surface comprising amine monomers” is unclear. Claims 10 – 13, 15, 16, 23, 24, 26 – 34, 36 – 38, 41, 44, 46, 47, and 123 – 126 are also rejected as they depend from claim 1 and do not clarify the grounds of rejection.
Regarding claim 11, it is unclear how the polymeric surface can be polystyrene because claim 1 requires a positively charged polymerized amine surface and polystyrene is not positively charged, nor does it contain amine groups.
Regarding claim 126, it is unclear if “cells” refers to microglia, HPCs, or another cell type.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 10, 11, and 123 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 1 has been amended to recite “in two-dimensional culture on a positively charged plasma polymerized amine surface comprising amine monomers”. A plain reading of these limitations is that the surface is a positively charged polymerized amine surface.
Claim 10 fails to further limit claim 1 because claim 1 requires that the surface is a polymeric surface. Therefore, recitation of “polymeric surface” in claim broadens the “surface” of claim 1 to any polymeric surface.
Claim 11 fails to further limit claim 1 and instead broadens the surface of claim 1 because polystyrene does not contain amine groups and polystyrene is negatively charged.
Claim 123 fails to further limit claim 1 because claim 1 requires two-dimensional culture in step (a).
Claim 126 fails to further limit claim 37 because recitation of “cells” in claim 126 broadens “microglia” recited in claim 36 to any cell.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Interpretation
For the purpose of applying prior art, “positively charged plasma polymerized amine surface comprising amine monomers” of claim 1 and “polymeric surface” of claim 10 are interpreted as a positively charged polymerized amine surface prepared by plasma polymerization of amine monomers based on Applicant’s specification at para. 00176 that states “the cell culture surface may be coated with a plasma polymerized film. The source of the plasma polymerization is one or more monomers.”
For the purpose of applying prior art, “the polymeric surface is a polystyrene surface” of claim 11 is interpreted as the cell culture surface that is coated with a positively charged amine surface is polystyrene based on Applicant’s specification at para. 00176 and because polystyrene is negatively charged while claim 1 requires a positively charged surface.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
Claim(s) 1, 10, 11, 12, 13, 15, 23, 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; previously cited), hereinafter Salvagiotto as evidenced by FujiFilm (FujiFilm, iCell Endothelial Cells, 01434 and iCell Endothelial Cells, 11713; https://www.fujifilmcdi.com/icell-endothelial-cells-01434-getc01434 and https://www.fujifilmcdi.com/icell-endothelial-cells-11713-getc11713, Accessed 08/27/2026) in view of Irwin (Irwin, Elizabeth F., et al. Biomaterials 32.29 (2011): 6912-6919.), hereinafter Irwin, in view of Burns (Burns, Norman L. et. al. Journal of Colloid and Interface Science 178.1 (1996): 116-122.), hereinafter Burns.
Regarding claims 1 and 23, Salvagiotto teaches an in vitro 2D (“two-dimensional culture”) 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 1, right col. last para.; page 2, left col. para. 2 – 4; Figure 1A; page 3, left col. para. 1, 2, and last para. and right col. para. 1; page 5, right col. last para.; page 6; page 7, left col. para. 1). Salvagiotto teaches the culture conditions for producing HPCs also favored the generation of CD31+ endothelial progenitors (page 3, left col. para. 1). Salvagiotto teaches the endothelial cells are positive for CD31 (page 3, left col. para. 1) but is silent on whether the endothelial cells also express CD144 and CD105. However, Salvagiotto teaches the protocol of hematopoietic differentiation from hESC/hiPSCs was established during the development of iCell endothelial cells (page 5, right col. last para.) and iCell endothelial cells are iPSC-derived endothelial cells that are positive for expression of CD31, CD144, and CD105 as evidenced by FujiFilm (page 1 and 6) (“CD31+CD144+CD105+ endothelial cells” of claim 1 and 23). Salvagiotto does not teach the surface is “a positively charged plasma polymerized amine surface comprising amine monomers” or “the absence of extracellular matrix proteins” of claim 1.
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 the endothelial cells are positive for CD31 (page 3, left col. para. 1) but is silent on whether the endothelial cells also express CD144 and CD105. However, Salvagiotto teaches the protocol of hematopoietic differentiation from hESC/hiPSCs was established during the development of iCell endothelial cells (page 5, right col. last para.) and iCell endothelial cells iPSC-derived endothelial cells that are positive for expression of CD31, CD144, and CD105 as evidenced by FujiFilm (page 1 and 6). 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 the surface is “a positively charged plasma polymerized amine surface comprising amine monomers” or “the absence of extracellular matrix proteins” of step (a) of claim 1 or “the surface is a polymeric surface” of claim 10, or “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. However, Salvagiotto teaches culturing the iPSCs on murine collagen IV and human fibronectin used in the method allowed for cell attachment and the method is efficient and results in a pure population of HPCs that avoids the use of feeder cells and serum (Abstract; page 3, right col. last para.). Salvagiotto teaches hESCs and iPSCs can be used in the method to produce HPCs (page 2, left col. para. 2; page 3, left col. para. 1 – 3; page 5, right col. last para.; page 6). 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 media reagents of non-human origin used in the method was bFGF and teaches a humanized version culturing method by substituting BIT (bovine serum albumin, human recombinant insulin, and human transferrin) with HIT (human serum albumin, human recombinant insulin and human transferrin (page 2, left col. last para.; page 3, right col. last para.; page 6, left col.). Salvagiotto teaches HIT sustained hematopoietic and endothelial differentiation from hESCs under hypoxic conditions (page 2, left col. last para.; page 3, left col. para. 1). 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 “a positively charged plasma polymerized amine surface comprising amine monomers” and “in the absence of extracellular matrix proteins” of claim 1 and “polymeric surface” of claim 10 and “polystyrene surface” of claim 11 and “in the absence of” the recited extracellular matrix proteins of claim 15, Irwin teaches a method of culturing hESCs in defined media in the absence of extracellular matrix proteins (“in the absence of extracellular matrix proteins” of claim 1 and claim 15) on a positively charged polymerized amine surface (N-(3-Aminopropyl)methacrylamide hydrochloride, APMAAm) (“a positively charged” and “polymerized amine surface” of claim 1 and “polymeric surface” of claim 10) that is prepared by polymerizing APMAAm on polystyrene tissue culture plates (claim 11) by photoinitiated polymerization using a UV light source(page 6912, right col. last para.; page 6913, left col. para. 1 and 5; page 6914, right col.). Irwin teaches the APMAAm surface was as effective as a Matrigel coated surface in supporting proliferation of hESCs and the hESCs adapted to the APMAAm surface (page 6916, left col. para. 2 and right col. para. 1). Irwin teaches that hESCs cultured on the APMAAm surface retained pluripotency throughout 10 passages and hESCs cultured on the APMAAm surface could be differentiated into all three germ layers (page 6914, right col. last para.; page 6916, left col. para. 1 and right col. para. 2). Irwin teaches a completely defined hESC culture system exploiting a synthetic polymer interface would minimize cost, bring the practice in line with current culture methods, and aid in standardizing the process (page 6912, right col. para. 1). Irwin teaches the primary advantages of the hESC cell culture system that it does not require the prior attachment of peptides or proteins to promote cell attachment, it is scalable, it is low cost, and free of complex, undefined culture conditions (page 6918, right col. para. 4). Irwin teaches Matrigel contains collagen IV (page 6912, left col. para. 2 and right col. para. 1). Irwin teaches they believe the method has the potential to be used for both self-renewal of hiPS, and directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions (page 6918, right col. para. 1). One would have been motivated to combine the teachings of Salvagiotto and Irwin to substitute the extracellular matrix of Salvagiotto (murine collagen IV and human fibronectin) with a polymerized amine surface to lower the cost and time associated with prior attachment of extracellular matrix proteins for the production of HPCs from iPSCs.
Irwin does not teach the polymerized APMAAm amine surface was “plasma polymerized” of claim 1. Irwin teaches bovine serum albumin from the media played a key role in hESC attachment to the APMAAm surface (Abstract; page 6917, right col. para. 1; page 6918, left col. para. 1 and right col. para. 2). Therefore, one would have been motivated to identify a polymerized amine surface to which human serum albumin adsorbs in order to mediate iPSC attachment to produce HPCs under the xenogenic-free conditions for potential clinical applications as taught by Salvagiotto (page 3, right col. last para.; page 6, left col.).
Regarding “plasma polymerized” of claim 1, Burns teaches plasma polymerization of 1,2-diaminocyclohexane (DACH) onto polystyrene results in a positively charged polymerized amine surface that adsorbs human serum albumin (Abstract; page 117, left col. para. 1 and 3; page 120, left col. para. 3; Figure 3). Burns teaches adsorption of human serum albumin is not dependent on the age of the surface most likely due to the ability of the protein to adjust its conformation during adsorption (page 120, left col. para. 5; page 121, left col. para. 3 and 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 an in vitro method for differentiating iPSCs in two-dimensional culture on an extracellular matrix protein coated surface to HPCs and differentiate the HPCs to endothelial cells that are positive for expression of CD31, CD144, and CD105 as evidenced by FujiFilm with the teachings of Irwin regarding a method of culturing stem cells on a positively charged polymerized amine surface that was as effective as an extracellular matrix coated surface regarding cell proliferation where bovine serum albumin played a key role in cell attachment to the amine surface and where the method has the potential to be used for directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions with the teachings of Burns regarding a positively charged plasma polymerized amine surface that adsorbs human serum albumin to arrive at the claimed in vitro method for differentiating induced pluripotent stem cells (iPSCs) comprising: (a) culturing the iPSCs in two-dimensional culture on a positively charged plasma polymerized amine surface comprising amine monomers or negatively charged surface in the absence of extracellular matrix proteins to produce hematopoietic precursor cells
(HPCs), wherein the positively charged amine surface does not comprise polylysine; and (b) differentiating the HPCs to CD31+CD144+CD105+ endothelial cells. One would have been motivated to combine the teachings of Salvagiotto, Irwin, and Burns in a low cost, efficient, and xenogenic-free method of producing HPCs from iPSCs and further differentiating the HPCs to endothelial cells for clinical use in cell therapy 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 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 and Irwin teaches a completely defined hESC culture system exploiting a synthetic polymer interface would minimize cost, bring the practice in line with current culture methods, and aid in standardizing the process. One would have a reasonable expectation of success in combining the teachings and substituting the extracellular matrix surface of Salvagiotto with the positively charged plasma polymerized amine surface of Burns for adhesion of iPSCs and differentiation to HPCs in the humanized method of differentiating hESCs/iPSCs to HPCs under xenogenic-free conditions as taught by Salvagiotto because Salvagiotto teaches HIT sustained hematopoietic and endothelial differentiation from hESCs and Irwin teaches hESCs cultured on the polymerized amine surface retain pluripotency and can be differentiated into the three germ layers and Irwin teaches the method has the potential to be used for directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions and Irwin teaches bovine serum albumin adsorption plays a key role in stem cell attachment to the positively charged polymerized amine surface and Burns teaches human serum albumin adsorbs onto the positively charged plasma polymerized amine surface.
Claim(s) 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Salvagiotto (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829; previously cited), hereinafter Salvagiotto as evidenced by FujiFilm (FujiFilm, iCell Endothelial Cells, 01434 and iCell Endothelial Cells, 11713; https://www.fujifilmcdi.com/icell-endothelial-cells-01434-getc01434 and https://www.fujifilmcdi.com/icell-endothelial-cells-11713-getc11713, Accessed 08/27/2026) in view of Irwin (Irwin, Elizabeth F., et al. Biomaterials 32.29 (2011): 6912-6919.), hereinafter Irwin, in view of Burns (Burns, Norman L. et. al. Journal of Colloid and Interface Science 178.1 (1996): 116-122.), hereinafter Burns as applied to claims 1, 10, 11, 12, 13, 15, 23, 47, 123, and 124 above, and further in view of Burton (WO-2018067826-A1; previously cited), hereinafter Burton which is cited on the IDS filed 10/09/2023.
Salvagiotto in view of Irwin and Burns make obvious the limitations of claim 1 as set forth above. Salvagiotto does not teach “engineering the iPSCs to have disrupted expression of TREM2, SNCA, or 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 63, para. 00244 – 00245; page 65, para. 00247; Figure 1A; page 74, para. 00276; page 75, para. 00277). Burton teaches the iPSCs with MeCP2 knockout were found to more efficiently produce HPCs by about 2 – 7 fold (page 16, para. 0077; page 44 – 45, para. 00182; page 75, para. 00277).
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 an in vitro method for differentiating iPSCs in two-dimensional culture on an extracellular matrix protein coated surface to HPCs and differentiate the HPCs to endothelial cells with the teachings of Irwin regarding a method of culturing stem cells on a positively charged polymerized amine surface that was as effective as an extracellular matrix coated surface regarding cell proliferation with the teachings of Burns regarding a positively charged plasma polymerized amine surface that adsorbs human serum albumin with the teachings of Burton regarding iPSCs with MeCP2 knocked out increasing the efficiency in differentiating iPSCs to HPCs to arrive at the claimed method further comprising engineering the iPSCs to have disrupted expression of MeCP2 prior to step (a). One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, and Burton in an efficient method of producing high levels of HPCs from iPSCs and further differentiating the HPCs to endothelial cells for clinical use in cell therapy and one would have a reasonable expectation of success in combining the teachings as Burton teaches differentiation of MeCP2 knockout iPSCs to HPCs increases efficiency by about 2 – 7 fold.
Claim(s) 24 and 125 is/are rejected under 35 U.S.C. 103 as being unpatentable over Salvagiotto (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829; previously cited), hereinafter Salvagiotto as evidenced by FujiFilm (FujiFilm, iCell Endothelial Cells, 01434 and iCell Endothelial Cells, 11713; https://www.fujifilmcdi.com/icell-endothelial-cells-01434-getc01434 and https://www.fujifilmcdi.com/icell-endothelial-cells-11713-getc11713, Accessed 08/27/2026) in view of Irwin (Irwin, Elizabeth F., et al. Biomaterials 32.29 (2011): 6912-6919.), hereinafter Irwin, in view of Burns (Burns, Norman L. et. al. Journal of Colloid and Interface Science 178.1 (1996): 116-122.), hereinafter Burns as applied to claims 1, 10, 11, 12, 13, 15, 23, 47, 123, and 124 above, and further in view of Chong (Chong, Mark SK, et al. Biomaterials 30.12 (2009): 2241-2251), hereinafter Chong.
Salvagiotto in view of Irwin and Burns make obvious the limitations of claims 1 and 23 as set forth above. Salvagiotto does not teach “culturing the HPCs on a carboxyl surface in the presence of endothelial differentiation media” of claim 24. However, Salvagiotto teaches after 6 days of differentiation of the iPSCs hematopoietic and CD31+ endothelial progenitors were detected (page 3, right col. para. 1; Figure 4A).
Regarding “CD31+CD144+CD105+ endothelial cells” of claim 24 and “at least 90% CD31+CD144+CD105+ endothelial cells” of claim 125, Salvagiotto teaches the endothelial cells are positive for CD31 (page 3, left col. para. 1) but is silent on whether the endothelial cells also express CD144 and CD105. However, Salvagiotto teaches the protocol of hematopoietic differentiation from hESC/hiPSCs was established during the development of iCell endothelial cells (page 5, right col. last para.) and iCell endothelial cells iPSC-derived endothelial cells that are >90% positive for expression of CD31, CD144, and CD105 (claim 125) as evidenced by FujiFilm (page 1 and 6).
Regarding “culturing the HPCs on a carboxyl surface in the presence of endothelial differentiation media” of claim 24, Chong teaches a method of culturing endothelial progenitor cells (EPC) on a carboxyl surface in EPC medium where the EPCs express CD31 and CD144, and von Willebrand Factor and Ac-LDL uptake indicating endothelial phenotype and function (Figure 2 – 3; page 2242, left col. para. 1 and last para. and right col. para. 4; page 2243, right col. para. 2 – 3). Chong teaches to promote adhesion of the EPCs onto the carboxyl surface, the surface was modified with CD34 antibodies and the significant loading of carboxyl groups on the surface allows for biomolecule conjugation (page 2244, right col. para. 1; page 2246, left col. para. 2; page 2249, right col. para. 2); page 2250, left col. para. 2. Chong teaches CD34 antibodies offer the additional advantage of cell selection (page 2250, left col. para. 2). Chong teaches EPCs are highly proliferative but this is lost in terminally differentiated cells and capture of CD34 positive cells may thus improve the healing and tissue integration process (page 2250, left col. para. 2). Chong teaches culturing EPCs on one side of a carboxyl surface with immobilized CD34 antibodies and culturing umbilical cord perivascular cells (UCPVCs) on the opposite side of the surface (without CD34 antibodies) resulted in confluent monolayers for both cell types where the EPCs expressed CD144 (VE-Cadherin) as a model of a blood vessel (page 2242, left col. para. 1; page 2246, left col. last para. and right col. para. 1; Figure 7; page 2248, left col. para. 1). Chong teaches cell layers can synergistically interact across the carboxyl surface in an organized fashion akin to native tissue and hence result in improved function of the engineered tissue (page 2250, left col. para. 3). Chong teaches the possible utility of the carboxyl surface for the generation of cell-sheet constructs, with minimal synthetic material, which would be suitable not only for vascular applications, but for the engineering of layered tissue such as skin, cornea or myocardium (page 2250, left col. last para. and right col. para. 1). Chong teaches EPC which are required for seeding of the intima surface, have poor adhesive qualities on synthetic surfaces and this can be addressed by the placement of CD34 antibodies, as demonstrated in coronary stents (page 2241, right 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 an in vitro method for differentiating iPSCs in two-dimensional culture where after 6 days of differentiation HPCs and CD31+ endothelial progenitors were detected and differentiate the HPCs to endothelial cells that are positive for expression of CD31, CD144, and CD105 as evidenced by FujiFilm with the teachings of Irwin regarding a method of culturing stem cells on a positively charged polymerized amine surface where the method has the potential to be used for directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions with the teachings of Burns regarding a positively charged plasma polymerized amine surface that adsorbs human serum albumin with the teachings of Chong regarding adhering and culturing CD31+CD144+ endothelial progenitors on a carboxyl surface in the presence of EGM-2 media to produce cells with endothelial phenotype and function to arrive at the claimed method wherein step (b) comprises culturing the HPCs on a carboxyl surface in the presence of endothelial differentiation media to produce CD31+CD144+CD105+ endothelial cells. One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, and Chong in a method of producing highly proliferative endothelial cells for vascular engineering as Chong teaches the possible utility of the carboxyl surface for the generation of cell-sheet constructs, with minimal synthetic material, which would be suitable not only for vascular applications, but for the engineering of layered tissue such as skin, cornea or myocardium and Chong teaches EPCs are highly proliferative but this is lost in terminally differentiated cells and capture of CD34 positive cells may thus improve the healing and tissue integration process. One would have a reasonable expectation of success in combining the teachings as Salvagiotto teaches the method produces HPCs and endothelial progenitor cells and Chong teaches endothelial progenitor cells can be cultured on the surface to produce a model of a blood vessel and Chong teaches cell layers can synergistically interact across the carboxyl surface in an organized fashion akin to native tissue and hence result in improved function of the engineered tissue and Chong teaches coronary stents use CD34 antibodies for capture of endothelial progenitor cells.
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; previously cited), hereinafter Salvagiotto as evidenced by FujiFilm (FujiFilm, iCell Endothelial Cells, 01434 and iCell Endothelial Cells, 11713; https://www.fujifilmcdi.com/icell-endothelial-cells-01434-getc01434 and https://www.fujifilmcdi.com/icell-endothelial-cells-11713-getc11713, Accessed 08/27/2026) in view of Irwin (Irwin, Elizabeth F., et al. Biomaterials 32.29 (2011): 6912-6919.), hereinafter Irwin, in view of Burns (Burns, Norman L. et. al. Journal of Colloid and Interface Science 178.1 (1996): 116-122.), hereinafter Burns as applied to claims 1, 10, 11, 12, 13, 15, 23, 47, 123, and 124 above, and further in view of Lippmann (Lippmann ES, et. al. Nat Biotechnol. 2012 Aug;30(8):783-91), hereinafter Lippmann.
Salvagiotto in view of Irwin and Burns make obvious the limitations of claim 1 and 23 as set forth above. Salvagiotto does not teach “further comprising differentiating the endothelial cells to brain microvascular endothelial cells (BMECs) or lymphatic endothelial cells under conditioned-media free conditions” of claim 26. However, Salvagiotto teaches after 6 days of differentiation of the iPSCs hematopoietic and CD31+ endothelial progenitors were detected (page 3, right col. para. 1; Figure 4A).
Lippmann teaches a method of culturing iPSCs with unconditioned medium to produce cells with endothelial cell morphology which are cultured with defined endothelial cell (EC) medium followed by subculture on extracellular matrix for expansion and purification to produce BMECs (page 3, last para.; page 4, para. 1; page 6, para. 1; Figure 2a and 3; page 10 last para.). Lippmann teaches the BMECs possess significant blood brain barrier (BBB) character (page 9, para. 1). Lippmann teaches hPSC-derived BMEC model could be used for studies of brain development, disease mechanisms, and drug delivery (page 10, para. 2). Lippmann teaches the BBB plays an important role in brain health and is often compromised in disease (Abstract). Lippmann teaches a renewable source of human BBB endothelium could prove enabling for brain research and pharmaceutical development (Abstract). Lippmann teaches a robust in vitro BBB model of human origin would be of high utility for conducting high-throughput screening of brain-penetrating molecules or for study of BBB developmental, regulatory and disease pathways in humans (page 2, para. 2). Lippmann teaches human BBB models have been established by culturing primary human BMECs isolated at autopsy or freshly resected brain specimens but issues with BMEC availability limit the universal use of these human BBB models (page 2, 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 an in vitro method for differentiating iPSCs in two-dimensional culture where after 6 days of differentiation HPCs and CD31+ endothelial progenitors were detected and differentiate the HPCs to endothelial cells that are positive for expression of CD31, CD144, and CD105 as evidenced by FujiFilm with the teachings of Irwin regarding a method of culturing stem cells on a positively charged polymerized amine surface where the method has the potential to be used for directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions with the teachings of Burns regarding a positively charged plasma polymerized amine surface that adsorbs human serum albumin with the teachings of Lippmann regarding a method of culturing iPSC-derived cells with endothelial cell morphology with an endothelial cell medium to produce BMECs to arrive at the claimed method further comprising differentiating the endothelial cells to brain microvascular endothelial cells (BMECs) under conditioned-media free conditions. One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, and Lippmann in a method of producing human BMECs as a BBB model as Lippmann teaches a renewable source of human BBB endothelium could prove enabling for brain research and pharmaceutical development and Lippmann teaches a robust in vitro BBB model of human origin would be of high utility for conducting high-throughput screening of brain-penetrating molecules or for study of BBB developmental, regulatory and disease pathways in humans and Lippmann teaches human BBB models have been established by culturing primary human BMECs isolated at autopsy or freshly resected brain specimens but issues with BMEC availability limit the universal use of these human BBB models. One would have a reasonable expectation of success in combining the teachings as Salvagiotto teaches the method produces endothelial cell progenitors and Lippmann teaches BMECs are produced by culturing cells with endothelial cell morphology with EC medium and Lippmann teaches the BMECs possess significant BBB character.
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; previously cited), hereinafter Salvagiotto as evidenced by FujiFilm (FujiFilm, iCell Endothelial Cells, 01434 and iCell Endothelial Cells, 11713; https://www.fujifilmcdi.com/icell-endothelial-cells-01434-getc01434 and https://www.fujifilmcdi.com/icell-endothelial-cells-11713-getc11713, Accessed 08/27/2026) in view of Irwin (Irwin, Elizabeth F., et al. Biomaterials 32.29 (2011): 6912-6919.), hereinafter Irwin, in view of Burns (Burns, Norman L. et. al. Journal of Colloid and Interface Science 178.1 (1996): 116-122.), hereinafter Burns as applied to claims 1, 10, 11, 12, 13, 15, 23, 47, 123, and 124 above, and further in view of Kopher (Kopher, Ross A., et al. Bone 47.4 (2010): 718-728.; previously cited, hereinafter Kopher in view of Chua (Chua KN, et. al. Biomaterials. 2006 Dec;27(36):6043-51; previously cited), hereinafter Chua.
Salvagiotto in view of Irwin and Burns make obvious the limitations of claim 1 as set forth above.
Salvagiotto teaches that after 6 days of differentiation of hESCs the CD34 progenitor marker appeared and early CD34+ cells are a heterogeneous population consisting of hematopoietic, endothelial, and mesenchymal progenitor cells (page 3, left col. para. 2). Salvagiotto teaches at day 6 the culture contains cells that are CD34+ and cells that are CD34- in Figure 2A. Salvagiotto teaches at day 6 of differentiation of hESCs and iPSCs, the culture contains CD34+ HPCs and CD31+ endothelial progenitor cells (page 3, left col. para. 2 and right col. para. 1).
Salvagiotto, Irwin, and Burns do not teach “MSC” 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 MSCs are positive for CD73” of claim 31.
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 function as MSC progenitor cells that can be differentiated into CD34-CD73+ MSCs (Abstract; page 719, left col. para. 3; 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 differentiation of hESCs results in CD34+CD73+ cells and CD34-CD73+ cells, both of which were cultured in media to support MSC proliferation (claim 28) (page 720, right col. last para.; page 719, left col. para. 4). Kopher teaches the resulting cells express CD73, CD44, and CD105 (claim 30) with more than 90% positive for CD73 (claim 31) and function as MSCs (Figure 1A – E; page 721, left col.; page 724, left col. para. 2; Figure 5). 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, right 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 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 two progenitor cell populations expressing CD34 have differentiated into functional MSCs (page 726, right col. last para.). Kopher teaches MSCs are currently under study to aid in several therapies including bone and tissue repair, and hESC-derived MSCs will provide improved opportunities for regenerative medicine (page 726, right col. para. 2; Abstract; page 718, left col.). Kopher teaches that fetal- and adult-derived MSCs appear to have some limitations including donor availability (page 718, right col. para. 1). Kopher teaches the MSCs underwent osteogenic differentiation in vivo (page 719, right col. last para.; page 720, left col. para. 1; page 721, right col.; page 722; page 723, 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. However, both Salvagiotto and Kopher teach that pluripotent stem cells can be differentiated to a population of CD34+ cells that contain hematopoietic, endothelial, and mesenchymal progenitor cells, and Kopher teaches these CD34+ cells can be differentiated to MSCs. One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, and Kopher in a xenogenic-free method to produce MSCs from pluripotent stem cells for use in regenerative medicine such as bone and tissue repair as Kopher teaches there are limitations to obtaining adult-derived MSCs including donor availability and pluripotent stem cell-derived MSCs will provide improved opportunities for regenerative medicine. However, as the method of Kopher includes culturing the CD34+ cells on Matrigel, one would have been motivated to identify a xenogeneic-free cell culture surface for culturing pluripotent stem cell-derived CD34+ cells to obtain MSCs.
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 CD34+ 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 an in vitro method for differentiating iPSCs in two-dimensional culture where after 6 days of differentiation HPCs and CD34+ and CD34- cells were detected and early CD34+ cells are a heterogeneous population consisting of hematopoietic, endothelial, and mesenchymal progenitor cells with the teachings of Irwin regarding a method of culturing stem cells on a positively charged polymerized amine surface where the method has the potential to be used for directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions with the teachings of Burns regarding a positively charged plasma polymerized amine surface that adsorbs human serum albumin with the teachings of with the teachings of Kopher regarding a method of differentiating CD34+ MSC progenitors to MSCs with the teachings of Chua regarding expanding CD34+ HPCs on an amine surface to arrive at the claimed method where the method comprises differentiating the HPCs to MSCs. One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, Kopher, and Chua in a xenogenic-free, 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 CD34+ mesenchymal progenitors could be cultured to obtain MSCs and Kopher teaches the resulting MSCs demonstrated osteogenic differentiation in vivo and Chua teaches an amine surface showed the best performance for expansion of HPCs.
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 (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829; previously cited), hereinafter Salvagiotto as evidenced by FujiFilm (FujiFilm, iCell Endothelial Cells, 01434 and iCell Endothelial Cells, 11713; https://www.fujifilmcdi.com/icell-endothelial-cells-01434-getc01434 and https://www.fujifilmcdi.com/icell-endothelial-cells-11713-getc11713, Accessed 08/27/2026) in view of Irwin (Irwin, Elizabeth F., et al. Biomaterials 32.29 (2011): 6912-6919.), hereinafter Irwin, in view of Burns (Burns, Norman L. et. al. Journal of Colloid and Interface Science 178.1 (1996): 116-122.), hereinafter Burns as applied to claims 1, 10, 11, 12, 13, 15, 23, 47, 123, and 124 above, and further in view of Kopher (Kopher, Ross A., et al. Bone 47.4 (2010): 718-728.; previously cited, hereinafter Kopher in view of Loibl (Loibl, Markus, et al. BioMed research international 2014.1 (2014): 395781.), hereinafter Loibl as evidenced by Rojas (Duarte Rojas, Juan Manuel, et. al. Biomedicines 12.1 (2024): 140.), hereinafter Rojaas.
Salvagiotto in view of Irwin and Burns make obvious the limitations of claim 1 as set forth above.
Salvagiotto, Irwin, and Burns do not teach “MSC” of claim 28. However, Salvagiotto teaches that after 6 days of differentiation of hESCs the CD34+ progenitor marker appeared and early CD34+ cells are a heterogeneous population consisting of hematopoietic, endothelial, and mesenchymal progenitor cells and the HPCs can be further differentiated to endothelial cells (page 3, left col. para. 2). Salvagiotto teaches at day 6 the culture contains cells that are CD34+ and cells that are CD34- in Figure 2A. Salvagiotto teaches at day 6 of differentiation of hESCs and iPSCs, the culture contains CD34+ HPCs and CD31+ endothelial progenitor cells (page 3, left col. para. 2 and right col. para. 1).
Regarding “differentiating the HPCs to MSCs” of claim 28, Kopher teaches hESC-derived CD34+CD73- cells function as MSC progenitor cells that can be differentiated into CD34-CD73+ MSCs (Abstract; page 719, left col. para. 3; 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 differentiation of hESCs results in CD34+CD73+ cells and CD34-CD73+ cells, and the CD73+ cells were sorted and differentiated into CD73+CD44+CD105+ MSCs by culturing in media to support MSC proliferation (page 719, left col. para. 4; page 720, right col. last para; page 721, left col. para. 1; Figure 1A – E). 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 fetal- and adult-derived MSCs appear to have some limitations including donor availability (page 718, right col. para. 1). Kopher teaches MSCs are currently under study to aid in several therapies including bone and tissue repair, and hESC-derived MSCs will provide improved opportunities for regenerative medicine (page 726, right col. para. 2; Abstract; page 718, left col.). Kopher teaches the MSCs underwent osteogenic differentiation in vivo (page 719, right col. last para.; page 720, left col. para. 1; page 721, right col.; page 722; page 723, left col.). Kopher does not teach “differentiating the MSCs to pericytes” of claim 32 or “seeding cells consisting of MSCs in the presence of pericyte medium comprising FGFβ, EGF, and IGF-1, in the absence of extracellular proteins” of claim 33 or “the pericytes are positive for NG2, PDGFRβ, and/or CD146” of claim 34. However, both Salvagiotto and Kopher teach that pluripotent stem cells can be differentiated to a population of CD34+ cells that contain hematopoietic, endothelial, and mesenchymal progenitor cells, and Kopher teaches the CD34+ cells can be differentiated to MSCs and CD34-CD73+ cells can be isolated and differentiated to MSCs. One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, and Kopher in a xenogenic-free method to produce MSCs from pluripotent stem cells for use in regenerative medicine such as bone and tissue repair as Kopher teaches there are limitations to obtaining adult-derived MSCs including donor availability and pluripotent stem cell-derived MSCs will provide improved opportunities for regenerative medicine. However, as the method of Kopher includes culturing the isolated CD34+CD73+ and CD34-CD73+ cells on Matrigel, one would have been motivated to identify a xenogeneic-free cell culture surface for culturing pluripotent stem cell-derived CD34+CD73+ and CD34-CD73+ cells to obtain MSCs.
Regarding “differentiating the MSCs to pericytes” of claim 32 or “seeding cells consisting of MSCs in the presence of pericyte medium comprising FGFβ, EGF, and IGF-1, in the absence of extracellular proteins” of claim 33 or “the pericytes are positive for NG2, PDGFRβ, and/or CD146” of claim 34, Loibl teaches a method of single culture of depleted-MSCs (“consisting of MSCs” of claim 33) in IMDM-PL (Platelet Lysate growth factors) (“pericyte medium” of claim 33) in the absence of extracellular matrix proteins (“absence of extracellular proteins” of claim 33) results in cells that express CD146, NG2, and PDGFR-β (claim 32 and 34) and Loibl teaches pericytes express CD146 (page 2, left col. last para. and right col. para. 4 – 5; Figure 1c; Supplementary Table 1; Supplementary Figure 1; page 4, right col. last para.; page 6, left col. para. 1; page 6, right col. para. 8). Loibl teaches depleted-MSCs cells were depleted from CD133+, CD34+, and CD146+ cells and are free of pericytes and endothelial progenitor cells (page 2, right col. para. 4; Figure 2b; page 6, right col. para. 8). Therefore, Loibl’s method of culturing cells consisting of depleted-MSCs in IMDM-PL media produces pericytes (page 8, left col. para. 1 and right col. para. 1). The platelet lysate of Loibl contains bFGF, EGF, and IGF-1 as evidenced by Rojas (“FGFβ, EGF, and IGF-1” of claim 33) (page 3, para. 5; page 5, para. 4; page 7, para. 3 – 4; Figure 2).
Loibl teaches tissue engineering techniques for the regeneration of large bone defects require sufficient vascularization of the applied constructs to ensure a sufficient supply of oxygen and nutrients (Abstract; page 1, left col. para. 1). Loibl teaches the capacity for bone tissue regeneration is limited and fails in large bone defects (page 1, left col. para. 1). Loibl teaches the gold-standard strategy remains autologous bone implants, associated with an additional surgical procedure at the harvesting site causing an increase of operation time, pain, and risk for infection at the donor site (page 1, left col. para. 1). Loibl teaches the approach of engineering bone tissue not only depends on the presence of osteogenic cells at the healing site but also requires an adequate vascularization of the applied biomaterial and therefore osteoblasts and endothelial cells or their progenitors play a crucial role for successful engraftment of cell seeded biomaterials (page 1, left col. para. 1 and right col. para. 1). Loibl teaches pericytes represent a subpopulation of MSCs in bone marrow, contributing to microvessel maturation, stability, structure, and function and establish important direct cell-cell contact with endothelial cells of immature blood vessels (page 2, left col. para. 2). Loibl teaches some studies suggest that pericytes may serve as guiding structures aiding outgrowth of endothelial cells to form early capillary sprouts (page 2, left col. para. 2). Loibl teaches coculture strategies may promote angiogenesis for cell-based tissue engineered bone grafts (page 9, left col. para. 4). Loibl teaches direct coculture of endothelial progenitors and MSCs produce pericytes that express CD146 and NG2 (Figure 1a – b; page 6, right col. para. 4).
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 an in vitro method for differentiating iPSCs in two-dimensional culture where after 6 days of differentiation HPCs and CD34+ and CD34- cells were detected and early CD34+ cells are a heterogeneous population consisting of hematopoietic, endothelial, and mesenchymal progenitor cells where the HPCs were further differentiated to endothelial cells, with the teachings of Irwin regarding a method of culturing stem cells on a positively charged polymerized amine surface where the method has the potential to be used for directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions with the teachings of Burns regarding a positively charged plasma polymerized amine surface that adsorbs human serum albumin with the teachings of Kopher regarding a method of differentiating CD34+ MSC progenitors to MSCs with the teachings of Loibl regarding a method of differentiating MSCs to pericytes to arrive at the claimed method wherein the method comprises differentiating the HPCs to MSCs and further comprises differentiating the MSCs to pericytes. One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, Kopher, and Loibl in a xenogenic-free, defined method of producing endothelial cells, MSCs, and pericytes from pluripotent stem cells for use in bone tissue repair 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 such as bone repair and Loibl teaches the capacity for bone tissue regeneration is limited and fails in large bone defects and Loibl teaches the gold-standard strategy remains autologous bone implants, associated with an additional surgical procedure at the harvesting site causing an increase of operation time, pain, and risk for infection at the donor site and Loibl teaches the approach of engineering bone tissue not only depends on the presence of osteogenic cells at the healing site but also requires an adequate vascularization of the applied biomaterial and therefore osteoblasts and endothelial cells or their progenitors play a crucial role for successful engraftment of cell seeded biomaterials. One would have a reasonable expectation of success in combining the teachings as Kopher teaches the MSCs underwent osteogenic differentiation in vivo and Loibl teaches pericytes represent a subpopulation of MSCs in bone marrow, contributing to microvessel maturation, stability, structure, and function and establish important direct cell-cell contact with endothelial cells of immature blood vessels and Loibl teaches direct coculture of endothelial progenitors and MSCs produce pericytes that express CD146 and NG2.
Claim(s) 36, 37, 38, 41, 44, and 126 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 (Salvagiotto, Giorgia, et al. PloS one 6.3 (2011): e17829; previously cited), hereinafter Salvagiotto as evidenced by FujiFilm (FujiFilm, iCell Endothelial Cells, 01434 and iCell Endothelial Cells, 11713; https://www.fujifilmcdi.com/icell-endothelial-cells-01434-getc01434 and https://www.fujifilmcdi.com/icell-endothelial-cells-11713-getc11713, Accessed 08/27/2026) in view of Irwin (Irwin, Elizabeth F., et al. Biomaterials 32.29 (2011): 6912-6919.), hereinafter Irwin, in view of Burns (Burns, Norman L. et. al. Journal of Colloid and Interface Science 178.1 (1996): 116-122.), hereinafter Burns as applied to claims 1, 10, 11, 12, 13, 15, 23, 47, 123, and 124 above, and further in view of Blurton-Jones (WO2018160496-A1; Filed 02/26/2018; Published 09/07/2018), hereinafter Blurton-Jones which is cited on the IDS filed 10/09/2023 as evidenced by FujiFilm2 (FujiFilm2, iCell Endothelial Cells, 01434 and iCell Microglia, 01279; https://www.fujifilmcdi.com/icell-microglia-01279-gmgl01279, Accessed 08/28/2026), hereinafter FujiFilm2 in view of Muffat (Muffat J, et. al. Nat Med. 2016 Nov;22(11):1358-1367; previously cited), hereinafter Muffat.
Salvagiotto in view of Irwin and Burns make obvious the limitations of claim 1 as set forth above. Salvagiotto, Irwin, and Burns do not teach “microglia” of claim 36 or “neutrally charged surface or ultralow attachment surface” of claim 37 or “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 or “at least 90% for CD33 and at least 50% positive for CD11b” of claim 126.
Regarding claim 36, Blurton-Jones teaches a method of culturing iPSCs to produce hematopoietic progenitors (iHPCs) and differentiating the iHPCs to microglia (iMGLs) (page 46 – 47, para. 0162; page 29, para. 0141; Figure 1A, 1Bi, 1Bii; page 29 – 30, para. 0142 – 0143; Figure 1A; page 47, para. 0163).
Regarding claim 37, Blurton-Jones teaches the iHPCs were plated in Matrigel-coated 6-well plates and cultured in the presence of microglia differentiation media (page 29, para. 0142; page 47, para. 0163) but does not teach the surface is “neutrally charged” or “ultralow attachment”.
Regarding claim 38, Blurton-Jones teaches that the microglia differentiation media comprises MCSF, IL34, and TGFβ1 (Figure 1A; page 47, para. 0163).
Regarding claim 41, Blurton-Jones teaches in that iMGLs are positive for CD45, CD11b, and CD33 (page 6, para. 0031 – 0033; page 9, para. 0051; page 42, para. 0154; page 72, claim 47; Figure 1D – 1F, 4C, 17A).
Regarding claim 44, Blurton-Jones teaches day 38 iMGLs exhibited high purity and day 38 iMGLs were transplanted into the cortex of MITRG mice and does not teach the iMGLs were purified prior to transplantation (page 30, para. 0143; page 44 – 45, para. 0158; page 47, para. 0163; page 58, para. 0193).
Regarding claim 126, Blurton-Jones teaches in Figure 3A middle panel that the iMGL are at least 50% positive for CD11b. Blurton-Jones does not teach the cells are at least 90% positive for CD33. However, iMGL generated from iHPCs by the method of Blurton-Jones are 92.7% positive for CD33 and CD11b as evidenced by FujiFilm2 (page 1, 3, and 6).
Blurton-Jones does not teach the surface is “neutrally charged” or “ultralow attachment” of claim 37. However, Blurton-Jones teaches there is a deficiency in the art of acquiring microglia cells to further investigate the roles microglia cells play in CNS development and neurological disorders (page 1, para. 0004). Blurton-Jones teaches microglia play a critical role in neurological disorders, including Alzheimer’s Disease, highlighting the need to improve our understanding of their function in both health and disease (page 17, para. 0090). Blurton-Jones teaches studying human microglia is challenging because of the rarity and difficulty in acquiring primary cells from human fetal or adult CNS tissue (page 18, para. 0090). Blurton-Jones teaches there is a pressing need to develop a renewable source of human microglia, such as from PSCs including iPSCs and ESCs (page 18, para. 0090). Blurton-Jones teaches in Example 3 that iMGLs act as surrogates of microglia in functional and physiological assays (page 36 – 37). Blurton-Jones teaches in Example 4 that iMGLs can be used to study Alzheimer’s disease as they phagocytose Aβ (page 38 – 39). Blurton-Jones teaches in Example 10 that iMGLs could potentially be used in studying human microglia in mouse CNS-disease models (page 44 – 45). One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, and Blurton-Jones in a xenogenic-free method to produce microglia from pluripotent stem cells for use in studying CNS development and neurological disorders as Blurton-Jones teaches there is a deficiency in the art of acquiring microglia cells to further investigate the roles microglia cells play in CNS development and neurological disorders and Blurton-Jones teaches studying human microglia is challenging because of the rarity and difficulty in acquiring primary cells from human fetal or adult CNS tissue and Blurton-Jones teaches there is a pressing need to develop a renewable source of human microglia, such as from PSCs including iPSCs and ESCs. However, as the method of Blurton-Jones includes culturing the iHPCs on Matrigel (page 47, para. 0163), one would have been motivated to identify a xenogeneic-free cell culture surface for culturing iHPCs to obtain microglia.
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 an in vitro method for differentiating iPSCs in two-dimensional culture to HPCs, with the teachings of Irwin regarding a method of culturing stem cells on a positively charged polymerized amine surface where the method has the potential to be used for directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions with the teachings of Burns regarding a positively charged plasma polymerized amine surface that adsorbs human serum albumin with the teachings of Blurton-Jones regarding a method of differentiating HPCs to microglia with the teachings of Muffat regarding differentiation of microglia on an ultra-low attachment surface and selective culturing of microglia on Primaria plates to arrive at the claimed method wherein the method comprises differentiating the HPCs to microglia. One would have been motivated to combine the teachings of Salvagiotto, Irwin, Burns, Blurton-Jones and Muffat in a xenogenic-free, defined method of producing microglia from pluripotent stem cells to CNS development and neurodegenerative diseases as Blurton-Jones teaches there is a deficiency in the art of acquiring microglia cells to further investigate the roles microglia cells play in CNS development and neurological disorders and Blurton-Jones teaches studying human microglia is challenging because of the rarity and difficulty in acquiring primary cells from human fetal or adult CNS tissue and Blurton-Jones teaches there is a pressing need to develop a renewable source of human microglia, such as from PSCs including iPSCs and ESCs. One would have a reasonable expectation of success in combining the teachings as Blurton-Jones teaches the iMGLs act as surrogates of microglia in functional and physiological assays and can be used to study Alzheimer’s disease as they phagocytose Aβ and could potentially be used in studying human microglia in mouse CNS-disease models 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.
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; previously cited), hereinafter Salvagiotto as evidenced by FujiFilm (FujiFilm, iCell Endothelial Cells, 01434 and iCell Endothelial Cells, 11713; https://www.fujifilmcdi.com/icell-endothelial-cells-01434-getc01434 and https://www.fujifilmcdi.com/icell-endothelial-cells-11713-getc11713, Accessed 08/27/2026) in view of Irwin (Irwin, Elizabeth F., et al. Biomaterials 32.29 (2011): 6912-6919.), hereinafter Irwin, in view of Burns (Burns, Norman L. et. al. Journal of Colloid and Interface Science 178.1 (1996): 116-122.), hereinafter Burns as applied to claims 1, 10, 11, 12, 13, 15, 23, 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; previously cited), hereinafter Kaupisch.
Salvagiotto in view of Irwin and Burns make obvious the limitations of claim 1 as set forth above. Salvagiotto, Irwin, and Burns do not teach the method is GMP compliant of claim 46. 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.).
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 an in vitro method for differentiating iPSCs in two-dimensional culture to HPCs and further differentiating the HPCs to endothelial cells and the method can be easily converted to xenogeneic-free conditions for potential clinical applications, with the teachings of Irwin regarding a method of culturing stem cells on a positively charged polymerized amine surface where the method has the potential to be used for directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions with the teachings of Burns regarding a positively charged plasma polymerized amine surface that adsorbs human serum albumin with the teachings of 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, Irwin, Burns, and Kaupisch in a xenogeneic-free, defined method for producing large quantities of highly pure endothelial cells for clinical therapies as 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 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
Applicant Argues: Applicant argues that there is no disclosure for producing CD31+CD144+CD105+ endothelial cells, microglia, MSCs or pericytes in Salvagiotto as in the instant claims.
Response to Arguments: This argument has been fully considered but is not persuasive because Salvagiotto teaches the hESC/hiPSCs produce HPCs that include endothelial progenitors and endothelial cells and the method for producing HPCs was established during the development of iCell endothelial cells (page 5, right col. last para.) which express CD31, CD144, and CD105 as evidenced by FujiFilm. Regarding microglia, Blurton-Jones teaches producing microglia from HPCs. Regarding MSCs, both Salvagiotto and Kopher teach pluripotent stem cell differentiation methods that produce a population of CD34+ cells that contain HPCs, EPCs, and MSC progenitors, and Kopher teaches the MSC progenitors could be isolate cultured to produce MSCs. Regarding pericytes, Loibl teaches MSCs can be cultured to produce pericytes. Therefore, Salvagiotto discloses producing endothelial cells and the combinations of teachings make obvious the production of microglia, MSCs and pericytes.
Applicant Argues: Applicant argues that a person of ordinary skill in the art could not have predicted that the matrix proteins disclosed in Salvagiotto as necessary for cell attachment could have been removed from the method and that HPCs produced by culturing on a positively charged amine surface in the absence of ECM proteins could result in HPCs that can be differentiated to the claimed cells of claim 1. Applicant argues Muffat does not teach or suggest producing HPCs from iPSCs on positively charged amine plates in 2D culture without ECM proteins. Applicant argues Kaupisch does not teach or suggest producing HPCs from iPSCs on positively charged amine plates in 2D culture without ECM proteins. Applicant argues Burton does not teach or suggest the presently claimed methods of culture on a positively charged plasma polymerized amine surface to differentiation iPSCs to HPCs.
Response to Arguments: These arguments have been fully considered but are not persuasive because a person of ordinary skill in the art reading Irwin and Burns would predict that the ECM proteins could be removed and replaced with an amine surface and bovine (as taught by Irwin) or human serum albumin (as taught by Burns) for cell attachment of iPSCs as Irwin teaches they believe the method has the potential to be used for both self-renewal of hiPS, and directed differentiation of hESCs and hiPSCs into specific lineages under the appropriate, defined media conditions and both Irwin and Burns teach serum albumin adsorbs to the amine surface and Irwin teaches bovine serum albumin mediates attachment of PSCs to the amine surface.
Applicant Argues: Applicant argues Abud, Ying, Boespflug, and Peters do not teach the limitations of the amine surface of amended claim 1.
Response to Arguments: These arguments are moot as the previous claim rejections citing the teachings of Abud, Ying, Boespflug, and Peters have been withdrawn.
Applicant Argues: Applicant argues Minami does not teach the limitation of amended claim 26.
Response to Arguments: This argument is moot as the previous claim rejection citing the teachings of Minami have been withdrawn.
Applicant Argues: Applicant argues that Kopher teaches ESCs were cultured on MEFs and Kopher relies on MEGs and Matrigel for differentiation of ESCs to MSC progenitor cells.
Response to Arguments: This argument is not considered persuasive because Kopher teaches maintaining the ESCs on MEFs and the claims do not recite any limitations regarding the maintenance of pluripotent stem cells and because claim 37 that requires the absence of extracellular matrix proteins is obvious as set forth above.
Applicant Argues: Applicant asserts that Chua does not teach or suggest the differentiation of iPSCs to HPCs using the aminated nanofiber mesh, merely the expansion of the primary human umbilical cord blood cells.
Response to Arguments: This argument is not considered persuasive because Salvagiotto in view of Irwin and Burns make obvious the differentiation of iPSCs to HPCs on a positively charged plasma polymerized amine surface as set forth above. Chua teaches expansion of CD34+ HPCs which are one of the CD34+ progenitor cells types produced by the method of Salvagiotto and Kopher. Chua provides motivation and a reasonable expectation of success for differentiating the CD34+ cells of Salvagiotto and Kopher on a defined, xenogeneic-free surface for obtaining MSCs.
Applicant Argues: Applicant argues that the method of Kumar is distinct from the claimed method.
Response to Arguments: This argument is moot as the previous claim rejections citing the teachings of Kumar have been withdrawn.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Z.M.B./Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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