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 .
DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 July 31, 2025 has been entered.
RESPONSE TO AMENDMENT
Status of Application/Amendments/claims
3. Applicant’s amendment filed July 31, 2025 is acknowledged. Claims 5-6, 12 and 15 are cancelled. Claims 1, 8 and 18-19 are amended. Claims 1-4, 7-11, 13-14 and 16-27 are pending in this application. Claims 23-27 are withdrawn without traverse (filed 04/26/2024) from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. Election was made as without traverse in the reply filed on April 26, 2024.
4. Claims 1-4, 7-11, 13-14 and 16-22 are under examination with respect to Kit for an EMP marker, PU.1 for a microglial marker, CD11B for a macrophage marker, CHIR99021 for a Wnt activator, IWP2 for a Wnt inhibitor, VEGF for a hematopoiesis-promoting cytokine, IL-6 for an interleukin, FGF2 for a FGF and M-CSF for a macrophage-promoting cytokine in this office action.
5. Applicant’s arguments filed on July 31, 2025 have been fully considered but they are not deemed to be persuasive for the reasons set forth below.
Claim Rejections/Objections Withdrawn
6. The objection to claims 1, 8, 10, 13 and 17-20 is withdrawn in response to Applicant’s amendment to the claims.
The rejection of claims 1-14 and 16-22 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112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement is withdrawn in response to Applicant’s amendment to the claims.
The rejection of claims 1-14 and 16-22 under 35 U.S.C. 103 as obvious over Takata et al. (Immunity, 2017; 47:183-198, as in IDS) in view of Douvaras et al. (US11149250) and Thomson et al. (US2016/0186137) is withdrawn in response to Applicant’s amendment to the claims and cancelation of claims 5-6 and 12.
New Grounds of Rejection Necessitated by the Amendment
The following rejections are new grounds of rejections necessitated by the amendment filed on July 31, 2025.
Claim Rejections - 35 USC § 103
7. 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 of this title, 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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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.
Claims 1-4, 7-11, 13-14 and 16-22 are rejected under 35 U.S.C. 103 as obvious over Takata et al. (Immunity, 2017; 47:183-198, as in IDS) in view of Sturgeon et al. (Nat. Biotechnol.2014; 32:554-561) Thomson et al. (US2016/0186137; was also issued as US10081792, cited previously) and Douvaras et al. (US11149250, cited previously).
Claims 1-4, 7-11, 13-14 and 16-22 as amended are drawn to an in vitro method for inducing differentiation of stem cells, comprising:
a) contacting stem cells with at least one activator of Nodal signaling (Nodal activator), at least one BMP and at least one activator of Wnt signaling that lowers GSK3b for activation of Wnt signaling (Wnt activator/GSK3b inhibitor) for 15-24 hours;
b) contacting the cells from (a) with at least one Nodal activator, at least one BMP and at least one Wnt inhibitor comprising a porcupine inhibitor for 2 days;
c) contacting the cells with at least one VEGF and at least one FGF for at least 1 day to obtain a population of cells expressing VE-cadherin;
d) contacting the cells with at least one hematopoiesis-promoting cytokine selected from the group consisting of VEGF, FGF, SCF, interleukins and combinations thereof, for at least 1 day to obtain a population of cells expressing at least one erythromyeloid progenitor (EMP) marker selected from the group consisting of Kit, CD41, CD235A, CD43 and combinations thereof;
e) contacting the cells with at least one hematopoiesis-promoting cytokine selected from the group consisting of SCF, interleukins, thrombopoietin (TPO) and combinations for at least 3 days to obtain a population of differentiated cells expressing at least one pre-macrophage marker selected from the group consisting of kit, CD45, CSF1R and combination thereof; and
f) inducing differentiation of the differentiated cells to cells expressing at least one microglial marker selected from the group consisting of CX3CR1, PU1, CD45, IBA1, P2RY12, TMEM119, SALL1, GPR34, C1QA, CD68 and combinations thereof.
Takata et al. teaches a method of generating microglia from mouse induced pluripotent stem cells (miPSC), comprising
A) generation miPSC-derived macrophage (miMac) from iPSCs by four phases:
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1) 1st phase (day 0-2), culturing miPSCs with BMP-4 (i.e. a BMP) and FGF-2 (i.e. a nodal activator) for between 0-2 day (i.e. a BMP and a nodal activator in step a) of instant claim 1);
2) 2nd phase (days 2-4), culturing the cells from step 1) above with BMP-4, FGF-2, Activin A (i.e. a Wnt inhibitor) (i.e. a BMP, a nodal activator and a Wnt inhibitor in step b) of instant claim 1), and VEGF (i.e. in step c) of instant claim 1);
3) 3rd phase (days 4-6), culturing the cells from step 2) above cells with VEGF to drive hematopoietic commitment, and with CSF-1 to support the emergence of primitive macrophages (i.e. hematopoiesis-promoting cytokines: VEGF and interleukins in step d) of instant claim 1);
4) 4th phase (day 6 onward), culturing the cells from step 3) IL-3 (i.e. interleukin/macrophage-promoting cytokine), SCF and CSF-1 (i.e. hematopoiesis-promoting cytokines in step e) of instant claim 1 and at least one macrophage-promoting cytokine in claim 20) to obtain the macrophage lineage. The EBs from the 3rd phase were cultured in Stempro Medium, supplemented with CSF-1 (50 ng/mL), IL-3 (10 ng/mL) and SCF (100 ng/mL) (i.e. at least one macrophage-promoting cytokine in claims 1 and 20) for another 5 to 7 days before use (Differentiation Day 6/Terminal Differentiation); and wherein CD41low-intc-kit+CD45lowF4/80–EMPs emerged (i.e. at least one EMP marker in step d) of instant claim 1) and by day 8 gave rise to CD45+CD11b+F4/80+CX3CR1+CD41- macrophage-like cells (i.e. at least one microglial marker in step f) of instant claim 1 and at least macrophage marker in claim 17); and
B) Isolating CD45+CD11b+F4/80+ miMACs (see p. 184-189).
Takata also teaches a method of generating microglia from human induced pluripotent stem cells (hiPSCs), comprising:
A) generation hiPSC-derived macrophage (hiMac) by the following steps:
1) 1st phase (days 0-6), incubating iPSC colonies with BMP-4 and VEGF, and with CHIR99021 (i.e. a Wnt activator that lowers GSK3b) during the first 2 days of differentiation (which relates to a BMP, a Wnt activator lowering GSK3b in step (a) of claims 1 and 18); adding FGF-2 (days 2-4) for hemangioblast formation was induced by (i.e. which relates to a BMP, a nodal activator (FGF2) and a Wnt activator that lowers GSK3b in steps a)-b) of instant claim 1) and then maintaining the cells with VEGF and FGF-2 (days 4-6) to generate CD (i.e. which relates to VEGF and FGF2 in step c)-d) of instant claim 1).
2) the next phase (days 6-10), adding DKK1, a Wnt antagonist to inhibit Wnt signaling for the promotion of primitive hematopoiesis (i.e. a Wnt inhibitor in step a) of claim 1). Incubating the cells with SCF,FGF-2, IL-3 and IL-6 to obtain hematopoietic cells expressing CSF-1R (i.e. a pre-macrophage marker in step e of instant claim 1, which relates to at least one hematopoiesis-promoting cytokine in steps d)-e) of instant claims 1, 19-20).
3) From day 16, incubating the cells from step 2) with CSF-1 to obtain hiMac (i.e. which relates to hematopoiesis-promoting cytokines, interleukins in step e) of instant claim 1, at least macrophage marker by a macrophage promoting cytokine in instant claims 2-3 and 20).
B) Isolating CD45+CD11b+F4/80+ hiMACs (i.e. at least one macrophage marker in instant claims 2-3 and 17 (see p. 194; p. 193, figure 6; p.e4).
Takata teaches that culturing and differentiating human iPSCs (HD33i) comprising: culturing the cells for 16 days in Stempro Medium, supplemented with the following cytokines during the differentiation process:
1) Differentiation Day 0: 5 ng/mL BMP4 (i.e. a BMP), 50 ng/mL VEGF, and 2 uM CHIR99021 (i.e. a BMP and a Wnt activator lowering GSKb in step a) of instant claim 1);
2) Differentiation Day 2: 5 ng/mL BMP4 (i.e. a BMP), 50 ng/mL VEGF, and 20 ng/mL FGF2 (i.e. a Nodal activator) (i.e. a BMP, a Nodal activator in step a) of instant claim 1);
3) Differentiation Day 4: 15 ng/mL VEGF and 5 ng/mL FGF2 (i.e. i.e. step c) of instant claim 1);
4) Differentiation Day 6 to 10: 10 ng/mL VEGF, 10 ng/mL FGF2, 50 ng/mL SCF(i.e. a hematopoiesis-promoting cytokines in step d) of instant claim 1), 30 ng/mL DKK-1 (i.e. a Wnt inhibitor in step b) of instant claim 1), 10 ng/mL IL-6, and 20 ng/mL IL-3; (i.e. at least one hematopoiesis-promoting cytokine for at least 1 day in step d) of instant claim 1);
5) Differentiation Day 12 and 14: 10 ng/mL FGF2, 50 ng/mL SCF, 10 ng/mL
IL-6, and 20 ng/mL IL-3 (i.e. at least one hematopoiesis-promoting cytokine for at least 1 day in step d)-e) of instant claim 1)
6) From Differentiation Day 16, the cells were switched to SF-Diff supplemented with 50ng/mL CSF-1 (i.e. at least one macrophage-promoting cytokine), and full medium change was done every 3 days up to Differentiation Day 25; and
Isolating CD45+CD11b+CD163+ CD14+CX3CR1+ hiMAC (i.e. at least one pre-macrophage marker, at least one microglial marker in step e)-f) of instant claim 1) (see p. 194; p. 193, figure 6; p.e4).
But Takata does not explicitly teach a combination of a Nodal activator, a BMP and a Wnt activator lowering GSK3b in step a), the Wnt inhibitor is a porcupine inhibitor including IWP2 and is added after 15-24 hours after step a) of claim 1 or CD235A marker or other microglial markers recited in claim 1 or KDR in claims 8-10 or the concentration ranges or the culturing duration that are exactly identical to the claimed ranges or combinations recited in claims 21-22.
Sturgeon et al. teach that generation of hematopoietic primitive progenitors (KDR+CD235a+) depends on stage-specific activin-nodal signaling and inhibition of the Wnt–b-catenin pathway (see abstract). Sturgeon teaches that treatment of PSC with CHIR99021 (a Wnt activator/GSKb inhibitor) prevents the development of the primitive hematopoietic program while specifying the definitive program, whereas treatment of PSC with IWP2 prevents the specification of the definitive hematopoietic program while promoting the primitive hematopoietic program. Sturgenon teach that inhibiting the Wnt–β-catenin pathway by a Wnt-b inhibitor, IWP2, at day 2-3 of differentiation cultures of hiPSC or MSC-iPS1 led to a 2-fold increase in the size of the CD235a+ population and in the size of the CD43+ population compared to the DMSO-treated control; whereas addition of the GSK-3 inhibitor CHIR99021 (CHIR), a Wnt agonist (Wnt activator), during the same time frame inhibited development of the CD235a+ population (see p. 258, 2st col., 1st paragraph to p. 259, 1st col.). Sturgeon teaches that primitive hematopoiesis from a KDR+CD235a+ hematopoietic primitive progenitors can be obtained by differentiation embryoid bodies in the presence of BMP4, followed by stage-specific addition of bFGF, VEGF and hematopoietic cytokines and activin-nodal signaling was manipulated between days 2 and 3 (p. 555, figure 1; p. 558-559, figures 4-5). Sturgeon teaches that CD235a, a hematopoietic potential marker, was first expressed at day 3 of differentiation on a subset of KDR+ mesoderm, 24 h after the emergence of the T+ (also known as T brachyury) and CD235a is only expressed on hPSC-derived CD34+ cells, and the CD34+ cells emerges within 24hrs (p. 554, 2nd col., 2nd paragraph; p. 259, 2nd col., 2nd paragraph).
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Thomson et al. (US2016/0186137) teaches a method of making a composition comprising human microglial cells, wherein the method comprises:
i) obtaining human hematopoietic precursor cells (HPCs) by culturing human pluripotent stem cells (hPSCs) in a culture medium comprising FGF2, TGFb1, and a ROCK inhibitor under normoxic conditions for about 24 hours to obtain HPCs (paragraphs [0029]-[0030]);
ii) obtaining human myeloid progenitors by culturing hPSC-derived HPCs obtained from step i) in a culture medium comprising FGF2, a VEGF, TPO, SCF, IL-6, and IL-3 for about 3-5 days to obtain human myeloid progenitors (paragraphs [0032]);
iii) obtaining human primitive macrophages by culturing human myeloid progenitors from step ii) in a culture medium comprising insulin and GM-CSF, a hematopoietic cytokine to obtain human microglial cells comprising at least 80% CD45+/CD11b+/CD14+ primitive macrophages; or
i) obtaining human hematopoietic precursor cells (HPCs) by a method of directing differentiation of human pluripotent stem cells (hPSCs) into HPCs comprises:
i. culturing the hPSCs under hypoxic conditions for about 40 hours to about 48 hours in a chemically defined culture medium (DM1) comprising one or more of the following factors: a ROCK inhibitor (e.g. Y-27632), BMP4 (50ng/ml) (i.e. a BMP), Activin A (12.5ng/ml), and a GSK3b inhibitor selected from CHIR99021, LiCl, and BIO (i.e. a Wnt activator that lowers GSK3b for activation of Wnt signaling) (see para. [0034], [0054]-[0057], [0087]-[0090]; Examples 1-2, Table 2), which relates to steps a) and b) of instant claim 1;
ii. on Day 4, culturing the cultured hPSCs (cultured as described above) under hypoxic conditions in a culture medium (DM2) comprising a FGF including FGF2 (50ug/ml), a VEGF including VEGF-A (50ug/ml), and an inhibitor of TGFβ-mediated signaling (TGF-β inhibitor) including SB-431542 (10uM); and A-83-01 as in claim 18 (see para [0091]-[0092]), which relates to step (c) of instant claim 1;
iii. On Day 6, directing differentiation of hPSC-derived HPCs into myeloid progenitors by culturing hPSC-derived HPCs under normoxic conditions in a chemically defined growth medium (DM3) comprising a FGF including FGF2 (50ng/ml), a VEGF including VEGF-A (50ng/ml), thrombopoietin (TPO) (50ng/ml), and at least one cytokine selected from SCF (50ng/ml), IL-6 (50ng/ml), and IL-3 (10ng/ml) as in claims 18-22, or a mixture thereof or culturing hPSC-derived HPCs under normoxic conditions in a myeloid differentiation culture medium (DM4) comprising one or more hematopoietic cytokines including GM-CSF (also known as CSF2) (200ng/ml), IL-3, or IL-5 for 2-5 days as in claims 18-22 (paragraphs [0037]-[0038]; [0054]-[0057], [0095]-[0096]; Examples 1-2, Tables 1-5), which relates to steps (d) and (e) of instant claim 1; and
iv. obtaining microglia/macrophage precursors, After 2-5 days of myeloid progenitor expansion, culturing the cells in a macrophage differentiation medium (DM5) comprising IMDM, FBS (10%), IL-1β (10ng/ml) and M-CSF (20ng/ml) as in claims 18-22 (paragraph [0066]-[067]) to obtain CD45+, CD14+ and CD11b+ (paragraph [0097]), which meets the limitation of step (f) of instant claim 1.
Douvaras et al. (US11149250) teach a method for generating microglial cells comprising:
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a) culturing pluripotent stem cells (PSCs) in a cell culture medium under conditions that induce myeloid differentiation, leading to the generation of CD14+ and/or CX3CR1+ microglial progenitor cells, wherein culturing comprises contacting PSCs in a first composition comprising mTeSR Custom medium containing 80 ng/ml BMP4 for approximately 4 days; wherein PSCs differentiation was induced with mTeSR Custom medium containing 80 ng/ml BMP4; At day 4, the cells were induced with 25 ng/ml bFGF, 100 ng/ml SCF and 80 ng/ml VEGF in StemPro-34 SFM (with 2 mM GutaMAX); 2 days later, the medium was supplemented with 50 ng/ml SCF, 50 ng/ml IL-3, 5 ng/ml TPO, 50 ng/ml M-CSF and 50 ng/ml Flt3; and from day 14 with 50 ng/ml M-CSF, 50 ng/ml Flt3 and 25 ng/ml GM-CSF; and between days 25-50, CD14+ or CD14+CX3CR1+ progenitors were isolated and re-plated onto tissue culture-treated dishes or Thermanox plastic coverslips in Microglial Medium (RPMI-1640, with 2 mM GlutaMAX-I, 10 ng/ml GM-CSF and 100 ng/ml IL-34) and the medium was replenished every 3 to 4 days for at least 2 weeks to obtain microglial cells (iPSC-MG) expressing iPSC-MG expressing microglial markers including IBA-1, CD11c, TMEM119, P2RY12, CD11 b and CX3CR1 (i.e. at least microglial marker in claim 1) (figure 1A; col. Examples 1-3, claims 1-17).
Douvaras teaches a method for generating microglial cells comprising culturing pluripotent stem cells (PSCs) in a cell culture medium under conditions that induce myeloid differentiation, leading to the generation of CD14+ and/or CX3CR1+ microglial progenitor cells, wherein culturing comprises:
a)-i) Day 0-4: contacting the PSCs of the cell culture medium with a first composition comprising BMP4 (i.e. Wnt activator) in mTeSR1 media without lithium chloride, GABA, pipecolic acid, bFGF or TGFβ1 and culturing the PSCs in the presence of the first composition for approximately 4 days; to obtain KDR+CD235a+ (i.e. KDR:at least one mesoderm progenitor marker in claims 8 and 10; CD235a: at least one EMP marker in claims1 and 10) population of primitive hemangioblasts;
a)-ii) Day 4-6, contacting the cell culture medium with a second composition comprising one or more factors selected from the group consisting of bFGF, SCF, VEGF-A and a combination thereof, in a serum-free hematopoietic cell medium, including 25 ng/ml bFGF, 100 ng/ml SCF and 80 ng/ml VEGF in StemPro-34 SFM (with 2 mM GutaMAX) for approximately 2 days;
a)-iii) Day 6-14, contacting the cell culture medium with a third composition comprising one or more factors selected from the group consisting of SCF, IL-3, TPO, M-CSF, FLT3 ligand and a combination thereof (i.e. hematopoiesis-promoting cytokine), in a serum-free hematopoietic cell medium, including 50 ng/ml SCF, 50 ng/ml IL-3, 5 ng/ml TPO, 50 ng/ml M-CSF and 50 ng/ml Flt3 for approximately 8 days;
a)-iv) From Day 14, contacting the cell culture medium with a fourth composition comprising one or more factors selected from the group consisting of M-CSF, FLT3 ligand, GM-CSF and a combination thereof, in a serum-free hematopoietic cell medium including 50 ng/ml M-CSF, 50 ng/ml Flt3 and 25 ng/ml GM-CSF for approximately 11 to 36 days; to obtain CD45+ (i.e. pre-macrophage marker in claim 1) and/or CX3CR1+ (i.e. microglial marker in claim 1) microglial progenitor cells at Day 16, ,
a)-v) between Days 25-50, isolating CD14+ and CX3CR1+ microglial progenitor cells produced in (a).
b) culturing the CD14+ and CX3CR1+ microglial progenitor cells in a first microglial differentiation medium comprising IL-34, or comprising IL-34 and GM-CSF (i.e. macrophage promoting cytokine in claim 20), thereby generating microglial cells (iPSC-MG) expressing iPSC-MG expressing microglial markers including IBA-1, CD11c, TMEM119, P2RY12, CD11 b and CX3CR1 (i.e. microglial marker in claim 1); wherein the first microglial differentiation medium further comprises GM-CSF; and wherein the second microglial differentiation medium further comprises one or more factors selected from the group consisting of GM-CSF, NGF-β, CCL2 and a combination thereof; and wherein the CD14+ and/or CX3CR1+ microglial progenitor cells are cultured in the first or second microglial differentiation medium for approximately 15 days including a Microglial Medium (RPMI-1640, Life Technologies, with 2 mM GlutaMAX-I, 10 ng/ml GM-CSF and 100 ng/ml IL-34). Medium was replenished every 3 to 4 days for at least 2 weeks (see figures 1A and 9; col. 2, line 53-col.5, line 56; col. 11, lines 1-12; col. 12, lines 5-10; col. 12, line 45-col. 13, line 9; col. 14-26, Examples 1-3; col. 29-30, claims 1-17).
The teaching of Sturgeon provides motivation and an expectation of success in generation of hematopoietic primitive progenitors (KDR+CD235a+) by adding a Wnt-b inhibitor, IWP2, at day 2-3 or within 24hrs of differentiation cultures of hiPSC because including and adding the Wnt-b inhibitor, IWP2, at day 2-3 or within 24hrs of differentiation cultures of hiPSC promotes the generation of CD235a+ population and the generation of the CD43+ population, which lead to more production of KDR+CD235a+ hematopoietic primitive progenitors that are used in the Takata’s method for generation of microglial cells from stem cells .
The teachings of Thomson and Douvaras provide motivation and an expectation of success in generation of microglial cells from stem cells using different concentration ranges or the culturing duration for generation of human microglial cells from differentiation of stem cells into hematopoietic precursor cells (HPCs), differentiation of hPSC-derived HPCs into myeloid progenitors, differentiation of myeloid progenitors into generation of CD14+ and/or CX3CR1+ microglial progenitor cells and microglial cells.
A person of ordinary skill in the art would have recognized that selecting applying the known combination of a Nodal activator, a BMP and a Wnt activator lowering GSK3b including CHIR99021, the known Wnt inhibitor that is a porcupine inhibitor including IWP2 that is added within 24 hours after step a) of claim 1 to obtain primitive hematopoietic precursor cells with CD235A and/or KDR or CD43 recited in claims 1 and 8-10 or other microglial markers recited in claim 1 and the known concentration ranges or the culturing duration within the claimed ranges or combinations recited in claims 21-22 and the known technique of generating human microglial cells from differentiation of stem cells into hematopoietic precursor cells (HPCs), differentiation of hPSC-derived HPCs into myeloid progenitors, differentiation of myeloid progenitors into generation of CD14+ and/or CX3CR1+ microglial progenitor cells and microglial cells disclosed by Sturgeon, Douvaras and Thomson to the Takata’s method would have yielded the predictable result of generation of microglial cells by inducing differentiation of stem cells into differentiated cells expressing at least one erythromyeloid progenitor (EMP) marker including Kit, CD41, CD235A and CD43 or combinations thereof, expressing at least one pre-macrophage marker including kit, CD45, CSF1R and combination thereof and inducing differentiation of differentiated cells to cells expressing at least one microglial marker including CX3CR1, PU1, CD45, IBA1, P2RY12, TMEM119, SALL1, GPR34, C1QA, CD68, CD45 and combinations thereof, and resulted in an improved method.
Including and using the known combination of a Nodal activator, a BMP and a Wnt activator lowering GSK3b including CHIR99021, the known Wnt inhibitor that is a porcupine inhibitor including IWP2 that is added within 24 hours after step a) of claim 1 to obtain primitive hematopoietic precursor cells with CD235A and/or KDR or CD43 recited in claims 1 and 8-10 or other microglial markers recited in claim 1 and the known concentration ranges or the culturing duration within the claimed ranges or combinations recited in claims 21-22 disclosed by Sturgeon, Douvaras and Thomson in the method of Takata would induce differentiation of stem cells and increase generation of microglial cells, and would expand application of the method of Takata in generation and use of microglial cells for therapeutic or pharmaceutical purposes.
Further, even if the concentration ranges or the culturing duration are not exactly identical to the claimed ranges or combinations, the claimed method requires culturing the differentiated cells with neurons for at least 5 days or contacting the differentiated cells with at least one macrophage-promoting cytokine for at least about 5 days and culturing the cells with neurons for at least 5 days, contacting the cells with at least one Wnt activator for about 15-24hrs, or 20hrs, contacting the cells with at least one Wnt inhibitor for 2 days, which are overlapping with the range of Takata, Sturgeon, Douvaras and Thomson because Takata, Sturgeon, Douvaras and Thomson teach specific days and concentrations that are either within or overlapping with the claimed range as set forth above. Because the claimed range overlaps with the range disclosed by the prior art, a prima facie case of obviousness exists. Note that
In the case where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima faciecase of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990), See MPEP §2144.05-I.
“a prima facie case of obviousness exists where the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have the same properties. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985)” See MPEP §2144.05-I.
Thus, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to select and apply the known combination of a Nodal activator, a BMP and a Wnt activator lowering GSK3b including CHIR99021, the known Wnt inhibitor that is a porcupine inhibitor including IWP2 that is added within 24 hours after step a) of claim 1 to obtain primitive hematopoietic precursor cells with CD235A and/or KDR or CD43 recited in claims 1 and 8-10 or other microglial markers recited in claim 1 and the known concentration ranges or the culturing duration within the claimed ranges or combinations recited in claims 21-22 and the known technique of generating human microglial cells from differentiation of stem cells into hematopoietic precursor cells (HPCs), differentiation of hPSC-derived HPCs into myeloid progenitors, differentiation of myeloid progenitors into generation of CD14+ and/or CX3CR1+ microglial progenitor cells and microglial cells disclosed by Sturgeon, Douvaras and Thomson to the Takata’s method, and yield the predictable result of inducing differentiation of stem cells and increasing generation of microglial cells in application of the use for therapeutic or pharmaceutical purposes.
Further routine optimization of Takata’s, Sturgeon’s, Douvaras’s and Thomson’s concentrations or incubation time or duration would have led to the claimed range of 15-24hrs or 20hrs for Wnt activators, the claimed range of at least about 1-5 days or 2 days for Wnt inhibitors or the claimed range of 1-6uM for Wnt activators or the claimed range of 1-10uM for Wnt inhibitors, the claimed range of 1-400ng/ml for hematopoiesis-promoting cytokines and the claimed range of 1-200ng/ml for macrophage promoting cytokines because Takata, Sturgeon, Douvaras and Thomson teach that specific days and concentrations that are either within or overlapping with the claimed range as set forth above achieve inducing differentiation of stem cells into differentiated cells expressing at least one microglial marker desired in the Takata’s method. The person of ordinary skill in the art would have found it obvious to optimize within the range taught by Takata, Sturgeon, Douvaras and Thomson because Takata, Sturgeon, Douvaras and Thomson teach that this entire range can induce differentiation of stem cells into cells expressing at least one microglial marker, and also teaches how to optimize the culturing conditions, incubation time and concentrations. Note that
“[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” See In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)”; “The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.” see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382; In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969); Merck & Co. Inc. v. Biocraft Laboratories Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert.denied, 493 U.S. 975 (1989); In re Kulling, 897 F.2d 1147, 14 USPQ2d 1056 (Fed. Cir. 1990); and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997). See MPEP § 2144.05.
Conclusion
8. NO CLAIM IS ALLOWED.
9. The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Abud (PhD Dissertation, Univeristy of California, Irvine, 2017) teaches a method for generating microglial cells expressing CD35+/Iba1+ from differentiation of hematopoietic progenitor cells expressing CD235a that are differentiated from human hPSCs, comprising: 1. Differentiation of iPSCs to hematopoietic progenitor cells (iHPCs): a) Day0, culturing hPSCs in a cell culture medium comprising FGF2 (50ng/ml), BMP4 (50ng/ml; i.e. Wnt activator), Activin-A (12.5ng/ml), R1 (1uM) and LiCl (2mM); b) Day 2, culturing the hPSCs in a culture medium supplement with FGF2 (50ng/ml) and VEGF (50ng/ml); c) Day 4, media was changed to a medium comprising FGF2 (50ng/ml), VEGF (50ng/ml), TPO (50ng/ml), SCF (10ng/ml), IL-6 (50ng/ml) and IL-3 (10ng/ml) for 4 days; d)isolating iPSC-derived HPCs that are CD43+; 2. Differentiation of iHPCs to iMGLs: a) Day 0 or Day 10 from iPSC, culturing CD43+ iHPCs in iMGL differentiation medium (DMEM/F12, insulin (0.2mg/ml), holo-transferrin (0.011mg/ml), sodium selenite (13.4ug/ml), B27 (2%), N2 (0.5%), monothioglycerol (200uM), Glutamax, NEAA, insulin (5ug/ml) supplemented with M-CSF (25ng/ml), IL-34 (100ng/ml) and TGF-b1(50ng/ml); after 25 days (35 days from iPSC), iMGL were cultured in complete differentiation medium supplemented with CD200 (100ng/ml) and CXCL1 (100ng/ml) for additional three days; 3 co-culture iMGL with neurons (see p. 17-22; p. 24-27; p. 36-66).
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Chang-Yu Wang
April 3, 2026
/CHANG-YU WANG/Primary Examiner, Art Unit 1675