DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 01/07/2026 has been entered.
Claim Status
In the submission dated 01/07/2026, Claims 2 and 5 have been cancelled. claims 1, 8, 29 and 48 have been amended. Claims 31, 33, 36, 45, 54, 55, 60, 66, 67, 69, 72 and 77 remain withdrawn as being directed to un-elected inventions. Accordingly, claims 1, 8, 29, 48 and 51 are pending and under current examination.
Priority
The instant application is a national stage entry of PCT application PCT/US2020/029721, filed 04/24/2020. Acknowledgement is made of the applicant’s claim for benefit to prior-filed U.S. provisional patent applications 62/837,891, which is filed 04/24/2019.
Status of Prior Rejections/Response to Arguments
The rejection of claims 1, 2, 5 and 8 under 35 U.S.C. §103 over Studer et al. in view of Fujimori et al. and Zhou et al. is maintained:
The rejection of claims 1, 2, 5, 8, 29 and 48 under 35 U.S.C. §103 over Studer et al. in view of Fujimori et al. and Zhou et al., further in view of Nishiyama et al. is maintained:
The rejection of claims 1, 2, 5, 8, 29, 48 and 51 under 35 U.S.C. §103 over Studer et al. in view of Fujimori et al. and Zhou et al., further in view of Nishiyama et al., and further in view of Van Der Graaf et al., Linton et al., Walter et al. and McCoy et al. is maintained:
The cancellation of claims 2 and 5 renders the rejection thereto moot.
Regrading claims 1, 8, 29, 48 and 51, Applicant amends claim 1, adds the concentrations of the chemicals used in the method, as well as the limitations regarding the properties of the cells in the method, and asserts the cited references, alone or in combination, do not disclose each of the elements of the claims (Remarks, p9). Moreover, Applicant alleges that one of skill in the art would not be suggested or motivated by the cited references for altering the steps of the method disclosed therein, and have the methods of the instant claims as amended, to produce nociceptor-like cells capable of fictional features recited in the instant claims as amended (Remarks, p10).
Applicant’s argument is fully considered but not found persuasive. As discussed in the previous and current (regarding the amended claims) office action, Studer et al., Fujimori et al., Zhou et al., Nishiyama et al., Van Der Graaf et al., Linton et al., Walter et al., and McCoy et al. teach all the limitations of instant claims and render obviousness to instant claims. Applicant is reminded that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In instant case, Studer et al. teach the method of lineage specific differentiation of hESC and/or hiPSC to nociceptors (i.e. nociceptor cells) using novel culture conditions, Fujimori et al. teach culture method for hPSC differentiation, Zhou et al. teach the method of culturing iPSC, all of them are closely related to the method of differentiation of hESC and/or hiPSC to nociceptors, PHOSITA would have been taught or suggested by the references, and combined their own knowledge, to optimize or adapt the teachings of the references and have the method of producing cells capable of differentiating into nociceptor-like cells as recited in instant claims.
Applicant further alleges that one of skill in the art would readily appreciate the cited references, at most, disclose that the methods therein provide nociceptor-like cells that express the receptors or receptors encoded by SCN9A, SCN10A, SCN11A P2RX3, TRPV1, and TRPM8, which are not relevant to the nociceptor-like cells produced by the methods recited in the instant claims, as amended (Remarks, p10). The examiner submits that the receptor expression of the nociceptor-like cells is considered as an inherent property. Though the cited references teach the expression of other receptors but not the receptors as recited in instant claims, the expression of the receptors by the nociceptor-like cells is considered as an inherent property of the nociceptor-like cells produced by the same method of Studer et al. in view of Fujimori et al. and Zhou et al.. Where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). See MPEP §2112.
The rejection is maintained and modified to encompass the claims as currently written.
Modified 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.
Claims 1 and 8 stand rejected under 35 U.S.C. 103 as being unpatentable over Studer et al. (US 20160201032 A1, published in 2016) in view of Fujimori et al. (Stem Cell Reports. 2017 Nov 14;9(5):1675-1691) and Zhou et al. (Stem Cells Int. 2016;2016:4937689). The rejection is modified necessitated by Applicant’s amendment.
Studer et al. teach the linage specific differentiation of pluripotent or multipotent stem cells, which can include, but is not limited to, human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC), somatic stem cells, cancer stem cells, or any other cell capable of lineage specific differentiation. Specifically described are methods to direct the lineage specific differentiation of hESC and/or hiPSC to nociceptors (i.e. nociceptor cells) using novel culture conditions. The nociceptors made using the methods of their invention are further contemplated for various uses including, but limited to, use in in vitro drug discovery assays, pain research, and as a therapeutic to reverse disease of, or damage to, the peripheral nervous system (PNS) (Abstract).
Regarding claim 1, Studer et al. teach their invention provides a method for inducing directed differentiation of a stem cell, comprising: a) providing: i) a cell culture comprising human stem cells; and ii) a first signaling inhibitor, a second signaling inhibitor and a third signaling inhibitor, wherein said first inhibitor is capable of lowering transforming growth factor beta (TGFβ)/Activin-Nodal signaling, said second inhibitor is capable of lowering Small Mothers Against Decapentaplegic (SMAD) signaling and said third inhibitor is capable of lowering glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling; b) contacting said stem cell with said first and said second inhibitor for up to 48 (or even up to 96) hours in vitro (to produce inhibited stem cell); and c) further contacting said inhibited stem cell with said third inhibitor for up to an additional 192 hours (or even up to 240 hours) for inducing directed differentiation of a stem cell (parag 0016). Studer et al. teach in one embodiment, the method further comprises steps for making neural stem cell precursors and making nociceptor cells (see parag 0016). In one embodiment, said stem cell is a human induced pluripotent stem cell (parag 00016). In one embodiment, the stem cells are cultured in a monolayer (parag 0008). Studer et al. teach in one embodiment, said first inhibitor(s) is selected from the group comprising SB431542, LDN193189, and combination thereof (parag 0012), wherein the concentration of SB431542 is 10 µM (see parag 0270, which is in the range of 20 nM-40 µM as recited in instant claim). In one embodiment, said third inhibitor comprises DAPT and derivatives thereof. In one embodiment, said activator (activation of Wnt signaling) comprises CHIR99021 and derivatives thereof (parag 0012). Studer et al. teach Activation of Wnt signaling can be achieved with a variety of compounds including CHIR99021, LiCl, TDZD-8, recombinant Wnt or other molecules which activate Wnt signaling pathways. A preferred embodiment utilizes the composition comprising CHIR99021, DAPT, and SU5402 at a concentration of 0.3-100 µM (parag 0173). This teaching reads on “a method comprising: incubating for approximately 24-144 hours (e.g., 48 hours) attached monolayer cultures of human pluripotent stem cells in an effective amount or concentration of at least one compound SB431542, the effective concentration of SB431542 is 0.3-100 µM (i.e., 10 µM)” and “culturing the cells for 168-432 hours (e.g., 192 hours) in a second medium comprising an effective amount or concentration of at least one third compound CHIR99021” in instant claim. Studer et al. teach the concentration of CHIR99021 as 0.3-100 µM (parag 0173), which is overlap with the concentration of 20 nM-20 µM as recited in instant claim. In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. See MPEP 2144.05(I).
Furthermore, Studer et al. teach in one embodiment, the kit (which comprising the three inhibitors) further comprises a fourth inhibitor that lowers fibroblast growth factor (FGF) receptor family signaling, wherein said FGF receptor family signaling comprises vascular endothelial growth factor (VEGF) receptors, fibroblast growth factor (FGF) receptors and platelet-derived growth factor (PDGF) tyrosine kinase receptors. In one embodiment, said kit further comprises a fifth inhibitor capable of lowering Notch signaling (parag 0016). Studer et al. teach in one embodiment, said fourth inhibitor is
selected from the group consisting of SU5402 and derivatives thereof (parag 0016), wherein the concentration of SU5402 is 10 µM (parag 0270). In one embodiment, said fifth inhibitor is selected from the group consisting of N-N-(3,5-Difluorophenacetyl)-L-alanyl-S-phenylglycine t-butyl ester (DAPT) and derivatives thereof (parag 0015), wherein the concentration of DAPT is 10 µM (parag 0270). This teaching reads on “(a second medium) comprises an effective amount or concentration of at least one fifth compound DAPT, the effective concentration of DAPT is 5 nM- 50 µM (i.e., 10 µM), and an effective amount or concentration of at least one sixth compound SU5402, the effective concentration of SU5402 is 2 nM- 20 µM (i.e., 10 µM)” in instant claim.
As stated above, Studer et al.’s method shows the cell culture is from human induced pluripotent stem cell (hiPSC) to a neural crest lineage cell which is capable of turning to nociceptor cells or nociceptor cells (see parag 0016), wherein the human pluripotent stem cells they used are human embryonic stem cells (see i.e., Example I, parag 0268), reads on (d) in instant claim. Studer et al. teach the reagents used for instant claim, including using reagent SB431542 for inhibiting growth factor beta (TGFβ)/Activin-Nodal signaling (parag 0012), using CHIR99021 for activation of Wnt signaling (parag 0012), using DAPT for inhibiting Notch pathway (parag 0015), and using SU5402 for inhibiting VEGF and Notch signaling (parag 0016). Studer et al. also teach two culture periods: first period with inhibiting TGFβ for up to 48 (or even up to 96) hours (which is in the range of 24-144 hours as recited in instant claim), second period with activating Wnt signaling for up to an additional 192 hours (or even up to 240 hours) (which is in the range of 168-432 hours as recited in instant claim). Moreover, inhibiting VEGF and Notch signaling is together with activating Wnt signaling (see claim 30 of Studer et al.).
Instant claim differs from Studer et al. in the following four features:
1) for the first period of culture, instant claim 1 also includes using CHIR98014 or CHIR99021, wherein the effective concentration of CHIR98014 or CHIR99021 is 20 nM - 20 µM;
2) having the step of dissociating the incubated cells;
3) for the second period of culture, instant claim also includes using SB431542 for inhibiting TGFβ signaling; wherein the effective concentration of SB431542 is 20 nM - 40 µM; and
4) instant claim specifically points out the one or more nocispheres further comprise the nociceptor-like cells; and wherein the nociceptor-like cells detectably express one or more of OPRM1,OPRMK1, OPRD1, or OPRL1.
However, these were disclosed by Fujimori et al. and Zhou et al..
Fujimori et al. report a simple treatment with three small molecules—SB431542, dorsomorphine, and CHIR99021—that enhanced hPSC differentiation into three germ layers with a chemically transitional embryoid-body-like state (CTraS). Induction of CTraS reduced the innate differentiation propensities of hPSCs (even unfavorably differentiated hPSCs) and shifted their differentiation into terminally differentiated cells, particularly neurons (Abstract).
Zhou et al. determined that culturing iPSC-derived NPCs as three dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers GFAP and aquaporin 4 (AQP4) in the differentiated cells (Abstract).
Regarding 1) and 3), Fujimori et al. teach the effects of SB431542 (SB), dorsomorphin (DM), and CHIR99021 (CHIR). SB has been implicated in efficient neural conversion of human ESCs (hESCs) and hiPSCs via inhibition of SMAD signaling in combination with Noggin activity. Noggin, an inhibitor of BMP signaling, can be replaced by DM, which only enhances neural induction. CHIR is an inhibitor of glycogen synthase kinase 3 (GSK3) and activates the canonical Wnt signaling pathway (p1676, left column). Use of SB, DM, and CHIR has a synergistic inhibition of the GSK3, TGF-β, and BMP Signaling Pathways (p1676, right column). Fujimori et al. teach adding the three chemicals for whole period (e.g., figure 1E, three molecules from day 0-day 6) or SB and CHIR together (e.g., from day 1-day 6, see figure 1A-D) for differentiation of human pluripotent stem cells (hPSCs), the concentration of SB is 2 µM (see supplemental experimental procedures) which is in the range of 20 nM-40 µM, and the concentration of CHIR is 3 µM (see supplemental experimental procedures) which is in the range of 20 nM-20 µM. Fujimori et al. teach inhibiting TGFβ and activating Wnt during all the culture period for the iPSCs differentiation.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Studer et al.’s method for inducing directed differentiation of human induced pluripotent stem cells to neural crest stem cells capable of making nociceptor cells or nociceptor -like cells, and involve inhibiting TGFβ signaling and activating Wnt signaling in the whole culture period, as taught by Fujimori et al.. The difference between instant claim and Studer et al.’s method is instant claim inhibiting TGFβ and activating Wnt the whole culture period. Given that Fujimori et al. teach using inhibiting of TGFβ and activating Wnt effectively enhances the differentiation of hPSCs (see figure 1), one of ordinary skill in the art would have substituted Studer et al.’s method of inhibiting TGFβ signaling for the first culture period and activating Wnt signaling in the second culture period, and inhibit TGFβ and activate Wnt signaling in the whole culture period for hPSC differentiation. This simple substitution of one known element (inhibiting TGFβ and activating Wnt signaling in the whole culture period for hPSC differentiation) for another known element (Studer et al.’s method of inhibiting TGFβ for the first culture period and activating Wnt signaling in the second culture period) is likely to be obvious when predictable results are achieved. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (see MPEP § 2143, B.).
Regarding 2) the step of dissociating the incubated cells, Zhou et al. teach 2D monolayer culture of Neural progenitor cells (NPCs) and terminal differentiation and 3D neurospheres culture of NPCs and terminal differentiation (p2, right column, Materials and Methods). For 3D neurosphere culture, Monolayer NPCs from all three lines (N S5, N S8, and RT S11) were dissociated into single cells with Accutase. A total of 1 × 106 cells/mL were plated on the low attachment dish in neural maintenance medium and maintained for 7 days as neurosphere cultures (p2, right column). The monolayer NPCs were induced from human iPSCs by dual inhibition of SMAD signaling with 10 𝜇M SB431542 and 100 ng/mL Noggin (p2, right column). Herein the 3D neurospheres culture is comprising the monolayer culture of human iPSCs, then dissociate the cultured cells to culture the dissociated cells in neurospheres, which is the same procedure as instant claim.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Studer et al.’s method of monolayer culture of hPSCs for inducing directed differentiation of human induced pluripotent stem cells to neural crest stem cell capable of making nociceptor cells or nociceptor (-like) cells, and culture monolayer NPCs from human iPSCs, dissociate the NPCs, then use neurosphere culture to further culture and differentiate NPCs as taught by Zhou et al.. The skilled artisan would have been motivated to dissociate the monolayer cultured NPCs to neurosphere culture since Zhou et al. teach the neurospheres generated from 3D free-floating aggregates of NPCs with a certain spatial degree of complexity better mimic the main features of brain tissue and can therefore be considered as more relevant in vitro models (p7, left column). Zhou et al. also teach neurosphere culture is better for the grow and maintenance of NPCs: neurosphere diameter increases from day 4 to day 7, which indicates the ability of cell proliferation within the 3D culture system (p7, left column), given that better proliferation of NPCs leads to better source of cell differentiation, one of ordinary skill in the art would be motivated to choose this culture method (culture monolayer NPCs from human iPSCs, dissociate the NPCs, then use neurosphere culture to further culture and differentiate NPCs) for iPSCs culture and differentiate to e.g., nociceptor-like cells. There would be a reasonable expectation of success of culturing monolayer NPCs from human iPSCs, dissociating the NPCs and using neurosphere culture to further culture and differentiate NPCs, since Zhou et al. teach the culture method (p2, right column, Materials and Methods).
Regarding 4), the limitations are considered as inherent properties of the cells produced by the method. Where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). See MPEP §2112.
In sum, inhibiting TGFβ and activating Wnt signaling in the whole culture period and monolayer culture human iPSCs, dissociate the monolayer cells, then use neurosphere culture to further culture and differentiate, as recited in instant claim, are all considered as an alternative way or an optimization of a previous culture method.
Regarding claim 8, Studer et al. teach contacting stem cell (hPSC) with said first and said second inhibitor for up to 48 (or even up to 96) hours in vitro, further contacting said inhibited stem cell with said third inhibitor for up to an additional 192 hours (or even up to 240 hours). In one embodiment, said second inhibitor is a small molecule selected from the group consisting of LDN193189, derivatives thereof and mixtures thereof (see parag 0016). Studer et al. also also teach a small molecule BMP inhibitor, LDN-193189, was used in place of Noggin to inhibit BMP (parag 0279-0280). This teaching reads on “d) the second medium is not supplemented with additives inhibiting bone morphogenic protein (BMP) protein pathways” in instant claim.
Claims 1, 8, 29 and 48 stand rejected under 35 U.S.C. 103 as being unpatentable over Studer et al. (US 20160201032 A1, published in 2016) in view of Fujimori et al. (Stem Cell Reports. 2017 Nov 14;9(5):1675-1691) and Zhou et al. (Stem Cells Int. 2016;2016:4937689), further in view of Nishiyama et al. (Neurosci Res. 2016 Jun;107:20-9). The rejection is modified necessitated by Applicant’s amendment.
The teaching of Studer et al. in view of Fujimori et al. and Zhou et al. is set forth above.
Regarding claim 29, following the discussion of claim 1, Studer et al. teach inducing directed differentiation of a stem cell, wherein said differentiated stem cell is selected from the group consisting of a neural crest stem cell, a neural crest lineage cell and a neuronal lineage cell which are nociceptor-like cells capable of making nociceptor cells (see parag 0016). Studer et al. do not teach cryopreserving the neural crest stem cell, a neural crest lineage cell or a neuronal lineage cell. However, it is prima facie obvious in view of Nishiyama et al..
Nishiyama et al. developed an advanced slow-freezing technique using a programmed freezer with a magnetic field called Cells Alive System (CAS) and examined its effectiveness on human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs) (Abstract).
Regarding claim 29, Nishiyama et al. use Cells Alive System (CAS) which is known to have a novel freezing technology and it has been used in the cryopreservation of various kinds of cells, including murine osteoblasts, rat bone tissue fragments, rat mesenchymal stem cells (MSCs), human periodontal ligament cells, and human ES cells, and cryopreserve hiPSC-NS/PCs and examined the effects of cryopreservation process on hiPSC-NS/PCs (p21, left column). Since herein the iPSC differentiated cells are capable of differentiating into nociceptor-like cells may be neural crest stem cells, a neural crest lineage cell and a neuronal lineage cell (see parag 0016), they are one type of hiPSC-NS/PCs and can be cryopreserved by Nishiyama et al.’s method, which reads on “b) cryopreserving one or more of the cells capable of differentiating into the nociceptor-like cells” in instant claims.
Regarding claim 48, as discussed above, the iPSC differentiated cells are capable of differentiating into nociceptor-like cells may be neural crest stem cells, a neural crest lineage cell and a neuronal lineage cell (see parag 0016), and they are in the form of neurospheres. Nishiyama et al. teach cell freezing, a total of 2 or 5 × 106 cells were placed as neurospheres in a cryo-tube (p21, left column). Nishiyama et al. teach they found that frozen-thawed hiPSC-NS/PCs were comparable with non-frozenones at the transcriptome level (Abstract). This teaching reads on step “a) after culturing the dissociated cells, cryopreserving the one or more nocispheres and thawing of the cryopreserved one or more nocispheres” in instant claim.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Studer et al.’s method for inducing directed differentiation of human induced pluripotent stem cells to neural crest stem cell capable of making nociceptor cells or nociceptor (-like) cells, and further cryopreserve the nocispheres comprising cells capable of differentiating into the nociceptor-like cells (e.g., neural crest stem cells) as taught by Nishiyama et al.. The skilled artisan would have been motivated to cryopreserve the nocispheres comprising cells capable of differentiating into the nociceptor-like cells (e.g., neural crest stem cells) for future use and still obtain cells which are viable and comparable with non-frozen ones, which is beneficial for further research and clinical study. There would be a reasonable expectation of success of cryopreserving and thawing the nocispheres comprising cells capable of differentiating into the nociceptor-like cells (e.g., neural crest stem cells) since Nishiyama et al. teach the method of cryopreserving hiPSC-NS/PCs (p21, left column).
Claims 1, 8, 29, 48 and 51 stand rejected under 35 U.S.C. 103 as being unpatentable over Studer et al. (US 20160201032 A1, published in 2016) in view of Fujimori et al. (Stem Cell Reports. 2017 Nov 14;9(5):1675-1691) and Zhou et al. (Stem Cells Int. 2016;2016:4937689), further in view of Nishiyama et al. (Neurosci Res. 2016 Jun;107:20-9), and further in view of Van Der Graaf et al. (US 10123529 B2, patented in 2018), Linton et al. (J Med Chem. 2005 Nov 3;48(22):6779-82), Walter et al. (US 20160096800 A1, published in 2016) and McCoy et al. (US 20100221823 A1, published in 2010).
The teaching of Studer et al. in view of Fujimori et al. and Zhou et al., and further in view of Nishiyama et al. is set forth above.
Regarding claim 51, Studer et al. in view of Fujimori et al. and Zhou et al., and further in view of Nishiyama et al. do not teach the cryopreservation medium. However, the cryopreservation medium components are prima facie obvious in view of Van Der Graaf et al., Linton et al., Walter et al. and McCoy et al..
Van Der Graaf et al. teach compounds comprising derivatives of Chroman 1(see Col 7, Table 1). Van Der Graaf et al. teach the chroman 1 derivatives compounds protect the cells against cell injury, which may eventually lead to cell death via necrosis or apoptosis. The compounds according to the invention provide protection against cell injury. The protection can be provided during storage. Cells stored with compounds according to the invention have a decreased cell death compared with cells stored without the compound (Col 6, L44-51). In one embodiment, the above compounds protect mammalian cells, such as cultured cell lines (e.g., from human origin), stem cells, primary cells, blood platelets, blood cells, and tissue cells (Col 6, L52-55).
Linton et al. teach apoptosis, or programmed cell death, is a highly regulated biological process involved in maintaining normal tissue homeostasis. Dysregulation of apoptosis can lead to loss of cells and caspases are responsible for the disassembly and phagocytosis of an apoptotic cell (p6779, left column). Linton et al. teach compound IDN-6556 (Emricasan) is a very potent, irreversible pan caspase inhibitor having potent anti-apoptotic activity in vitro and in vivo (p6781, right column).
Walter et al. teach a method of treating an integrated stress response-associated disease in a patient in need of such treatment (parag 0010). Walter et al. teach ISRIB can influence cell fate by reducing viability of cells subjected to ER-stress and blocking multiple stress effectors (see parag 0403, 0404). Walter et al. teach ISRIB is used to block translational regulation by the four eIF2α kinases PERK (activated by ER stress), PKR (activated by viral infection), HRI (activated by heme deficiency) and GCN2 (activated by amino acid starvation) (see parag 0225).
McCoy et al. teach methods for mammalian cell culture, wherein the methods make use of media containing polyamines, such as putrescine, Spermidine, and spermine (parag 0002). McCoy teaches cell viability, viable cell density and expression of the protein of interest are improved in medium may also comprise combinations of spermine, spermidine and putrescine (see parag 0008).
It would have been prima facie obvious to one of ordinary skill before the effective filing date of the claimed invention to modify Studer et al.’s method for inducing directed differentiation of human induced pluripotent stem cells to neural crest stem cell capable of making nociceptor cells or nociceptor (-like) cells, and further cryopreserve the nocispheres comprising cells capable of differentiating into the nociceptor-like cells (e.g., neural crest stem cells) with a cryopreservation medium comprising Chroman 1 and/or a derivative thereof as taught by Van Der Graaf et al., Emricasan and/or the derivative thereof as taught by Linton et al., trans-ISRIB as taught by Walter et al. and polyamines comprising putrescine, spermine and spermidine as taught by McCoy et al.. The skilled artisan would have been motivated to use such cryopreservation medium to improve cell survival since: (1) Van Der Graaf et al. teach the chroman derivative can be used to protect against cell injury; (2) Linton et al. teach emricasan has potent anti-apoptotic activity; (3) Walter et al. teach trans-ISRIB blocks multiple stress effectors and can be combined with polyamines, and (4) McCoy et al. teach cells grown with polyamines (spermine, spermidine, putrescene) improves their viability. There would be a reasonable expectation of success of having this cryopreservation medium since Van Der Graaf et al. teach the chemical of Chroman 1 derivative, Linton et al. teach the chemical of Emricasan, Walter et al. teach the chemical of trans-ISRIB and McCoy et al. teach polyamines (spermine, spermidine, putrescene), the skilled artisan would readily know how to add the components together in the cryopreservation medium.
Conclusion
No claims are allowed.
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/Q.G./Examiner, Art Unit 1633
/FEREYDOUN G SAJJADI/Supervisory Patent Examiner, Art Unit 1699
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