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 .
Election/Restrictions
Applicant's election with traverse of Group I (claims 1-8) in the reply filed on 7/27/2026 is acknowledged. The traversal is on the ground(s) that Becari et al does not teach the special technical feature of the claims. Becarri does not teach the different cells of the spheroid and their gastruloid does not form all the cell types particular those of the central nervous system. This is not found persuasive because Applicant is not reading limitations into the claims special technical feature of the claims set that are not required in all claims. The special technical feature only requires the ES-like cell core, EPI-L cells, and XEN-like cells, which Becarri disclose. The remainder of the feature are not present in all the claims and thus are not part of the technical feature.
The requirement is still deemed proper and is therefore made FINAL.
Claims10-13, 18-22, 34, and 37-38 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 7/27/2026.
Claims 1-8 are under consideration in this office action.
Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Specification
The disclosure is objected to because of the following informalities: Figure Legend Title and legends starting on page 14 of the published application refer to color drawings. However, the drawings provided are in black and white. As such, all the color or coloring disclosures cited cannot be discerned.
The disclosure is objected to because of the following informalities: Page 15, [0218], col 2, last two lines of the paragraph, the disclosure states, “q, Gene set enrichment categories”. The “q” in this recitation appears to be a typographical error and should recite “k” to be consistent with the figure 12.
The disclosure is objected to because it contains embedded hyperlinks and/or other form of browser-executable code on page 43, line 8; p. 44, lines 21 and 24 of the original specification. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
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.
Claims 1-8 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 base claim 1, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 1 recites the broad recitation “vertebrates cells”, and the claim also recites “mammalian cells” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. It is not apparent how one can arrive at structures limited to “mammalian cells” using “vertebrates cells” as claimed. As such, the scope of the cell origin is not apparent.
Also, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 1 recites the broad recitation “organized 3D cell structures” as the end product in the preamble , and the body of the claim also recites “spheroids…composed of …3D self-assembled cell types” as the end product which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Claim 1(ii) recites, “an agent inhibiting small ubiquitin-like modified (SUMO) conjugation (SUMO inhibitor)”. This recitation is indefinite it states inhibiting SUMO conjugation without specifying to what SUMO is conjugating. Conjugation is solely binding. If Applicant is intending to inhibit the action of SUMO or inhibit SUMO signaling pathways, amending the recitation to state “SUMO inhibitor” would be remedial. The scope of the above recitation is not apparent.
Claim 1(v) recites, “the spheroids are composed of at least three 3D self-assembled cell types encompassing from the center to the periphery of the spheroid embryonic stem-like cells (ES-like cells), form the core of the spheroid on a monolayer of epiblast-like cells (EPI-L cells) and surrounded by extraembryonic endoderm cells (XEN-like cells) wherein the cell types in the spheroid lack pluripotency.”. The relational structure between the EPI-L cells and the core, ES-like cells, and spheroid is not apparent. The claim states the “cell types…”from the center to the periphery of the spheroid”. Then the claim states ES-like cells “form the core of the spheroid on a monolayer of epiblast-like cells” It is clear that the ES-like cells are intended to form the core or the spheroid”. However, it states the “spheroid on a monolayer of” EPS-L, does that means the core is cultured on a monolayer? the whole spheroid is cultured on a monolayer of EPI-L cells? How can the core or the spheroid be cultured on a monolayer and then surrounded by XEN-like cells? Further how would this represent the three cell types in a spherical formation? As such, the metes and bounds of the organized 3D cell structure of the claims are not apparent.
Claim also recites that the spheroid comprises “embryonic-like stem cells” and “epiblast-like cells” but further states “the spheroids lack pluripotency”. Lacking pluripotency by definition in the means that the cells are not embryonic stem cells, epiblast cells, or “like” these cell types. As such, the metes and bounds of the spheroid lacking pluripotency when it comprises embryonic-like cells and epi-blast like cells that pluripotent stem cells by definition are not apparent.
Claims 2-8 depend upon claim 1 and therefore also comprise the indefinite subject matter discussed above.
Regarding claim 2, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 2 recites the broad recitation “a first culture medium suitable for ESC culture and maintenance of pluripotent state”, and the claim also recites “the first ESC culture medium” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. “A first culture medium suitable for ESC culture and maintenance of pluripotent state” is not limited to an embryoid stem cell culture medium. For example, DMEM medium is “suitable for ESC culture and maintenance of pluripotent state” regardless of it has any factors that would maintain pluripotent state. It does not even need to have any embryonic stem cell specific elements and can be just DMEM. In contrast, “ESC culture medium” has to at least have factors present for support of embryonic stem cells culture or differentiation. As such it is a narrower limitations that the medium that is recited as “suitable”.
Similarly, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 2 recites the broad recitation “a second culture medium suitable for ESC culture and differentiation of cells”, and the claim also recites “the second ESC culture medium” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. For reasons discussed above this recitation is indefinite.
Regarding claim 8, the claim recites, “the method is performed without providing a morphogen substance to the cells”. First “the cells” lacks sufficient antecedent basis and is indefinite because the base claim recites multiple different cells. As such, which cells are “the cells”? Second, the negative proviso of “not providing a morphogen substance” in the method does not make sense given that the method of the base claim treats with a SUMO inhibitor, which is a morphogen substance and possible differentiation factors in the second medium which are also morphogen substances.
Claim Objections
Claim 4 is objected to because of the following informalities: the claim recites, “the time between two consecutive steps of hypoSUMOlation treatment”. However, the previous recitations of two steps of hypoSUMOlation treatment do not require them to be “consecutive”. As such, the recitation lacks sufficient antecedent basis. Appropriate correction is required.
Claim 5 is objected to because of the following informalities: Claim 5 recites the dangling preposition, “carried out”. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-8 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the following:
An in vitro method of preparing a spheroid comprising embryonic-like structure comprising:
(i) providing a first culture comprising a mouse embryonic stem cell (ESC) in a first ESC medium comprising serum and LIF;
(ii) adding a SUMO E1 enzyme inhibitor to the first culture to provide a first hypoSUMOlation treatment to the first culture;
(iii) at least three days after (ii) adding a SUMO E1 enzyme inhibitor to provide a second hypoSUMOlation treatment to the first culture, wherein the SUMOR E1 enzyme inhibitor in the first and second hypoSUMOlation treatments are conducted not more than 48 hours;
(iv) culturing the cells of the first culture in a second ESC medium comprising N2B27 and LIF to provide a second culture after the second hypoSUMOlation treatment;
(v) recovering spheroids comprising a core of embryonic stem-like cells (ES-like cells) in contact with epiblast-like cells (EPI-L cells), all surrounded by extra-embryonic endoderm cells (XEN-like cells), wherein the ES-like cell express Nanog, Sox2, Esrrb, and Sall1, EPI-L cells express Pou3f1, Pim2, Lef1, and XEN-like cells express Sox17, Gata6, Gata4, and Col14a1.
The specification does not reasonably provide enablement for the following:
Any vertebrate or mammalian species of pluripotent or multipotent cell other than a mouse pluripotent stem cell;
Any first media suitable for ESC culture and maintenance of pluripotency;
Any agent inhibiting any SUMO conjugate;
Any second media suitable for ESC culture and differentiation;
Any organized 3D cell structure comprising spheroids with ES-like cells, EPI-L cells, and XEN-like cells expressing any markers and that are not pluripotent.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make and use the claimed invention, if not, whether an artisan would require undue experimentation to make and use the claimed invention and whether working examples have been provided. When determining whether a specification meets the enablement requirements, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make and use the invention based on the content of the disclosure is “undue”.
Nature of Invention: An in vitro method of forming a spheroid with embryo like cell types using patterned modulation of SUMOylation.
Breadth of the Claims: The claims recite providing a mammalian 3D cells structure using vertebrate cells. Vertebrate cells are a genus of cells that original from any tissue in a vertebrate (or spined) animal. Thus the breadth of the claimed starting cell type is any cell of any tissue from any mammal, bird, reptile, marsupial. The cells is further limited by being a multipotent or pluripotent stem cell. Pluripotent cells included embryonic stem cels, epiblast cells, germ cells, some primordial stem cells, some hematopoietic stem cells, embryonic carcinoma cells, as well as induced pluripotent stem cells. Mutipotent cells are any cells that are not totipotent or pluripotent and have the ability to give rise to multiple lineage cells, multiple cells of one lineage, bipotent, unipotent, progenitor cells, and precursor cells. Also they are sometimes call adult stem cells. As such, the starting cell material encompasses a large number of cells species from many different animal species. The claims also recite, “medium suitable for ESC culture or maintenance”. The breadth of this medium is a base medium capable of supporting ESC culture and maintenance including or lacking factors that maintain pluripotency. The claims also recite, “medium suitable for ESC culture and differentiation”. The breadth of this medium is a base medium capable of supporting ESC culture and maintenance including or lacking factors that maintain pluripotency and including or lacking factors that induce pluripotent or multipotent cell differentiation. The claims recites “an agent inhibiting…SUMO…conjugation”. The breadth of this recitation encompass an inhibitor or antagonist that blocks or reduces conjugation of any factor in the SUMO signaling pathway. The claims also recite “spheroids are composed of…from center to periphery…ES-like cells…forming the core of the spheroid…on a monolayer of…EP-L cells…and surrounded by…XEN-like cells…wherein the cell types in the spheroid lack pluripotency. The claims do not recite any markers of structurally defining elements of the three cell types list. Thus the three listed cell types can comprise any marker expression, and any other structural/functional “likeness” to ES cells, epiblast cells and extraembryonic endoderm cells. The claims cells are also recited as lacking pluripotency, which means that lack the ability to self-renew, lack the ability to give rise to all cells of the body, and lack pluripotent marker expression.
Specification Guidance: The specification provides the following guidance (citations from the pregrant publication):
[0003] In vitro recapitulation of early mammalian embryogenesis is a very active field of research.sup.1-13. Two main strategies have been developed to build mouse or human embryo-like structures (ELS), either by combining embryonic and extraembryonic stem cells.sup.1-3, which spontaneously self-organize to mimic post-implantation embryos, or by challenging embryonic stem cells (ESCs) with a pulse of Wingless-type integration site protein (WNT) agonist to recapitulate early organogenesis.sup.4-8. However, a large portion of the central nervous system is missing from these structures, particularly brain derivatives.
[0004] By contrast, the inventors have been able to design methods suitable to prepare 3D cell structures that may enable to obtain various degrees of early organogenesis, until elongated ELS characterized by a head-to-tail body axis with mid-hindbrain neuronal cell types as well as Schwann cell precursors, concomitant with somitogenesis.
[0007] The methods of the invention stem from the determination by the inventors that repeated transient inhibition of small ubiquitin-like modifier (SUMO) conjugation (so-called hypoSUMOylation) in mouse ESCs was suitable and sufficient to form organized 3D cell structures encompassing characterized epiblast-containing spheroids. These epiblast-like cells of spheroids were then capable of undergoing lineage-specific differentiation into all three embryonic germ layers when cultured in different culture formats including 2D plates or 3D microfluidics, especially in an optimized droplet-microfluidic platform. Mechanistically, the steps (i.e. rounds) of hypoSUMOylation gradually increased the global level of DNA methylation, which in turn repressed transcription of Nanog and other pluripotency-associated genes, facilitating cell fate determination.
[0011] The invention accordingly relates to a method of in vitro preparing organized 3D cell structures of mammalian cells wherein the method comprises: [0012] i. Providing a population of pluripotent or multipotent vertebrate cells, in particular mammalian cells, in particular rodent cells, especially mouse cells in a first culture medium suitable for ESC culture and maintenance of pluripotent state, [0013] ii. On the cells in the first culture medium of i., performing two steps of hypoSUMOylation treatment with an agent inhibiting small ubiquitin-like modifier (SUMO) conjugation (SUMO inhibitor), [0014] wherein the second step of hypoSUMOylation treatment is separated in time from the first step of hypoSUMOylation treatment by at least 3 days, in particular by less than 20 days, in particular by 3 to 15 days or 5 to 15 days and more particularly by 3 to 5 days and [0015] wherein each step of hypoSUMOylation treatment with the SUMO inhibitor is conducted for not more than 48 h, preferably is conducted for 48 h, and optionally recovering cells obtained after one or two steps of hypoSUMOylation treatment, [0016] iii. Culturing the cells obtained after the second hypoSUMOylation step of treatment with the SUMO inhibitor according to ii. in a second culture medium suitable for ESC culture and differentiation of cells, wherein the first and the second culture medium have different composition, [0017] iv. Optionally repeating at least once the step of hypoSUMOylation treatment with the SUMO inhibitor, wherein each repeat step of hypoSUMOylation treatment is carried out as in ii. and is performed separated in time from the immediately previous one as in ii., [0018] v. Recovering spheroids, wherein the spheroids are composed of at least three 3D self-assembled cell types encompassing from the center to the periphery of the spheroids embryonic stem-like cells (ES-like cells), forming the core of the spheroid on a monolayer of epiblast-like cells (EPI-L cells) and surrounded by extraembryonic endoderm cells (XEN-like cells) and in particular wherein the cell types in the spheroids lack pluripotency.
[0027] In a particular embodiment of the methods of the invention, the inhibitor of SUMOylation is a selective small-molecule inhibitor of SUMO E1 enzyme wherein the effect of hypoSUMOylation on DNA hypermethylation is partially mediated by Sall4 transcription factor known to regulate DNA methyltransferase Dnmt3a (Zhang. J. et al).
[0028] In a particular embodiment of the methods of the invention, the inhibitor of SUMOylation is a selective small-molecule inhibitor of SUMO E1 enzyme, in particular is ML-792. ML-792 (CAS No.: 1644342-14-2) is a molecule of the following formula (provided by Takeda) that is a selective inhibitor of SUMO-activating enzyme that interferes with the first step of the SUMOylation of proteins cascade catalyzed by the SUMO activating enzyme (SAE), SUMO E1. ML-792 belongs to adenosine sulfamate (AdoS) inhibitors of E1 enzymes and inhibits SUMO1 conjugation to target proteins that are known in the art as chromatin-associated proteins.
[0064] Spheroids are expected to be obtained from day 15 from the onset of the first step of hypoSUMOylation treatment (that may coincide with the first day or the culture of the cells in the culture medium suitable for ESCs) if at least 2 steps of hypoSUMOylation treatment have been carried out that have enabled increase in DNA methylation of the cells. The spheroids are composed of at least three distinct self-assembled cell types encompassing from the center to the periphery of the spheroids embryonic stem-like cells (ES-like cells or ES-L), forming the core of the spheroid on a monolayer of epiblast-like cells (EPI-L cells) and surrounded by extraembryonic endoderm cells (XEN-like cells). The spheroids may alternatively be described as a core of ES-like cells, resting on epiblast-like cells and surrounded by XEN-like cells. The harvested spheroids are especially adherent spheroids.
Working Examples: The specification describes the following:
[0231] Cell Culture
[0232] Mouse ES-R1 cells.sup.14 were used for most experiments and were maintained in serum+Lif medium (KnockOut DMEM supplemented with 15% ES cell qualified FBS, 1% GlutaMAX, 1% MEM non-essential amino acids, 1% penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, 10 ng/mL Lif (Miltenyi Biotec #130-099-895)) on gelatin-coated plates in a humidified incubator (37° C., 5% CO.sub.2). The Sox1::eGFP-T::mCherry double reporter CGR8 mouse ES cells (gift from David M. Suter, Swiss Federal Institute of Technology, Lausanne, Switzerland, ref.56) were maintained on Mitomycin C-treated mouse embryonic fibroblast feeder cells in serum+Lif medium, and the MERVL::tdTomato-Dppa2/4 DKO mouse ES-E14 cells (gift from Wolf Reik, Babraham Institute, Cambridge, UK.sup.57) were cultured in serum+Lif medium on gelatin-coated plates. Mouse ES-D3 cells (from ATCC) were used for droplet-microfluidic platform validation experiments and were cultured in ESLIF medium as previously described.sup.10.
[0233] For 2i culture, ES-R1 cells were maintained in N2B27+Lif medium (1:1 Neurobasal medium and Advanced DMEM/F-12, 1% N-2 supplement, 2% B-27 supplement, 0.05% Bovine albumin fraction V, 1% GlutaMAX, 1% penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, 10 ng/mL Lif) supplemented with 1 μm PD0325901 (Miltenyi Biotec #130-103-923) and 3 μm CHIR99021 (Miltenyi Biotec #130-103-926).
[0234] For retinoic acid differentiation assays, cells were seeded in 6-well plates in medium without Lif (serum medium for D1and D8, N2B27 medium for D18) and treated with 1 μM of Retinoic Acid (Sigma #R2625). Medium was refreshed every day for a total of 5 days of treatment. Cells were collected every 24 h for RNA extractions.
[0235] For 5-azacytidine experiments, cells were seeded in 6-well plates in serum+Lif medium and treated with 0.1 μM of 5-azacytidine (Abcam #ab142744). Medium was refreshed every day for a total of 5 days of treatment.
Generating Adherent Spheroids
[0238] Using ML-792 as hypoSUMOylation agent (SUMO E1 enzyme inhibitor): ESCs (ES R1 cells-129X1×129S1—ATCC) were dissociated with Trypsin-EDTA and plated (2.5 million cells in 100 mm dish) in serum+Lif medium supplemented with 2.5 μM ML-792 (Takeda Pharmaceuticals International Co.). Medium was replaced the next day to refresh treatment. After 48 h of treatment, plates were rinsed twice with PBS then cells were allowed to recover in serum+Lif. 5 days after the end of the first round, cells were similarly counted and plated for a 2.sup.nd round of ML-792. After 48 h of treatment, plates were rinsed twice with PBS then cells were allowed to recover in N2B27+Lif medium. 6 to 8 days later, cells form three-dimensional adherent spheroids, which can be maintained in culture or frozen for subsequent use (cell stocks in ES cell qualified FBS with 10% DMSO). [0239] Using TAK-981 as hypoSUMOylation agent (SUMO E1 enzyme inhibitor): The protocol used is similar to the protocol disclosed herein when using ML-792 as a hypoSUMOylation agent in so far as cell line used (ES R1 cells-129X1×129S1: the R1 cell line was established in August 1991, from a 3.5 day blastocyst produced by crossing two 129 substrains (129S1/SvImJ and 129X1/SvJ).), steps of thawing and cell culture procedure (thawing performed on feeders, culture performed on gelatin and passaging) incubator settings (37° C., 5% CO.sub.2). By contrast the following specific parameters are taken into consideration with TAK-981:
[0240] On Day 1: first round. 500 000 ES R1 cells were plated per well well of a gelatin-coated 6-well plate in 2 ml of serum+Lif medium, supplemented with 0.1 μM of TAK-981. On Day 2, medium was replaced (aspirated) to refresh treatment (2 mL of Serum+Lif medium, supplemented with 0.1 μM of TAK-981 were added). After 48 h of treatment, on Day 3, the medium was aspirated and wells were rinsed twice with PBS then cells were allowed to recover in 2 ml of added serum+Lif. 6 days after the end of the first round, cells were similarly counted and plated for a 2.sup.nd round of TAK-981 (for example starting on Day 9 from the start of first round). On Day 9, 500 000 ES R1 cells were seeded per well on a gelatin-coated 6-well plate in 2 mL of Serum+Lif medium, supplemented with 0.1 μM of TAK-981 On day 10, the medium was aspirated and 2 ml of serum+Lif medium were added, supplemented with 0.1 μM of TAK-981 After 48 h of treatment (on Day 11), the medium was aspirated and wells were rinsed twice with PBS then cells were allowed to recover in 2 ml of N2B27+Lif medium that were added. Culture was carried out in N2B27+Lif medium. 3 to 5 days later (Day 14 to 16), cells form three-dimensional adherent spheroids, which can be maintained in culture or frozen for subsequent use (cell stocks in ES cell qualified FBS with 10% DMSO).
DISCUSSION
[0292] Here, we describe a new strategy to mimic mouse embryo development in absence of exogenous morphogens, that relies on transient waves of hypoSUMOylation to generate, from sole ESCs, self-organized ELS with all three germ layers. Crosstalk between the spheroid cell types is sufficient to trigger a series of morphogenetic events characteristic of natural gastrulation and early organogenesis that enable the emergence of additional cell populations regionalized in complex structures. However, important cell types from various stages of development are missing, including PGCs and forebrain tissue. Future work is needed to optimize culture conditions, such as adding a WNT agonist or using rotating culture platforms, to improve embryo-like morphology.sup.4,48. Importantly, D18 spheroids are stable after freeze/thaw cycles, allowing generation of ELS in only 7 days with one culture medium. Our protocol is thus highly scalable and adaptable. The added value of the droplet-microfluidic platform in boosting lineage diversity requires further investigation to determine whether this system modifies the physicochemical microenvironment (e.g. pH gradient) to favor axial elongation of gastruloids similar to natural embryos.sup.49.
[0293] Brief suppression of SUMOylation both decreased the heterogeneous expression of Nanog and increased the global level of DNA methylation, consistent with events of the peri-implantation stage.sup.50,51. This could suggest that a physiological wave of hypoSUMOylation may occur at gastrulation to facilitate cell fate determination, as demonstrated at the 2C-stage.sup.24,25. Finally, adjusting this protocol for human ESCs may provide new insights into early stages of development and the role of SUMO as a barrier to cell fate change. Overall, our work lays foundations for exploring epigenetic drugs as tools to control the balance between cell fate robustness and lineage commitment, with the ultimate goal of reconstructing complex multicellular architecture.
Thus while the specification and working examples provide specific guidance to mouse pluripotent cells that use two highly specific media, one species of inhibitor to SUMO E1 enzyme, to produce a spheroid comprising ES-like cells, EPI-L cells, and XEN-like cells characterized by each having specific marker expression, the specification fails to provide enabling guidance to the breadth of any vertebrate species of pluripotent and multipotent cells, a homogenous population thereof, any inhibitors of SUMO conjugation that leads to the claimed spheroid lacking pluripotency. It is noted that the status of “pluripotency” is not measured in the cells of the spheroids, but are only characterized by the presence of characteristic markers. To this end, while the ES-like cells in the working example do not express Oct4 the do express other markers of pluripotency. Given that these pluripotency markers are present and not other measures of pluripotency were provided to demonstrate “lack pluripotency”, the specification does not provide enabling guidance to demonstrate “lack pluripotency”.
State of the Art: The state of the art producing in vitro derived 3D organoid or spheroid structure comprising distinct embryonic cell population structuring such as gastrulation, ES-like cells, EPI-L cells and XEN-like cells is highly unpredictable. As Applicant notes in the response, the prior art of Beccari et al (previously made of record) describe gastrulated embryonic like spheroid structure comprises multiple embryonic cell types. However, Becarri’s structure miss a large portion of the nervous system, particularly brain derivative and the genes associated with either extraembryonic structures or anterior plate derivative were not or were poorly expressed (page 2, Figure 2).
Regarding “homogenous” pluripotent or multipotent cells, while these cells can be provided with a high degree of homogeneity using cells sorting and selection technology known in the prior art, a means to provide a 100% homogenous, pure pluripotent or multipotent cell population is still unpredictable and the rationale. Cahan and Daley (Nat Rev Mol Cell Biol 2013. 14(6):357-368. Authors manuscript pp. 1-23) report: “not all pluripotent cell lines are equal in their capacity to differentiate into desired cell types in vitro. Genetic and epigenetic variations contribute to functional variability between cell lines and heterogeneity within clones. These genetic and epigenetic variations could ‘lock’ the pluripotency network resulting in residual pluripotent cells or alter the signalling response of developmental pathways leading to lineage bias. The molecular contributors to functional variability and heterogeneity in both embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are only beginning to emerge… recent comparisons of pluripotent stem cells have uncovered molecular variations that correlate with their functional variability (defined as in vitro differentiation capacity). A pressing question is the extent to which variability and heterogeneity will affect the usefulness of pluripotent stem cells, regardless of the derivation method..” See page 1 and abstract. Cahan and Daley further describe factors that provide for variability such as loss of potency in a clonal cell line and factor that result in poor quality and stability of pluripotent stem cells. Many of these same factors also apply to multipotent stem cells.
Regarding differentiation of pluripotent and multipotent stem cells, Fowler et al (Wiley Interdisip Rev Dev Biol 2020 9(3):e368. Author’s Manuscript pp. 1-36) reports, “Too many choices can be problematic. This is certainly the case for human pluripotent stem cells (hPSCs): they harbor the potential to differentiate into hundreds of cell-types, yet it is highly challenging to exclusively differentiate hPSCs into a single desired cell-type. This review focuses on unresolved and fundamental questions regarding hPSC differentiation and critiquing the identity and purity of the resultant cell populations… While many in vitro differentiation protocols purport to “mimic development”, the exact number and identity of intermediate steps that a pluripotent cell takes to differentiate into a given cell-type in vivo remains largely unknown. Consequently most differentiation efforts inevitably generate a heterogeneous cellular population, as revealed by single-cell RNA sequencing and other analyses. The presence of unwanted cell-types in differentiated hPSC populations does not portend well… approaches to differentiate 3-dimensional “organoids” from hPSCs intentionally generate heterogeneous cell populations. While this is intended to mimic the rich cellular diversity of developing tissues, whether all such organoids are spatially organized in a manner akin to native organs (and thus, whether they fully qualify as “organoids”) remains to be fully resolved.” See abstract.
As such, the means of both providing a homogenous population and also directing differentiation of stem cells to multiple populations is hindered by variability and integrity of the starting cell type as well has their lack of a strict nature to direct to the desired cell fate predictably.
Regarding the use of any vertebrate species of cell, Fowler et al reports, “A common critique of current “developmentally-guided” hPSC differentiation efforts is that such strategies are not actually guided by human development; rather, they are informed by our knowledge of how model organisms such as mouse, frog, fish or chicken develop (as emphasized by this very review). Yet it is evident that a human is not a mouse. Human and mouse evolutionarily diverged tens of millions of years ago…, since which their exact mode of development—including the molecular identity of various embryonic progenitors and the signals that specify them—have diverged…” Fowler continues, stating, “This issue of evolutionary differences is not simply theoretical”, describing differences in timing, factors needed to support pluripotency and differentiation being different between human and mice and further detailing a hindered ability to provide germinal layer tissue cultures in species such as pigs, monkeys and human (p. abstract, 17-18).
As such, embryonic organoid techniques developed in mice do not predictably translate into techniques for other species of vertebrates and mammals.
Regarding SUMOylation inhibitors, Backett and Blagg (Curr Med Chem 2021. 28(20):3892-3912. Author’s Manuscript pp.1-33) teaches, “The enzymatic pathway of SUMOylation is very similar to ubiquitinylation and involves an activating enzyme, a conjugating enzyme, ligases and deconjugating enzymes. SUMOylation modulates the function of a number of proteins associated with various pathways.” See abstract. “As previously stated, the SUMOylation pathway is analogous to the ubiquitinylation pathway, with the primary difference being the number of isozymes identified for each step. 40 ubiquitin E2 enzymes have been identified in humans, however there is only one human SUMO E2 enzyme, Ubc9.3,15 Similarly, 79 functional deubiquitinating enzymes have been characterized as well as more than 600 ubiquitin E3 ligases, compared to six SENPs and ten SUMO E3 ligases.4,16 The number of enzymes required to regulate these pathways is related to the number of proteins modified by each post-translational process, as more than 10,000 substrates have been shown to be modified by ubiquitin, whereas 3,000–6,000 proteins have been identified as substrates for SUMOylation.17 Another key difference is that multiple isoforms of SUMO have been discovered as compared to the single ubiquitin polypeptide. Three isoforms of SUMO (SUMO1–3) are expressed in humans, and there are key differences among them. SUMO2 and SUMO3 share 97% sequence homology and are often denoted as SUMO2/3 as many detection methods cannot distinguish between them.3 Furthermore, it has been demonstrated that proteins that are SUMOylated with SUMO2/3 are generally associated with cellular stress.18 Whereas, SUMO1 is only 50% similar to SUMO2/3 and primarily modulates the physiological state of proteins.19 While all isoforms use the same enzymatic machinery, the underlying mechanisms that dictate the specificity between the isoforms remain unclear.” See page 2.
As such, teaching only one species of inhibitors that inhibit one specific enzyme (E1) in mouse pluripotent cells does not provide enablement guidance to the breadth of any agent inhibiting SUMO conjugation. The art teaches it is a multifactor pathway with divergent functions across various pathways. Further given the great degree of variability and unpredictability described in this type of organoid culture method, the description of an inhibitor of SUMO E1 enzyme to do predictably enable inhibitors of all SUMO cognates inhibitors.
Overall the state of the prior art in this field is highly unpredictable, leading to understandings that methods developed in one species of cell using one specific combinations of media and steps does not predictably arrive at the extrapolation to the broader elements as claimed.
Amount of Experimentation: A high degree exploratory experimentation to determine if a method within the confines of the claimed method would be applicable to any other cells species, to multipotent cells, to any media, using any inhibitor of SUMO conjugation. This level of experimentation goes beyond routine optimization experimentation and thus would be considered undue.
Thus the breadth of the claims is not enablement. The specification solely provide specific guidance to providing mouse pluripotent cells, cultured first in one specific ESC medium comprising serum and LIF, treatment with inibitors that inhibit one specific enxyme in the SUMOylation pathway, and producing one specific organoid to ES-like cells, EPI-L cells, and XEN-like cells with one specific marker express that denotes their characterization of each of these cell types. The prior art does not supplement the lack of enabling guidance by the specification for the broader embodiments. Further, the art teaches a high degree of unpredictability in embryonic development spheroid culture and application of the method to the broader embodiment would be undue because it goes far beyond routine optimization into determining IF the breadth method would predictable function.
No claims are allowed.
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MARCIA S. NOBLE
Primary Examiner
Art Unit 1632
/MARCIA S NOBLE/ Primary Examiner, Art Unit 1632
