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 .
Priority
The instant application is a national stage entry under 35 U.S.C. § 371 of PCT/US2023/013997 (filed 02/27/2023). Acknowledgement is made of Applicants’ claim for benefit of U.S. Application 63/314,958 (filed 02/28/2022).
Election/Restrictions
Applicant’s election without traverse of Group I in the reply filed on 07/21/2026 is acknowledged.
Claims 1-31 are pending in the instant application; claims 1-26 read on the elected invention.
Claim Objections
Claims 5-26 are objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim cannot depend from any other multiple dependent claim. See MPEP § 608.01(n). Accordingly, the claims have not been further treated on the merits.
Claim Interpretation
The following comments are made to establish broadest reasonable interpretation for the record.
Regarding claim 1: This claim is directed to a method for in vitro differentiation of pluripotent stem cells. As supported by the instant specification (par. 0044), the term pluripotent stem cell is interpreted as including e.g., induced pluripotent stem cells, embryonic stem cells.
Lines 5-6 of the instant claim recite, “…step-wise differentiating the cells into germ layers, progenitor cell types, and differentiated cell types…”. In the absence of clear guidance or definitions in the specification, this limitation is interpreted as: a first step comprising differentiation of the pluripotent stem cells to one or more germ layers (mesoderm, endoderm, and ectoderm); a second step comprising further differentiation of the cells to one or more progenitor cell types; and a third step comprising differentiation of the cells to a fully (e.g., terminally) differentiated cell type.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-4 are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Okazaki, et al. (US 2020/0102543), as evidenced by Dame, et al. (Methods Mol Biol. 2020), Red-Horse and Siekmann (Bioessays. 2019), and Bowen (“Microanatomy of the Digestive Tube.” 2001).
Okazaki, et al. (hereinafter Okazaki) teaches a gut organoid comprising a tissue structure that encloses a cavity (Abstract).
Regarding claims 1, 4: Okazaki teaches a method for the generation of self-organized gut organoids on a cell culture substrate on which cell-adhesive regions are patterned, comprising inducing gut morphogenesis via a cellular patterning-bulging phase and a self-morphogenesis phase, wherein human embryonic stem cells (hESCs) or human iPS cells (hiPSCs) are assembled on circular-patterned cell-adhesive regions on the glass surface of a cell culture substrate in differentiation induction medium; cells accumulate in the cell-adhesive regions and form hemispheric dome-like structures after 7 days in culture, followed by the subsequent formation of self-organized enlarged cavitated structures (par. 0182). These gut organoids have a complex tissue architecture generated via all three germ layers (par. 0193), as well as differentiated cells such as absorptive enterocytes, goblet cells, enteroendocrine cells, Paneth cells (par. 0184). The gut organoids also contain LGR5-expressing cells (par. 0185); as evidenced by Dame, et al., LGR5+ cells are actively cycling stem cells and give rise to differentiated cell lineages along the gastrointestinal tract (pg. 2; par. 1); i.e., LGR5-expressing cells are progenitor cells. Additionally, the differentiated cells (e.g., enterocytes) will have necessarily differentiated from cells of a germ layer via a progenitor cell.
Red-Horse and Siekmann evidences establishment of the vasculature follows unique developmental processes, guided by distinct mechanisms important for obtaining proper hierarchical structure and optimal organ function (pg. 2; par. 1). Although all hierarchical in nature, arteries in different organs and different organisms exhibit slightly different structures (pg. 2; par. 2); thus, Red-Horse and Siekmann evidences the organization and necessary structures for spatially organized and hierarchal branching of vessels will differ among organ systems.
Okazaki teaches H&E staining of the motile gut organoids show the gut architecture consists of mucosa and submucosa (Fig. 2B), wherein the intestinal mucosa is formed by the epithelium and lamina propria (par. 0184). As evidenced by Bowen, the submucosa contains blood and lymphatic vessels (pg. 1; par. 6); within the mucosa is the lamina propria, which contains blood vessels and lymphatics supplying the epithelium (pg. 2; par. 3). Thus, the mucosa comprising lamina propria and submucosa structures of the motile gut organoids of Okazaki read on the spatially organized and hierarchical branching of vessels limitation recited in claim 1.
Thus, the method of Okazaki anticipates:
the method for in vitro differentiation of pluripotent stem cells to vascularized organoids comprising spatially organized and hierarchical branching of vessels, the method comprising: initiating a culture with geometrically patterned pluripotent stem cells (PSC); and step-wise differentiating the cells into germ layers, progenitor cell types, and differentiated cell types with defined agents acting on differentiation pathways limitations recited in claim 1; and
the wherein the PSC are human cells limitation recited in claim 4.
Regarding claims 2-3: Following the above discussion, Okazaki states Figs. 1A-1D explain the generation of motile gut organoids from human pluripotent stem cells on a cell culture substrate on which cell-adhesive regions are patterned (par. 0172); Fig. 1B shows many patterned hydrophilic polymer-coated circular cell-adhesive regions 1.5 mm in diameter formed on a cell culture substrate (par. 0023). An embodiment is disclosed wherein a stencil mask is used (par. 0104), as well as an embodiment wherein the substrate is precoated with matrix to promote adhesion of the stem cells to cell-adhesive regions (par. 0114). This anticipates:
the wherein the geometric patterning of PSC comprises coating a specific region of from about 1 mm to about 10 mm on the surface of a culture vessel with a matrix, where the PSC are allowed to attach to the matrix at the initiation of culture limitation recited in claim 2; and
the wherein the coating step comprises adhering a stencil comprising a hole of from about 1 mm to about 10 mm, and pouring a matrix solution over the stencil limitation recited in claim 3.
Claims 1 and 4 are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Wilson (WO 2022/169684).
Wilson teaches hiPSC disease models to identify DNA mutations causing early life diseases (Abstract).
Regarding claims 1, 4: Wilson teaches a method comprising inducing stem cells, such as human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) to develop into a target tissue of interest; e.g., cardiac muscle (par. 009). In an embodiment, the hiPSC-derived organoid comprises cells from multiple lineages; e.g., the three embryonic germ layers differentiated from hiPSCs (par. 075). The organoid may further comprise vasculature (par. 075), blood vessels, and angiogenic vessel formation (par. 089); under broadest reasonable interpretation, this reads on the spatially organized and hierarchical branching of vessels limitation recited in claim 1.
In an embodiment, organoid differentiation comprises spatial patterning of the hESCs or hiPSCs (par. 075), wherein geometric confinement is sufficient to trigger self-organized patterning (par. 077). The method may comprise use of a chemically defined differentiation medium for differentiating the stem cells into cardiomyocytes, i.e., a fully differentiated cell type (par. 068); in an embodiment, the organoid comprises vascular precursor cells (par. 0112).
Thus, Wilson anticipates:
the method for in vitro differentiation of pluripotent stem cells to vascularized organoids comprising spatially organized and hierarchical branching of vessels, the method comprising: initiating a culture with geometrically patterned pluripotent stem cells (PSC); and step-wise differentiating the cells into germ layers, progenitor cell types, and differentiated cell types with defined agents acting on differentiation pathways limitations recited in claim 1; and
the wherein the PSC are human cells limitation recited in claim 4.
Claim Rejections - 35 USC § 103
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.
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 are rejected under 35 U.S.C. 103 as being unpatentable over Wilson (WO 2022/169684), in view of Sahni, et al. (J Vis Exp. 2016) and Werley, et al. (PLoS One. 2017).
The teachings of Wilson are set forth above; claims 1 and 4 are anticipated by the same.
Shani, et al. (hereinafter Sahni) teaches stencil micropatterning for human pluripotent stem cells (Abstract).
Werley, et al. (hereinafter Werley) teaches the effect of island geometry on action potential waveform and calcium dynamics in human induced pluripotent stem cells cultured in patterned islands of different sizes and shapes (Abstract).
Regarding claims 2-3: Following the above discussion, Wilson teaches an embodiment wherein the patterning is performed using a stencil, wherein the hiPSCs are seeded onto a previously deposited Matrigel™ matrix within stencil holes (par. 079). Wilson does not teach the remaining limitations recited in the instant claims.
However, Sahni teaches a polydimethylsiloxane (PDMS) stencil with features (i.e., holes) up to 2,000 µm for micropatterning hESCs (pg. 2; pars. 1-2).
Werley teaches a compliant polyacrylamide substrate doped with acryl-NHS, and patterns of fibronectin were transferred to the gel surface via a PDMS stamp, whereupon covalent reaction with the NHS irreversibly bonded the fibronectin to the gel; the bare polyacrylamide between the islands of fibronectin was highly cytophobic (pg. 7; par. 4). hiPSC-derived cardiomyocytes (hiPSC-CM) were seeded on square islands of edge length 50, 100, 250, 500 and 1000 µm; i.e., 0.05, 0.1, 0.25, 0.5, and 1 mm (pg. 8; par. 2). As island size increased, increases in the amplitudes of the Ca2+ and voltage transients and the upstroke velocity of the voltage were observed (starting at pg. 10; par. 3). In comparison to the 50 µm islands, in the 1 mm islands, Ca2+ transient amplitudes were 1.23-fold larger, voltage amplitudes were 1.81-fold larger, and the upstroke velocity of the voltage was 1.51-fold faster; these parameters are associated with increased functional maturity (pg. 11; par. 1).
Werley teaches the mechanism of island size-dependent maturation was investigated (pg. 13; par. 4); as optical electrophysiology parameters were similar between the 500 µm and 1 mm islands, confluent culture was used for ‘large islands’, while 100 µm islands were used for ‘small islands’ (pg. 12; par. 2). Region 1 comprised small islands plated far from their neighbors; region 2 comprised small islands coupled by a thin bridge to a large syncytium; region 3 comprised small islands separated by a thin gap from a bigger island; region 4 comprised small islands with both paracrine and electrical coupling to a nearby large island; and region 5 comprised cells embedded in a large syncytium (pgs. 14, 15). Cells grown in larger islands matured primarily through short-range interactions with neighboring cells; thus, nearest-neighbor contacts are important for the maturation of cardiomyocytes (pg. 15; par. 2).
Therefore, it would have been prima facie obvious to a person having ordinary skill in the art to have used the PDMS stencil of Sahni as the stencil for the spatial patterning of stem cells in the method of Wilson. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’. One would be motivated to do so for the increased amplitudes of the Ca2+ and voltage transients and the upstroke velocity of the voltage disclosed by Werley associated with increased functional maturity (pgs. 10, 11), as short-range interactions with neighboring cells is important for the maturation of cardiomyocytes (pg. 15). Further, Wilson discloses the use of a stencil (par. 079); Sahni discloses use of the PDMS stencil with human pluripotent stem cells (pg. 2); and Werley discloses use of the PDMS stamp with hiPSC-derived cardiomyocytes (pgs. 7, 8). Thus, the skilled artisan would have more than a reasonable expectation of success. This renders obvious:
the wherein the geometric patterning of PSC comprises coating a specific region of from about 1 mm to about 10 mm on the surface of a culture vessel with a matrix, where the PSC are allowed to attach to the matrix at the initiation of culture limitations recited in claim 2; and
the wherein the coating step comprises adhering a stencil comprising a hole of from about 1 mm to about 10 mm, and pouring a matrix solution over the stencil limitations recited in claim 3.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to GINA PRONZATI whose telephone number is (571)270-5725. The examiner can normally be reached Monday - Friday 9:00a - 5:00p ET.
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/GINA PRONZATI/Examiner, Art Unit 1633
/ALLISON M FOX/Primary Examiner, Art Unit 1633
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