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 .
Applicant's amendments to the claims filed on 10-16-2025 and Applicant's arguments filed on 05-15-2026 have been received and entered. Claims 2-3 have been amended. Claims 1-19 are pending.
This is a Non-Final office action.
Election/Restrictions
Applicant’s election without traverse of Group II (claims 2, 3, and 17) in the reply filed on 04-08-2024 is acknowledged.
Claims 1, 4-16, 18-19 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 04-08-2024.
Claims 2, 3, and 17 are under consideration.
Priority
This application is a 371 of PCT/US2019/057929 filed on 10/24/2019 that claims priority from US provisional application 62/749,947 filed on 10/24/2018.
Withdrawn- Claim Rejections - 35 USC § 103
Claims 2-3, 17 are rejected under 35 U.S.C. 103 as being unpatentable over Lu et al (Regen. Med. (2013) 8(4), 413–424, https://doi.org/10.2217/rme.13.36) (Applicant own work) in view of Amit et al (Pub. No.: US 2019/0284526 A1, Provisional application No .62/363. Upon further consideration, the previous rejections of claims are hereby withdrawn. Applicants' arguments with respect to the withdrawn rejections are thereby rendered moot. The claims are however subject to new rejections over the prior art of record, as set forth below.
New - 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 2-3, 17 are rejected under 35 U.S.C. 103 as being unpatentable over Lu et al (Regen. Med. (2013) 8(4), 413–424, https://doi.org/10.2217/rme.13.36) (Applicants’ own work) in view of Abbasalizadeh et al (Tissue Eng Part C Methods . 2012 Nov;18(11):831-51. doi: 10.1089/ten.TEC.2012.0161. Epub 2012 Jun 13.)
Claims interpretation:
The specification of the claimed invention teaches that the term "sphere" or "spheroid" means a three-dimensional spherical or substantially spherical cell agglomerate or aggregate (Page 12, lines 12-13). Since aggregate is clump of cells, cell clumps are interpreted as spheres.
According to the specification of the claim invention: "Hemogenic endothelial cells" refers to cells differentiated in vitro from PSCs that acquire hematopoietic potential and can give rise to multilineage hematopoietic stem and progenitor cells (line 10-11, page 15). Therefore, hemogenic endothelial cells (HECs) are interpreted as endothelial cells derived from PSCs that have the potential to become a hematopoietic stem and progenitor cells.
According to step (b) of claims 2-3, second spheres are interpreted as first spheres with PSCs being differentiated into HECs.
According to the specification of the claim invention: Scaffolds mimic the extracellular matrix of the native tissue, recapitulate the in vivo milieu and allow cells to influence their own microenvironments (line 34-35, page 11) …. the scaffold can be selected to mimic the in vivo niche to promote lineage specification such as NK cells, T lymphocytes, etc. (line 10-11, page 12). Therefore, “Scaffolds” in claims 2-3 are interpreted as Matrigel matrix which contains extracellular matrix or feeder-free culture systems.
Regarding to preamble of claims 2, 3, Lu et al teaches an efficient method to reproducibly generate large numbers of hemangioblasts (progenitor of hematopoietic cell lineages) from multiple hESC lines using an in vitro feeder and serum-free differentiation system. These cells can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column).
Regarding to claims 2(a) and 3(a), Lu et al teach hESCs and iPSCs were grown on feeder-free culture, both hESCs and iPSCs were plated and grown on Matrigel in mTeSR1 medium (Page 414, right column, 2nd para.). Once collected, uniform cell clumps were created by gently pipetting the cell suspension to form clumps less than 100 µm in diameter (Page 414, right column, last para.).
However, Lu et al does not specifically teaches the use of carrier-free 3-dimensional (3D) sphere culturing under continuous agitation while monitoring sphere size, wherein the first spheres have an average size of about 60-150 micrometers. Abbasalizadeh et al cure the deficiency.
Abbasalizadeh et al teach “bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor” (title). Abbasalizadeh et al teach “The cell lines were passaged and maintained in a microcarrier-free suspension culture as three-dimensional cell aggregates based on our previously established protocol” (Page 832, right column, 2nd para.) and “Single-cell inoculated dynamic culture and impact of enzymatic dissociation medium type on hPSC expansion rate …… Cell aggregates were treated with ROCKi 1 h prior to enzymatic dissociation. The bioreactor was agitated at 35 rpm (increased to 40 rpm after 24 h) and medium refreshing began after 48 h of culture with the same medium and no ROCKi. After 24–48 h, small cell aggregates (40– 80 mm) were observed in the bioreactor for both cell lines; aggregate sizes gradually increased due to cell division during the 10 days of culture (140–200 mm)” (Page 834, right column, last para.). Abbasalizadeh et al teach “Mass production of size-controlled cell aggregates in suspension dynamic condition. After optimization of single-cell inoculation and passaging conditions, we employed different combinations of agitation rates (35–60 rpm) and medium viscosities ……. to generate homogenous size-controlled aggregates of hPSCs in a dynamic suspension culture” (Bridging paragraph between page 836 to 837).
Thus, it is indicating that sphere culturing under continuous agitation while monitoring sphere size was recognized in the prior art to be a result-effective variable. A person of ordinary skill in the art would have been motivated to perform the monitoring sphere size a plurality of times to obtain desired sphere size out of the course of routine optimization.
Abbasalizadeh et al teach “1.9 x 109 undifferentiated hPSCs could be generated within 30 days in four spinner flasks that contain 200 mL of HFF-CM with the use of this culturing strategy and scale-up platform (Fig. 11).”
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Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Lu et al by using carrier-free sphere culturing under continuous agitation while monitoring sphere size as taught by Abbasalizadeh et al as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated to do so because of the following reasons:
One of ordinary skill in the art would be motivated to culture pluripotent stem cells in carrier-free and feeder-free sphere suspension culture under continuous agitation while monitoring sphere size because Abbasalizadeh et al teach that “The generation of size-controlled aggregates is a very important parameter that should be taken into consideration in bioprocess development. Homogenous aggregate formation will facilitate and expedite the scale-up process due to the more effective enzymatic dissociation and subsequently lower cell loss after each passaging, higher expansion rates, and finally higher pluripotency and differentiation potentials. Here, we have shown that size-controlled aggregates could be generated by using precise control of the agitation rates and addition of shear protectants. Our proposed method is more scalable than other technologies developed for cell aggregate size control, such as microwell mediated control, hanging drops, and microprinting technologies. In addition, we have demonstrated that the hydrodynamic culture condition of a bioreactor is a very important parameter for hPSC culture scale-up because of the very high sensitivity of hPSCs to shear stress. Importantly, increasing the agitation rates to improve nutrient delivery and homogenizing culture environment and subsequent aggregate formation should be well balanced in conjunction with limiting shear stress in dynamic conditions to minimize adverse effect on cell proliferation and pluripotency” (Page 844, left column, 3rd para. to right column).
One of ordinary skill in the art would be motivated to perform carrier-free suspension cultures instead of culture of cells adhered on the microcarriers because Abbasalizadeh et al teach that “Cell attachment of the microcarriers has been improved by surface modification or coating with undefined animal-derived or defined extracellular matrices, such as Matrigel, or synthetic peptides. However, microcarrier based cultures still suffer from critical scale-up challenges, such as massive cell loss due to difficulties with cell seeding and harvesting during passages, in addition to the inefficiency of these culture systems to expand undifferentiated cells over a prolonged time. Different protocols exist for scalable, carrier-free suspension cultures of undifferentiated hPSCs as cell aggregates for an extended time, while maintaining their self-renewal capabilities. This has been achieved by the successful single-cell inoculated suspension culture of hPSCs using a combination of conditioned or commercially defined medium (e.g., mTeSR; Stemcell Technologies), mostly supplemented with basic fibroblast growth factor (bFGF) and Rho kinase inhibitor (ROCKi) treatment” (Page 831, left to right column).
One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Abbasalizadeh et al provides proof of principle for culturing pluripotent stem cells by sphere culturing under continuous agitation while monitoring sphere size and Abbasalizadeh et al were successful in generating human pluripotent stem cells for large-scale production in a pluripotent state when cultured in a suspension culture devoid of substrate adherence.
Regarding to claims 2(b) and 3(b), Lu et al teaches Hemangioblast differentiation: Hemangioblast differentiation occurs via a two-step process. The first process is the 3.5-day EB stage, and at day 3.5–4 EBs were trypsinized. Day 7 hemangioblasts were collected and analyzed (Page 415, bridging left – right column). Further, Lu et al also teaches differentiation of hemangioblasts Figure 6: endothelial cell differentiation of hemangioblasts (hemogenic endothelial cells (HECs)) derived from human embryonic stem cells maintained on microcarriers (Page 420).
As mentioned above, Abbasalizadeh et al teach that “The generation of size-controlled aggregates is a very important parameter that should be taken into consideration in bioprocess development. Homogenous aggregate formation will facilitate and expedite the scale-up process due to the more effective enzymatic dissociation and subsequently lower cell loss after each passaging, higher expansion rates, and finally higher pluripotency and differentiation potentials. … that size-controlled aggregates could be generated by using precise control of the agitation rates and addition of shear protectants ….. Importantly, increasing the agitation rates to improve nutrient delivery and homogenizing culture environment and subsequent aggregate formation should be well balanced ….” (Page 844, left column, 3rd para.). Thus, a person of ordinary skill in the art would be motivated to perform precise control of the agitation rates to induce differentiation of the PSCs as taught by Abbasalizadeh et al.
Regarding to claims 2(c) and 2(d), Lu et al teaches endothelial cell culture & differentiation from hemangioblasts: Day 7 hemangioblasts were plated on fibronectin-coated dishes in endothelial cell growth medium. After 4 h, the media was changed and nonadherent cells were removed from culture. The media was changed daily. After 1 week, cells were dissociated with trypsin and collected. Cell number was determined and 125,000 cells were plated in 300 µl Matrigel in a four-well plate to determine angiogenesis functionality. (Page 415, right column, 3rd para.).
Regarding to claims 2(e) and claim 3 (c), Lu et al teach endothelial cell culture & differentiation from hemangioblasts: Day 7 hemangioblasts were plated on fibronectin-coated dishes in endothelial cell growth medium and the media was changed (Page 415, right column, 3rd para) and Hemangioblasts can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column). Lu et al teach that hematopoietic colony-forming erythroid cells, colony-forming granulocytes, colony-forming macrophages and colony-forming multilineage cells were formed 10–12 days after plating these cells in serum-free methylcellulose media containing a spectrum of hematopoietic cytokines (Figure 5). These adherent cells took up acetylated low-density lipoprotein and formed capillary vascular-like structures that also took up acetylated low-density-lipoprotein after replating on the Matrigel matrix for less than 24 h (Figure 6), confirming their endothelial lineage (Page 420, right column, 2nd para). These cells can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column).
Additionally, Abbasalizadeh et al teach homogenous size-controlled hPSC aggregates differentiation in carrier-free culture: “we evaluated the in vitro differentiation potential of hESC and hiPSC lines cultured in xeno-free dynamic suspension by replacing CM with non-CM, following prolonged expansion ….. Thus, we have shown that the developed scale-up platform can be used for mass production of homogenous size-controlled pluripotent hPSC aggregates in stirred suspension bioreactors under xeno-free conditions” (Page 840, right column, 1st para.). Thus, in view of cited references, it would be a matter of design choice for one of ordinary skill in the art to culture the HECs in carrier-free suspension cultures and differentiate into lymphoid lineage cells while permitting the lymphoid lineage cells to release (for claim 3 (c)).
Regarding to claim 17, Lu et al teaches the cells can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column, 2nd para.)
Conclusion
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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