Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
The amended claims filed on May 28, 2026, have been acknowledged. Claims 1 was amended. Claims 2-3, 5-9, 11, 13, and 15-36 were cancelled. Claims 45-47 are new. Claims 1, 4, 10, 12, 14, and 37-47 are pending and examined on the merits.
Applicant’s response has been considered. Rejections and/or objections not reiterated from the previous office action mailed December 1, 2025, are hereby withdrawn. The following rejections and/or objections are either newly applied or are reiterated and are the only rejections and/or objections presently applied to the instant application.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
Acknowledgment is made of Applicant’s claim for foreign priority under 35 U.S.C. 119(a)-(d).The applicant claims foreign priority from PCT/JP2019/012571 filed on March 25, 2019. While a certified copy of the foreign patent application PCT/JP2019/012571 is provided with the instant application, a certified English translation of said foreign patent application has not been provided.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1 and claims 45-47 are rejected under 35 U.S.C. 103 as being unpatentable over Risbud et al. (Spine 28: 2652-2659. 2003), Naqvi et al. (European Cells and Materials 37: 134-152. 2019; Published February 2019), Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota) Sakai et al. (Nature Communications 3: 1-11. 2012; referenced in IDS), and Wei-hong et al. (Chinese Journal of Tissue Engineering Research 18: 1718-1723. 2014), as evidenced by Sako et al. (Int. J. Mol. Sci. 22: 1-15. 2021). This is a new rejection made in response to Applicant’s amendments to claim 1. Any aspect of Applicant’s traversal that is relevant to the new rejection of record is addressed below.
Risbud teaches that young, male Wistar rats were killed with CO2 and the spinal columns were removed en bloc under aseptic conditions. Lumbar intervertebral discs comprising the vertebral end plates, anulus fibrosus, and nucleus pulposus were harvested and placed into multiwell tissue culture plates (Figure 1). The discs (i.e. non-digested tissue with an undestroyed tissue microenvironment) were maintained for 1 or 3 weeks in DMEM supplemented with 20% fetal calf serum, transforming growth factor-β1 (5 ng/mL), insulin-transferrin-selenium (10 μg/mL insulin, 5.5 μg/mL transferrin, and 0.5 ng/mL sodium selenite), antibiotics, and antimycotics with media changes being performed every 3 days. Additionally, NaCl was added to the DMEM to raise the osmolarity to 410 mOsm/kg. After incubation, nucleus pulposus cells were isolated (i.e. recovering the cultured Tie2+ stem cell population). (page 2653, column 1, paragraph 1-column 2, paragraph 2).
Risbud is silent regarding whether their nucleus pulposus explants were suspended in the culture medium.
However, Naqvi teaches that they collected intervertebral discs from mature bovine tails and cultured the discs comprising the nucleus pulposus for 28 d in 100 mL of high-glucose DMEM (HG-DMEM) supplemented with 10 % FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B, 1.5 mg/mL BSA and 40 μ g/mL L-proline under gentle agitation at 37 °C in normoxic (20 % oxygen) conditions, with medium changed twice weekly.
Wu teaches that a total of 10 NP [nucleus pulposus] tissue samples were obtained from patients who underwent microendoscopic discectomy for degenerative spine diseases. An explant culture method was employed to isolate NPSCs [nucleus pulposus stem cells] (Figure 6 shows that NPSCs express Tie2) from NP tissue. NP tissues were cut into 1 mm3 pieces and incubated at 37˚C in a 5% CO2 incubator without culture medium for 2 h to allow tissue attachment. Complete culture medium containing Dulbecco's Modified Eagle's Medium (DMEM)/F12 supplemented with 20% fetal bovine serum (FBS), 1% L‑glutamine and 1% penicillin‑streptomycin was added to the tissue culture dishes and incubated for an additional 17 days. The primary cells that had migrated out of the NP tissues and attached to dishes were passaged by a 2‑min treatment with 0.25% trypsin and 0.02% EDTA at 37˚C. The medium was replaced every 2 days. Cells were further passaged when they reached 80‑90% confluence.
The instant specification discloses that for each step at an "amplification culture stage", the culture medium was prepared by mixing 60 mL of DMEM (no glucose) and 40 mL of MEMa and by adding 20% of FBS immediately before use with/without basic FGF. The minced nucleus pulposus tissue was suspended in 3 mL of culture medium. Thereafter, the mixture was dispensed into one well of a 6-well culture dish (the culture surface was untreated), and cultured for 7 days (by WTC (whole tissue culture) method) (Specification paragraphs 0154-0156).
Although Risbud and Naqvi are silent regarding whether their intervertebral disc tissue is in suspension in their cultures, it is worth noting that they used similar media compositions to the one used in the instant specification and neither Risbud nor Naqvi identified treating their culture dishes to cause attachment of the tissue nor that their tissues were attached to the culture dish during the culture period. Furthermore, Naqvi teaches using 100 mL of their media with their explant tissue which would be enough media to allow for suspension culture to occur. Additionally, similar to the method of the instant specification, Wu teaches that nucleus pulposus tissue can be minced before explant culture and Sako evidences that human nucleus pulposus tissue minced into 3-5 mm pieces suspended in culture medium remain in suspension and not attached to the culture dish (which is also argued and identified in Applicant’s remarks on page 11, paragraphs 2-3). Therefore, although Risbud and Naqvi do not specifically state that they cultured their explant intervertebral disk tissue while suspended in their respective media compositions, one of ordinary skill in art would understand that minced nucleus pulposus tissue can be minced into 1 mm3 (smaller than the minced tissues of Sako) and placed in similar culture media as used by the Applicant at large volumes (up to 100 mL) that would lead to the nucleus pulposus tissue being in suspension. Therefore, it would have been well understood within the art that a suspension culture of explanted nucleus pulposus tissue could be used to culture the tissue for at least two weeks before isolating nucleus pulposus cells. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
The combined teachings of Risbud, Naqvi, and Wu do not teach wherein a Tie2 expression enhancer from the plant genus Cinnamomum is added to the culture media during culture of the non-digested tissue.
However, Ota teaches a method of culturing Tie2 positive progenitor cells with a Tie2 expression enhancer other than a growth factor. Baf3 cells, a murine pro [progenitor]-B cell line, Ba/F3 transduced with full-length murine TIE2(BaF/TIE2) were incubated in RPMI 1640 medium in the presence of IL-3 and 10% FCS. The cells were plated into a 6-well plate at 2x106 cells/1.5 mL/well and incubated overnight. A DMSO solution of the Cinnamomum cassia Blume hot water extract residue was added to the wells, followed by incubating for 10 minutes (column 8, lines 3-25). Although Ota teaches that the cinnamon extract activates TIE2, they do not specifically teach that the extract increases Tie2 expression, Figure 4 of Applicant’s specification shows that cinnamon extracts increase Tie2 expression. As such, the cinnamon extract of Ota is naturally a Tie2 expression enhancer. Finally, Applicant’s specification indicated that a “Tie2 expression enhancer” was known as a “Tie2 activator” in the art [0122], and Ota clearly demonstrated Tie2 phospho-activation.
Furthermore, Ota teaches that activation of Tie-2 is also known to induce a dormant state in cells other than vascular endothelial cells. For example, activation of Tie-2 in hematopoietic stem cells has been reported to induce dormancy in the hematopoietic stem cells. In other words, induction of Tie-2 makes it possible to maintain survival of hematopoietic stem cells in vitro for long periods of time (column 3, lines 35-45). Additionally, Ota teaches that the cinnamon extract Tie2 activator can be used as a drug for maintaining stem cells in vitro and in vivo by functioning as a therapeutic drug that induces quiescence in cancer cells (column 5, line 26-column 6, line 42).
Ota identifies angiotensin 1 as a known Tie-2 activation agent (column 5, lines 64-67) and that cinnamon extract was observed to cause phosphorylation of Tie-2 (i.e. Tie-2 activation) in the same manner as Angiotensin 1 (column 8, line 47-column 9, line 2).
Sakai teaches that soluble angiotensin 1 increases the rate of colony forming units (CFUs) of nucleus pulposus cells which was abolished when a Tie2 blocking antibody was added (page 5, column 2, paragraphs 2-3 and Figure 4). Therefore, although Tie2 activation causes the cells to enter a dormant state, they still undergo cell division and expansion and Tie2 activation actually increases the rate of cell division and expansion. Furthermore, Sakai teaches that Tie2 positive cells show the ability of self renewal which is lost with a decrease of Tie2 expression (abstract, Figure 6, and page 5, column 2, paragraph 5). Furthermore, Sakai shows a model of NP cells at different stages with Tie2 positive cells undergoing self-renewal in a dormant state (Figure 7).
Wu teaches that the expression of NP‑specific progenitor marker and NP cell marker Tie2, also referred to as CD 202b, is a cellular membrane receptor tyrosine kinase of the Tie family. Tie2 has been identified as a marker of NP precursor cells that were found to exhibit multipotency and self‑renewal capacity in animal and human NP. Wu teaches that Tie2 expression was tied to the rate of proliferation of nucleus pulposus cells in culture with higher proliferation rates when Tie2 expression is increased (H-NPSC cells) and lower proliferation when Tie2 expression is low (L-NPSC cells) (page 2200, column 2, paragraph 3 and Figures 6-7). This was also supported by the immunophenotypic results, which demonstrated an upregulation of the mature NP cell marker CD 24 in L‑NPSCs. NPSCs (i.e. L-NPSCs) from aged and degenerated NP tissues were associated with a low rate of proliferation and reduced differentiation potential, as well as downregulation of the NP progenitor marker Tie2 and higher expression of NP cell‑specific markers (page 2200, column 2, paragraph 3).
As such, enhancing Tie2 expression would cause the cells to adopt a more H-NPSC phenotype.
Furthermore, Wei-hong teaches that they cultured anulus fibrosis cells isolated from Sprague-Dawley rats in vitro with Cinnamon Twig Decoction Plus Pueraria and found that the Cinnamon Twig Decoction Plus Pueraria can repair the degeneration of anulus fibrosus cells (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the method of culturing nucleus pulposus tissue in suspension of the combined teachings of Risbud, Naqvi, and Wu with the method of culturing Tie2+ progenitor cells with a “Tie2 expression enhancer” from cinnamon bark hot water extract of Ota to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to combine with a reasonable expectation of success because Wu teaches that low expression of Tie2 was associated with a low rate of proliferation and reduced differentiation potential of NPSCs and Ota teaches that the cinnamon extract causes activation of Tie2 which has been reported to induce dormancy in the hematopoietic stem cells and cancer cells and Sakai showed that Tie2 activation through angiotensin 1 (Ota showed that cinnamon extract was observed to cause phosphorylation of Tie-2 (i.e. Tie-2 activation) in the same manner as Angiotensin 1) increases the rate of colony forming units (CFUs) of nucleus pulposus cells which was abolished when a Tie2 blocking antibody was added. Furthermore, Wei-hong identifies that Cinnamon Twig Decoctions have already been used with the related anulus fibrosus cells as a way of treating intervertebral disc degeneration. As such, it would have been obvious to combine the cinnamon bark extract of Ota with a culture of nucleus pulposus tissue to maintain/enhance Tie2 expression in the nucleus pulposus stem cells to maintain an H-NPSC phenotype and prevent their differentiation. As Cinnamon extracts have already been identified as a possible treatment option for intervertebral disk degeneration and have been cultured with anulus fibrosus cells, it would have also been obvious that cinnamon extracts could be cultured with nucleus pulposus cells, as well. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Response to Arguments
Applicant's arguments filed May 28, 2026, are acknowledged.
Applicant argues that the pending application provides unexpected results by demonstrating that the number of Tie2 positive NPSCs is effectively increased by culturing nucleus pulposus tissue extracts with cinnamon extract as shown in Figure 4. In contrast, Ota discloses that when the cinnamon extract is applied to Tie2-positive stem/progenitor cells derived from the NP tissue of the intervertebral disc, those cells in which Tie2 expression is enhanced should be induced to a dormant state. In fact, the Office continues to acknowledge that Ota discloses activation of Tie-2 is known to induce a dormant state in cells and can be used as a drug for maintaining stem cells in vitro and in vivo by functioning as a therapeutic drug that induces quiescence in cancer cells. Ota at col. 5, line 26 to column 6, line 42. Therefore, it would be counterintuitive for a PHOSITA to use cinnamon extract with the specific intent to increase proliferation and amplification. Furthermore, the model of Sakai also shows Tie2 positive cells undergoing self-renewal in a dormant state and would not be equated to significant amplification as disclosed in the pending application. Therefore, a person having ordinary skill in the art viewing the teachings of Ota and Sakai would have expected cinnamon extract to hinder proliferation and amplification, instead of increase amplification. Applicant also cites to “Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells” to define the term WTC. Applicant asserts the article has been included with the response (although this article does not appear to be attached with the remarks nor included in the IDS, it has been considered) (page 10, paragraph 1-page 11, paragraph 3).
Applicant's arguments and the cited art have been fully considered but they are not persuasive.
Although Applicant argues unexpected results, the claims are not commensurate in scope with the experimental results cited by the Applicant. MPEP 716.02(d) discloses that whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980) (Claims were directed to a process for removing corrosion at "elevated temperatures" using a certain ion exchange resin (with the exception of claim 8 which recited a temperature in excess of 100°C). Appellant demonstrated unexpected results via comparative tests with the prior art ion exchange resin at 110°C and 130°C. The court affirmed the rejection of claims 1-7 and 9-10 because the term "elevated temperatures" encompassed temperatures as low as 60°C where the prior art ion exchange resin was known to perform well. The rejection of claim 8, directed to a temperature in excess of 100°C, was reversed.). See also In re Peterson, 315 F.3d 1325, 1329-31, 65 USPQ2d 1379, 1382-85 (Fed. Cir. 2003) (data showing improved alloy strength with the addition of 2% rhenium did not evidence unexpected results for the entire claimed range of about 1-3% rhenium); In re Grasselli, 713 F.2d 731, 741, 218 USPQ 769, 777 (Fed. Cir. 1983) (Claims were directed to certain catalysts containing an alkali metal. Evidence presented to rebut an obviousness rejection compared catalysts containing sodium with the prior art. The court held this evidence insufficient to rebut the prima facie case because experiments limited to sodium were not commensurate in scope with the claims.).
Regarding the claims at issue in the instant application, claim 1 claims an amplification method comprising: culturing the cell population containing Tie2-positive stem/progenitor cells while present in a non-digested tissue and in an undestroyed tissue microenvironment, and while the tissue is suspended in a culture medium during an entirety of a culture period, the culture medium containing, as a Tie2 expression enhancer, an extract derived from a plant of the genus Cinnamomum; and recovering the cultured cell population containing the Tie2-positive stem/progenitor cells. The cited data from Figure 4 is different than what is claimed as there is a two part amplification (as identified in Table 2) comprising a first 14 day amplification using cinnamon extract followed by a 7 day amplification using basic FGF. Applicant does not cite any data that uses a single step of culturing nucleus pulposus tissue with cinnamon extract that lead to the purported unexpected results.
As such, the claims as written are not commensurate in scope with alleged unexpected results.
Additionally, it is not clear that these results would be unexpected as Sakai teaches that soluble angiotensin 1 increases the rate of colony forming units (CFUs) of nucleus pulposus cells which was abolished when a Tie2 blocking antibody was added (page 5, column 2, paragraphs 2-3 and Figure 4). Therefore, Tie2 activation is known to increase the rate of cell division and expansion. As such, using the cinnamon extract of Ota that is known to activate Tie2 expression would also be expected to increase the rate of cell division and expansion of Tie2 positive nucleus pulposus cells. Thus, it is not surprising that an amplification culture using a cinnamon extract that is a known Tie2 activator would increase the rate of cell division and expansion and amplify Tie2 expression.
Regarding Applicant’s arguments that one of ordinary skill in the art would have expected that the addition of cinnamon extract would have led to a dormant state that is not conducive to increase the proliferation and amplification of Tie2 positive cells in the nucleus pulposus tissue, this argument does not address the results from Sakai that are cited in the rejection above showing that that soluble angiotensin 1 increases the rate of colony forming units (CFUs) of nucleus pulposus cells which was abolished when a Tie2 blocking antibody was added (page 5, column 2, paragraphs 2-3 and Figure 4). These results show that Tie2 activation is known to increase the rate of cell division and expansion. As Ota identifies angiotensin 1 as a known Tie-2 activation agent (column 5, lines 64-67) and that cinnamon extract was observed to cause phosphorylation of Tie-2 (i.e. Tie-2 activation) in the same manner as Angiotensin 1 (column 8, line 47-column 9, line 2), one of ordinary skill in the art would understand that the cinnamon extract of Ota should exhibit similar increases in the rate of colony forming units (CFUs) of nucleus pulposus cells as it has a similar mechanism of action as angiotensin 1. Therefore, it would have been obvious to use the cinnamon extract of Ota to increase the rate of cell division and expansion of Tie2+ nucleus pulposus cells.
Claims 1 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Risbud et al. (Spine 28: 2652-2659. 2003), Naqvi et al. (European Cells and Materials 37: 134-152. 2019; Published February 2019), Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota) Sakai et al. (Nature Communications 3: 1-11. 2012; referenced in IDS), and Wei-hong et al. (Chinese Journal of Tissue Engineering Research 18: 1718-1723. 2014) as applied to claim 1 above and further in view of Chan et al. (The Spine Journal 10: 486–496. 2010). This is a new rejection made in response to Applicant’s amendment to claim 1. Applicant’s traversal has been addressed above.
The teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong are as discussed above.
The combined teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong do not teach wherein the non-digested tissue is a tissue obtained by thawing a cryopreserved tissue.
Chan teaches that they collected bovine caudal intervertebral discs (IVDs). Discs were kept for 24 hours in excess procion red solution (1% in PBS) under free-swelling condition, then snap frozen in liquid nitrogen (i.e. cryopreserved), dehydrated via -80°C acetone, and brought stepwise back to room temperature. Sections of 200 μm were cut with a rotating blade diamond saw. From each disc, the three most midsagittal sections including the nucleus pulposus were ground and polished on a micro grinding system to approximately 100 μm thickness. The fresh discs were cryopreserved in 20 mL high glucose (4.5 g/L) DMEM, containing 20 mM HEPES, 10% fetal bovine serum, 10% dimethyl sulfoxide, and 10% glycerol, with stepwise freezing and kept in liquid nitrogen for at least 1 week and up to 2 months before thawing for culturing (page 487, column 2, paragraph 2-page 488, column 1, paragraph 2). After thawing the frozen discs for 24 hours in the bioreactor, and culturing the discs for 7 days to examine cell viability and gene expression (page 488, column 1, paragraph 4). Chan teaches they confirmed the presence of viable cells in different compartments of the cryopreserved IVD, and these cells were maintained over the 7-day culture (page 495, column 1, paragraph 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined with the method of culturing nucleus pulposus tissue of the combined teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong with the cryopreservation of IVD tissue method of Chan to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to combine with a reasonable expectation of success because Chan successfully reduces to practice that IVD tissue comprising the nucleus pulposus can be cryopreserved while maintaining cell viability within the tissue following cryopreservation. Furthermore, Chan teaches that cryopreserved allogeneic intervertebral disc transplantation relieved pain and preserved motion, thus opening up a new treatment option for degenerative disc disease (abstract). Cryopreservation allows for tissue to remain viable for treatment for longer periods of time compared to fresh tissue. As such, as Chan teaches that cryopreserved IVD tissue maintains viable cells and has been successfully used as a treatment, it would have been obvious to use cryopreserved tissue to extend the time at which the tissue remains viable. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1, 10, 12, 14-15, and 39-41 are rejected under 35 U.S.C. 103 as being unpatentable over Risbud et al. (Spine 28: 2652-2659. 2003), Naqvi et al. (European Cells and Materials 37: 134-152. 2019; Published February 2019), Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota) Sakai et al. (Nature Communications 3: 1-11. 2012; referenced in IDS), and Wei-hong et al. (Chinese Journal of Tissue Engineering Research 18: 1718-1723. 2014), as applied to claim 1 above, and further in view of United States Patent Application No. 2003/0220692 (Shapiro). This is a new rejection made in response to Applicant’s amendment to claim 1. Applicant’s traversal has been addressed above.
Regarding claim 10, the teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong are as discussed above. Wu teaches a method of differentiating the NPSCs. Specifically, in one embodiment, Wu teaches the L-NPSCs are differentiated into NP cells (page 2198, column 2, paragraph 2 and Figure 6).
The combined teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong do not teach wherein the differentiation methods using cultureware having undergone cell attachment-increasing treatment.
Shapiro teaches a method of culturing precursor cells under conditions effective to cause the precursor cells to differentiate into nucleus pulposus cells. The precursor cells are cultured in a medium comprising fibronectin. The cells attach to the carrier through the interaction of fibronectin with integrin receptors located on the nucleus pulposus and precursor cell surfaces. Fibronectin is selectively adsorbed by the calcium phosphate layer that forms on the bioactive glass carrier. Fibronectin binds to hyaluronic acid, which in turn binds the CD44 receptors present on the surfaces of nucleus pulposus cells and precursor cells, thus serving to attach the cells to the surface-modified bioactive glass (paragraph 0076 and claims 63 and 80).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the method of differentiating NPSCs of Wu by using treated cultureware dishes to increase cell attachment and differentiate cells into nucleus pulposus cells, as identified by Shapiro, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because Wu successfully reduces to practice that nucleus pulposus stem cells can be differentiated into nucleus pulposus cells (NP cells) after isolating them from the nucleus pulposus tissue and Shapiro has successfully reduced to practice that treated culture dishes, such as with fibronectin, can be used for differentiating NPSCs into nucleus pulposus cells, and would have been obvious because these are adherent cells. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claims 12 and 14, Shapiro, as stated supra, teaches they used fibronectin to coat the carrier (paragraph 0076 and claims 63 and 80).
Regarding claim 39-40, Shapiro and Wu teach that nucleus pulposus cells express collagen type II (paragraph 0118 of Shapiro and page 2198, 2nd to last paragraph of Wu).
Regarding claim 41, as there is no time frame associated with the differentiation method of claim 10, the prepared cell population would inherently include differentiated target cells and undifferentiated Tie2 progenitor cells over the course of the differentiation culturing period as not all cells would immediately differentiate at the start of the culturing period.
Claims 1, 37-38, and 42-44 are rejected under 35 U.S.C. 103 as being unpatentable over Risbud et al. (Spine 28: 2652-2659. 2003), Naqvi et al. (European Cells and Materials 37: 134-152. 2019; Published February 2019), Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota) Sakai et al. (Nature Communications 3: 1-11. 2012; referenced in IDS), and Wei-hong et al. (Chinese Journal of Tissue Engineering Research 18: 1718-1723. 2014), as applied to claim 1 above, and further in view of United States Patent Application No. 2003/0220692 (Shapiro), van den Akker et al. (Arthritis Research & Therapy 16: 1-16. 2014), van den Akker et al. (BMC Musculoskeletal Disorders 17:1-13. 2016), and Baraniak et al. (Journal of the Mechanical Behavior of Biomedical Materials II: 63-71. 2012). This is a new rejection made in response to Applicant’s amendment to claim 1. Applicant’s traversal has been addressed above.
Regarding claims 37-38, the teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong are as discussed above. Wu teaches a method of differentiating the NPSCs into nucleus pulposus cells (page 2198, column 2, paragraph 2 and Figure 6).
The combined teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong do not teach wherein the differentiation method uses a culture medium containing an extracellular matrix-degrading agent.
Shapiro teaches a method of culturing precursor cells under conditions effective to cause the precursor cells to differentiate into nucleus pulposus cells. The precursor cells are cultured in a medium comprising fibronectin. The cells attach to the carrier through the interaction of fibronectin with integrin receptors located on the nucleus pulposus and precursor cell surfaces. Fibronectin is selectively adsorbed by the calcium phosphate layer that forms on the bioactive glass carrier. Fibronectin binds to hyaluronic acid, which in turn binds the CD44 receptors present on the surfaces of nucleus pulposus cells and precursor cells, thus serving to attach the cells to the surface-modified bioactive glass in a single layer (paragraphs 0076 and 0118 and claims 63 and 80). Shapiro teaches that the preferred bioactive molecule on the glass includes TGF-β (paragraph 0082).
Akker (2014) teaches that they generated cell cultures of immortalized nucleus pulposus mesenchymal stem cell-like cells (page 9, column 1, paragraph 1-column 2, paragraph 1). Akker teaches that NP-R (NP-responder; more immature stem cell phenotype) cells cultured on ACAN coated culture dishes (an attachment enhancing coating) showed poor attachment to the dish and readily formed floating spheroids within 24 hours (page 11, column 2, paragraph 2-page 12, column 12, paragraph 1 and Figure 6). Akker (2016) teaches that TGF-β stimulation increases collagen type II expression in NP-R cells (Figure 2).
Baraniak teaches a method of dissociating mesenchymal stem cell spheroids using a trypsin–collagenase–dispase solution coupled with mechanical agitation (page 65, column 1, paragraph 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of differentiating NPSCs of the combined teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong by differentiating the nucleus pulposus progenitor cells into nucleus pulposus cells using a culture medium containing an extracellular matrix-degrading agent, as identified by Shapiro and Baraniak, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Wu successfully reduces to practice that nucleus pulposus stem cells can be differentiated into nucleus pulposus cells (NP cells) after isolating them from the nucleus pulposus tissue and Shapiro teaches that they used treated culture dishes with fibronectin to cause attachment of the nucleus pulposus progenitor cells for differentiation on bioactive glass, such as with TGF-β. As Akker (2014) teaches that immature nucleus pulposus cells, such as nucleus pulposus progenitor cells, spontaneously generate spheroids during culture with an attachment enhancing coating, it would have been obvious to try to prevent spheroid formation to ensure that the cells stay dissociated and attach to the dish as a single layer for differentiation. Baraniak teaches that they used a trypsin-collagenase-dispase solution to dissociate mesenchymal stem cell spheroids (nucleus pulposus precursor cells are MSC-like). As such, it would have been obvious to use this kind of a solution to prevent spheroid formation. As Akker (2016) teaches that anabolic stimulating factors, such as TGF-β, cause collagen type II expression to increase, it would have been obvious to use a collagenase that targets collagen type II. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claims 42-43, Shapiro teaches that nucleus pulposus cells express collagen type II (paragraph 0118).
Regarding claim 44, as there is no time frame associated with the differentiation method of claim 37, the prepared cell population would inherently include differentiated target cells and undifferentiated Tie2 progenitor cells over the course of the differentiation culturing period as not all cells would immediately differentiate at the start of the culturing period.
Claims 1, 37-38, 42 and 44, are rejected under 35 U.S.C. 103 as being unpatentable over Risbud et al. (Spine 28: 2652-2659. 2003), Naqvi et al. (European Cells and Materials 37: 134-152. 2019; Published February 2019), Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota) Sakai et al. (Nature Communications 3: 1-11. 2012; referenced in IDS), and Wei-hong et al. (Chinese Journal of Tissue Engineering Research 18: 1718-1723. 2014), as applied to claim 1 above, and further in view of Almalki et al (Stem Cell Research & Therapy 7:1-12. 2016). T This is a new rejection made in response to Applicant’s amendment to claim 1. Applicant’s traversal has been addressed above. This rejection addresses the breadth of the extracellular matrix-degrading agent and is cited solely for this purpose.
Regarding claims 37-38, the teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong are as discussed above. Wu teaches a method of differentiating the NPSCs (expresses MSC markers; page 2193, column 1, paragraph 1) into chondrocytes.
Wu does not teach wherein the differentiation method uses a culture medium containing an extracellular matrix-degrading agent.
Almalki teaches the differentiation of MSCs is promoted by specific MMPs and/or TIMPs associated with a specific cell lineage. MMP-13 and MT-1MMP, in addition to MMP-2 and MMP-9, play a key role in the differentiation of MSCs to chondrocytes (page 9, column 2, paragraph 2). Almalki teaches that MMP-13 was found to have an important role in the later stages of chondrogenic differentiation of MSCs by degrading the main components of the cartilaginous matrix, aggrecan and type II collagen (page 5, column 1, paragraph 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of differentiating NPSCs into chondrocytes of the combined teachings of Risbud, Naqvi, Wu, Ota, Sakai, and Wei-Hong by differentiating the progenitor cells into chondrocytes using a culture medium containing an extracellular matrix-degrading agent such as MMP13 (which is known to degrade type II Collagen), as identified by Almalki, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Wu successfully reduces to practice that nucleus pulposus stem cells can be differentiated into chondrocytes after isolating them from the nucleus pulposus tissue and Almalki teaches that MMP-13 and MT-1MMP, in addition to MMP-2 and MMP-9, play a key role in the differentiation of MSCs to chondrocytes. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
In regard to claim, 42, chondrocytes express type II collagen
In regard to claim 44. as there is no time frame associated with the differentiation method of claim 37, the prepared cell population would inherently include differentiated target cells and undifferentiated Tie2 progenitor cells over the course of the differentiation culturing period as not all cells would immediately differentiate at the start of the culturing period.
Withdrawn Double Patenting
The prior double patenting rejections have been withdrawn in light of Applicant’s amendments to the claims of the 17/921,180 co-pending application to recite that they are culturing articular cartilage derived Tie2 positive cells.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KEENAN A BATES whose telephone number is (571)270-0727. The examiner can normally be reached M-F 7:30-5:00.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Doug Schultz can be reached on (571) 272-0763. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KEENAN A BATES/Examiner, Art Unit 1631
Prosecution Insights