Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on August 21, 2025, has been entered.
DETAILED ACTION
The amended claims filed on June 30, 2025, have been acknowledged. Claims 1 and 15 were amended. Claims 2-3, 5-9, 11, 13, and 17-36 were cancelled. Claims 37-44 are new. Claims 1, 4, 10, 12, 14-16, and 37-44 are pending and examined on the merits.
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.
Withdrawn Duplicate Claims, Warning
The prior objection to claim 15 under 37 CFR 1.75 as being a substantial duplicate of claim 10 is withdrawn in light of Applicant’s amendments to claim 15 to clarify the difference in the method steps of claims 10 and 15.
Withdrawn Claim Rejections - 35 USC § 102
The prior rejection of claims 1 and 3 under 35 U.S.C. 102(a)(1) as being anticipated by Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018) is withdrawn in light of Applicant’s amendments to claim 1 to require that the culture medium comprises an extract from a plant of the genus CInnamomum.
Withdrawn Claim Rejections - 35 USC § 102
The prior rejection of claims 1 and 4 under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018) as applied to claim 1 above and further in view of Chan et al. (The Spine Journal 10: 486–496. 2010) is withdrawn in light of Applicant’s amendments to claim 1 to require that the culture medium comprises an extract from a plant of the genus CInnamomum.
The prior rejection of claims 1, 10, 12, 15-16, and 41 under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), as applied to claim 1, above, and further in view of United States Patent Application No. 2014/0286912 (Silverman) is withdrawn in light of Applicant’s amendments to claim 1 to require that the culture medium comprises an extract from a plant of the genus CInnamomum.
The prior rejection of claims 1, 10, 12, 14-15, and 39-41 under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), as applied to claim 1 above, and further in view of United States Patent Application No. 2003/0220692 (Shapiro) is withdrawn in light of Applicant’s amendments to claim 1 to require that the culture medium comprises an extract from a plant of the genus CInnamomum.
The prior rejection of claims 1, 37-38, 42 and 44, under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), as applied to claim 1 above, and further in view of Almalki et al (Stem Cell Research & Therapy 7:1-12. 2016) is withdrawn in light of Applicant’s amendments to claim 1 to require that the culture medium comprises an extract from a plant of the genus CInnamomum.
The prior rejection of claims 1, 37-38, and 42-44 under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), 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) is withdrawn in light of Applicant’s amendments to claim 1 to require that the culture medium comprises an extract from a plant of the genus CInnamomum.
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.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018) and further in view of United States Patent No. 9,320,770 (Ota) and Sakai et al. (Nature Communications 3: 1-11. 2012; referenced in IDS). This is a new rejection that is substantially similar to a previous rejection.
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 (i.e. non-digested tissue with an undestroyed tissue microenvironment). 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 (i.e. recovering the cultured cell population).
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. The results of the present study demonstrated that Low‑NPSCs and High‑NPSCs expressed a low level of Tie2; however, L‑NPSCs displayed a significantly lower level compared with H‑NPSCs, suggesting that the number of progenitor cells among L‑NPSCs was decreased. 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 part of the method of Wu, as can be seen in Figure 7, the NPSCs undergo amplification , leading to an increase in Tie2 expression within the culture.
Wu does 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).
Furthermore, Wu also 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). As such, enhancing Tie2 expression would cause the cells to adopt a more H-NPSC phenotype.
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 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. 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. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota), and Sakai et al. (Nature Communications 3: 1-11. 2012) 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 that is substantially similar to a previous rejection.
The teachings of Wu, Ota, and Sakai are as discussed above.
The combined teachings of Wu, Ota, and Sakai 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 Wu, Ota, and Sakai 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, 15-16, and 41 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota), and Sakai et al. (Nature Communications 3: 1-11. 2012), as applied to claim 1, above, and further in view of United States Patent Application No. 2014/0286912 (Silverman). This is a new rejection that is substantially similar to a previous rejection.
Regarding claims 10 and 15, the teachings of Wu, Ota, and Sakai are as discussed above. Wu teaches a method of differentiating the NPSCs into osteogenic, adipocytic, or chondrogenic lineages.
The combined teachings of Wu, Ota, and Sakai do not teach wherein the differentiation methods using cultureware having undergone cell attachment-increasing treatment.
However, Silverman teaches a method of culturing nucleus pulposus cells on gelatin-coated flasks. Discarded adult human nucleus pulposus tissue from discectomy procedures was procured with IRB from consenting donors. Fibrous, annulus material and other tissue contaminants were removed via dissection. The remaining material was then washed three times with 2x antibiotic-antimycotic (ABAM) in PBS, and digested overnight in 300 units recombinant type-2 collagenase in DMEM/F12 with 1xABAM. The isolated cells were plated onto gelatin-coated flasks in expansion medium (DMEM/F12 with 10% FBS, 10 ng/mL EGF and 10 ng/mL FGF-2), and over time a subpopulation of cells attached to the plates, composed of stem/progenitor cells (i.e. Tie2-positive cells). These cells were expanded for up to 4 passages (paragraph 0120). Silverman teaches that NP-derived stem/progenitor cells (discogenic cells) underwent osteogenesis and adipogenesis (differentiation). Briefly, discogenic cells were dissociated for 15 minutes using TrypLE to form a single cell suspension and plated onto tissue-culture treated dishes (adherent cell culture plates) at 20,000 cells/cm2. Dishes of cells for osteogenic and adipogenic differentiation were maintained in DMEM with 10% FBS for 3 days, and then fed with the appropriate supplied differentiation media for 3 weeks. After differentiation, the monolayers were stained for calcification with Alizarin red or lipid content with Oil Red O (paragraph 0125 and Example 4). Silverman teaches that the receptacle may be treated to aid cell attachment and growth of disc tissue derived cells on gelatin or collagen-coated receptacles may allow differentiation of the discogenic cells (paragraphs 0058 and 0075).
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 Wu by using treated cultureware dishes to increase cell attachment, as identified by Silverman, 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 Silverman has successfully reduced to practice that treated culture dishes, such as with gelatin, can be used for differentiating NPSCs into adipogenic or osteogenic cells, which are adherent cells and therefore favor a surface capable of attachment. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claims 12 and 16, Silverman, as stated supra, teaches that gelatin (an extracellular matrix as defined by claim 16 of the instant application) can be used for treating the receptacle for differentiation (paragraph 0058 and 0075).
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, 10, 12, 14-15, and 39-41 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota), and Sakai et al. (Nature Communications 3: 1-11. 2012), as applied to claim 1 above, and further in view of United States Patent Application No. 2003/0220692 (Shapiro). This is a new rejection that is substantially similar to a previous rejection.
Regarding claims 10 and 15, the teachings of Wu, Ota, and Sakai 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 (p. 2198, 2nd to last para.).
The combined teachings of Wu, Ota, and Sakai 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 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 Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota), and Sakai et al. (Nature Communications 3: 1-11. 2012), 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 that is substantially similar to a previous rejection.
Regarding claims 37-38, the teachings of Wu, Ota, and Sakai are as discussed above. Wu teaches a method of differentiating the NPSCs.
Wu does 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 Wu, Ota, and Sakai 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 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 Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), United States Patent No. 9,320,770 (Ota), and Sakai et al. (Nature Communications 3: 1-11. 2012), as applied to claim 1 above, and further in view of Almalki et al (Stem Cell Research & Therapy 7:1-12. 2016). This is a new rejection that is substantially similar to a previous rejection. 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 Wu, Ota, and Sakai 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 Wu, Ota, and Sakai 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 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
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.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 1 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of copending Application No. 17/921,180 in view of Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018).
‘180 claims a method of culturing a cell population containing cartilage-derived Tie2-positive cells in a culture medium containing at least one kind of plant-derived extract having a Tie2 expression-enhancing effect (claim 1), such as a plant of the genus Cinnamomum (claim 2).
‘180 is silent as to the cartilage-derived Tie2-positive cells.
However, Wu teaches that a total of 10 NP [nucleus pulposus; a cartilage tissue] 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 (i.e. non-digested tissue with an undestroyed tissue microenvironment). 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 (i.e. recovering the cultured cell population).
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. The results of the present study demonstrated that Low‑NPSCs and High‑NPSCs expressed a low level of Tie2; however, L‑NPSCs displayed a significantly lower level compared with H‑NPSCs, suggesting that the number of progenitor cells among L‑NPSCs was decreased. 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).
Furthermore, Wu also 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). As such, enhancing Tie2 expression would cause the cells to adopt a more H-NPSC phenotype.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have used isolated non-digested nucleus pulposus tissue, as done by Wu, to culture cartilage-derived Tie2-positive cells in the culture method of claims 1-2 of ‘180 to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to use non-digested nucleus pulposus tissues with a reasonable expectation of success because Wu teaches that nucleus pulposus tissue are a known cartilage tissue with Tie2 positive cells that can be cultured in vitro to recover nucleus pulposus stem cells. Furthermore, 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) and reduced differentiation potential of NPSCs. As such, enhancing Tie2 expression would cause the cells to adopt a more H-NPSC phenotype. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
This is a provisional nonstatutory double patenting rejection.
Claims 1 and 4 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of copending Application No. 17/921,180 in view of Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), as applied to claim 1 above and further in view of Chan et al. (The Spine Journal 10: 486–496. 2010).
The teachings of ‘180 and Wu are as discussed above.
The combined teachings of ‘180 and Wu 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 ‘180 and Wu 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, 14-16, 39, and 41 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of copending Application No. 17/921,180 in view of Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), as applied to claim 1 above and further in view of United States Patent Application No. 2014/0286912 (Silverman).
The teachings of ‘180 and Wu are as discussed above. ‘180 claims wherein the culturing is performed in cultureware having a culture surface coated with a coating agent containing an extracellular matrix and/or a polyamino acid (claim 3), such as fibronectin (claim 4) or polylysine (claim 5). Furthermore, ‘180 claims a method of preparing a cell population containing chondrocytes differentiated from cartilage-derived Tie2-positive cells, the method comprising: comprising a step of performing the culture method according to claim 1 in order to enhance expression of Tie2 on cartilage-derived Tie2-positive cells in a cell population and induce differentiation into chondrocytes (claim 6) which express type II collagen (claim 7). ‘180 claims as part of their differentiation method, that wherein a cell population in which the cartilage-derived Tie2-positive cells also remain is obtained through the differentiation culture stage (claim 10). Wu teaches a method of differentiating the NPSCs into osteogenic, adipocytic, or chondrogenic lineages (Figure 8).
Although ‘180 claims using cultureware having undergone cell attachment-increasing treatment for the culture method of claim 1, ‘180 does not directly claim using cell attachment-increasing treatment as part of the differentiation method of claim 6.
However, Silverman teaches a method of culturing nucleus pulposus cells on gelatin-coated flasks. Discarded adult human nucleus pulposus tissue from discectomy procedures was procured with IRB from consenting donors. The isolated cells were plated onto gelatin-coated flasks in expansion medium (DMEM/F12 with 10% FBS, 10 ng/mL EGF and 10 ng/mL FGF-2), and over time a subpopulation of cells attached to the plates, composed of stem/progenitor cells (i.e. Tie2-positive cells). These cells were expanded for up to 4 passages (paragraph 0120). Silverman teaches that NP-derived stem/progenitor cells (discogenic cells) underwent chondrogenesis (differentiation) (paragraph 0126 and Example 5). Silverman teaches that the receptacle may be treated to aid cell attachment and growth of disc tissue derived cells on gelatin or collagen-coated receptacles may allow differentiation of the discogenic cells (paragraphs 0058 and 0075).
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 Tie2 positive cells of ‘180 and Wu by using treated cultureware dishes to increase cell attachment, as identified by Silverman, 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 and Silverman have successfully reduced to practice that Tie2 positive NPSCs can be differentiated into chondrogenic cells and that culture containers may be treated to aid cell attachment and growth of disc tissue derived cells on gelatin or collagen-coated receptacles to improve differentiation of the Tie2 positive cells. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1, 10, 12, 14-16, 39, and 41 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of copending Application No. 17/921,180 in view of Wu et al. (International Journal of Molecular Medicine 42: 2193-2202. 2018), 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).
Regarding claims 37-38, the teachings of ‘180 and Wu are as discussed above. ‘180 claims a method of preparing a cell population containing chondrocytes differentiated from cartilage-derived Tie2-positive cells, the method comprising: comprising a step of performing the culture method according to claim 1 in order to enhance expression of Tie2 on cartilage-derived Tie2-positive cells in a cell population and induce differentiation into chondrocytes (claim 6) which express type II collagen (claim 7). ‘180 claims as part of their differentiation method, that wherein a cell population in which the cartilage-derived Tie2-positive cells also remain is obtained through the differentiation culture stage (claim 10). Wu teaches a method of differentiating NPSCs into chondrocytes (Figure 8).
The combined teachings of ‘180 and Wu does 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 ‘180 and Wu by differentiating nucleus pulposus progenitor cells into nucleus pulposus cells using a culture medium containing an extracellular matrix-degrading agent, as identified by Shapiro and Baraniak, which can then be differentiated into chondrocytes, as shown by Wu, 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 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