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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 04/08/2026 has been entered.
1. Claims 1 – 18 remain pending. Claims 1 – 9 and 13 – 16 are under consideration.
Withdrawn Claim Objections
2. The objection of claims 1, 6, and 8 are withdrawn in view of Applicant’s amendment to the claims.
Withdrawn Claim Rejections
3. The rejection of claims 1 and 2 under 35 U.S.C. 102(a)(1) is withdrawn in view of Applicant’s amendment to the claim 1 to require 2D culturing.
4. The rejection of claims 1 and 2 under 35 U.S.C. 102(a)(2) is withdrawn in view of Applicant’s amendment to claim 1 to require 2D culturing.
5. The rejection of claims 1 – 3 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claim 1 to require 2D culturing.
6. The rejection of claims 1, 2, and 4 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claim 1 to require 2D culturing.
7. The rejection of claims 1, 2, and 5 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claim 1 to require 2D culturing.
8. The rejection of claims 1, 2, 6 – 9, and 13 – 16 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to the claim 1 and 6 to require 2D culturing
Claim Interpretation
9. For the purpose of applying prior art, “wherein when the method…” of claim 1 is interpreted as a conditional limitation that is not required when human pluripotent stem cells that are not aggregates or clusters are cultured.
10. For the purpose of applying prior art, “an adherent system” of step (b) of claim 6 is interpreted as the same as “culturing on a protein matrix” of step (c) of claim 6.
11. For the purpose of applying prior art, “sorting the cells” of step (d) of claim 6 is interpreted as sorting by any method.
12. For the purpose of applying prior art, “manually passaging” of claim 7 is interpreted as passaging without the use of enzymes.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
13. Claims 1, 2, and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Shamis (Shamis, Yulia, et al. Stem cell research & therapy 2.1 (2011): 10.), hereinafter Shamis which is cited on the IDS filed 09/02/2022 in view of Nagaoka (Nagaoka, Masato, et al. PLoS One 10.8 (2015): e0136350.), hereinafter Nagaoka in view of Flasza (Flasza, Marzena, et al. Regenerative medicine 2.6 (2007): 903-918.), hereinafter Flasza.
Regarding claim 1, Shamis teaches a method comprising culturing H9 human embryonic stem cells (“human pluripotent stem cells”) on MEFs (“(2D) adherent system”) in differentiation medium (“in the presence of a medium that is suitable for culturing fibroblasts”) to form EDK cells which display characteristics of dermal fibroblasts (Abstract; page 2, left col. last para. and right col. para. 1 – 2; Figure 1a and b; page 5, left col. para. 4 and right col. last para.; page 6, left col. and right col.; Figure 3). Shamis does not teach “in the absence of feeder cells” or “wherein said method meets good manufacturing practices (GMP) standards”.
Regarding claim 2, Shamis teaches the EDK cells display characteristics of dermal fibroblasts (Abstract; page 5, right col. last para.; page 6, left col. and right col.; Figure 3).
Regarding claim 5, Shamis teaches the medium is supplemented with BMP4 (page 2, right col. para. 2).
Shamis does not teach “in the absence of feeder cells” or “wherein said method meets good manufacturing practices (GMP) standards” of claim 1. However, Shamis teaches the use of pluripotent stem cells (PSCs) for future therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of hES- and hiPS-derived cells and will be essential to better predict their safety and stability following in vivo transplantation (page 1). Shamis teaches despite the critical impact of fibroblasts on tissue morphogenesis, homeostasis, and repair, gaps in our understanding of fibroblast lineage development has made the identification and reproducible isolation of specific fibroblast populations particularly challenging (page 9, right col. para. 2). Shamis teaches EDK cells secrete HGF which is a known to be secreted by dermal fibroblasts that supports epithelial development and repair (page 2, left col. last para.; page 6, right col.; Figure 3). Shamis teaches EDK cells could support tissue development and enable re-epithelization of wounded human skin equivalents (HSEs) that was linked to the expression and secretion of HGF (page 2, left col. last para. and right col. para. 1; page 6, left col. para. 1 and right col.; page 8, left col. and right col. para. 1; Figure 4; page 9, left col. para. 1 and right col. para. 1). Shamis teaches directed differentiation of hES-derived fibroblasts may allow these cells to be adapted for cell therapy and cutaneous regeneration (page 11, left col. para. 1). Shamis teaches the development of future cell-based treatments using hES-derived fibroblasts can potentially augment repair and improve overall tissue health because a decline in fibroblast numbers or function has been implicated in both skin aging and cancer development (page 11, left col. para. 1). Shamis teaches HSEs provide a complex tissue microenvironment that provides an important platform to further establish the EDKs stability, safety, and efficacy for future therapeutic transplantation (page 2, right col. para. 1). Shamis teaches they expect their findings will help address a central challenge facing clinical application of cells with properties of dermal fibroblasts for regenerative medicine by developing an efficient approach to reproducibly procure these cells from pluripotent stem cells in a way that offers predictable and effective tissue outcomes upon their therapeutic use (page 9, right col. para. 2).
Regarding “in the absence of feeder cells” of claim 1, Nagaoka teaches a method of differentiation of hPSCs cells to mesoderm by 2D adherent culturing on vitronectin variants (R-Fc and NC-Fc) or Matrigel in medium containing BMP4 (page 3, para. 2; page 4, para. 1; page 8, para. 2; Figure 3B). Nagaoka teaches hPSCs including hES and hiPSCs adhere to vitronectin variant-coated surfaces and Matrigel coated surfaces (page 6, para. 5; page 8, para. 1). Nagaoka teaches hPSCs cultured on the vitronectin variant-coated surface could form cartilage, kidney, striated muscle, and vasculature when transplanted in vivo (Figure 3C; page 10, para. 1; page 2, last para.; page 5, last para.; page 6, para. 1).
Nagaoka does not teach “wherein said method meets good manufacturing practices (GMP) standards” of claim 1. However, Nagaoka teaches the vitronectin variant is sufficient to maintain the pluripotency of hPSCs and facilitates the differentiation of the cells towards mesoderm and towards hepatocytes under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs (Abstract; page 14, para. 3). Nagaoka teaches the vitronectin variant is a highly defined cell adhesion substrate and offers an alternative to Matrigel (page 15, para. 3). Nagaoka teaches Matrigel is not a defined culture condition and is a relatively impure preparation with significant lot-to-lot variability that can affect the reproducibility of cell differentiation (Abstract; page 2, para. 1). Nagaoka teaches a concern with Matrigel is the possibility for contamination by pathogens, which raises serious safety issues for clinical approaches using stem cell-derived cells and may affect functional studies of these cells (page 2, para. 1). One would have been motivated to combine the teachings of Shamis and Nagaoka to substitute the feeder layer of Shamis with the vitronectin variant surface for culturing hPSCs of Nagaoka to better define the conditions in a reproducible method of producing dermal fibroblasts for subsequent clinical use as Shamis teaches they expect their findings will help address a central challenge facing clinical application of cells with properties of dermal fibroblasts for regenerative medicine by developing an efficient approach to reproducibly procure these cells from pluripotent stem cells in a way that offers predictable and effective tissue outcomes upon their therapeutic use.
Regarding “wherein said method meets good manufacturing practices (GMP) standards” of claim 1, Flasza teaches a GMP method for obtaining human dermal fibroblasts from neonatal foreskin and culturing human dermal fibroblasts to prepare a skin graft replacement (page 904, right col.; page 905, right col., para. 1 – 2; Figure 7). Flasza teaches all products used were manufactured in accordance with GMP standards in a licensed GMP manufacturing facility (page 904, right col. para. 1). Flasza teaches fixing the skin graft on full-thickness skin wounds on mice (page 907, right col. para. 3; page 911, right col. para. 2; Figure 6). Flasza teaches all wounds were completely healed by 28 days and no scarring or blistering was observed (page 911, right col. para. 2 – 3). Flasza teaches human dermal fibroblasts were detected at 28 days after graft application, suggesting persistence of applied cells in the healed wound (page 912, right col.).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Shamis regarding a method of differentiating human pluripotent stem cells to dermal fibroblasts in 2D adherent culture with the teachings of Nagaoka regarding a defined vitronectin-variant surface for adherent culture and differentiation of human pluripotent stem cells with the teachings of Flasza regarding a GMP method of obtaining, culturing, and producing a skin graft of human dermal fibroblasts to arrive at the claimed method for differentiating human pluripotent stem cells into fibroblasts, comprising a step of culturing the human pluripotent stem cells, or human pluripotent stem cell aggregates or clusters, in 2 dimensions (2D) on an adherent system in the presence of a medium that is suitable for culturing fibroblasts and in the absence of feeder cells, wherein when the method comprises culturing human pluripotent stem cell aggregates or clusters, then said method also comprises, prior to said culturing step, a step of forming and culturing said human pluripotent stem cell aggregates or clusters in 2D on an adherent system to support cell attachment and growth in the presence of a medium that is suitable for culturing human pluripotent stem cells; and wherein said method meets good manufacturing practices (GMP) standards. One would have been motivated to combine the teachings of Shamis, Nagaoka, and Flasza in a method to produce clinical-grade human dermal fibroblasts from hPSCs instead of skin biopsies for treating skin wounds as Shamis teaches directed differentiation of hES-derived fibroblasts may allow these cells to be adapted for cell therapy and cutaneous regeneration and Shamis teaches the development of future cell-based treatments using hES-derived fibroblasts can potentially augment repair and improve overall tissue health because a decline in fibroblast numbers or function has been implicated in both skin aging and cancer development. One would have a reasonable expectation of success in combining the teachings as Shamis teaches EDK cells could support tissue development and enable re-epithelization of wounded human skin equivalents and Nagaoka teaches the vitronectin variant is sufficient to maintain the pluripotency of hPSCs and facilitates the differentiation of the cells towards mesoderm and Flasza teaches the human dermal fibroblast skin graft could completely heal all wounds by 28 days and no scarring or blistering was observed and the human dermal fibroblasts were detected at 28 days after graft application, suggesting persistence of applied cells in the healed wound.
14. Claim(s) 3 is/are rejected under 35 U.S.C. 103 as being unpatentable over Shamis (Shamis, Yulia, et al. Stem cell research & therapy 2.1 (2011): 10.), hereinafter Shamis which is cited on the IDS filed 09/02/2022 in view of Nagaoka (Nagaoka, Masato, et al. PLoS One 10.8 (2015): e0136350.), hereinafter Nagaoka in view of Flasza (Flasza, Marzena, et al. Regenerative medicine 2.6 (2007): 903-918.), hereinafter Flasza as applied to claims 1, 2, and 5 above, and further in view of in view of Nazari (Nazari, Banafsheh, et al. The American journal of pathology 186.10 (2016): 2650-2664.), hereinafter Nazari.
The limitations of claim 1 are obvious in view of the combined teachings of Shamis, Nagaoka, and Flasza as set forth above. Shamis, Nagaoka, and Flasza do not teach “at least 90% papillary fibroblasts” of claim 3. However, Shamis teaches it is critical to fully characterize the properties of differentiated cells derived from hES cells by developing engineered, pre-clinical tissue models that will better predict their behavior following future therapeutic transplantation to humans (page 11, left col. last para.). Shamis teaches it is critical to fully-characterize the properties of differentiated cells derived from hES cells by developing engineered, pre-clinical tissue molecules that will better predict their behavior following future therapeutic transplantation to humans (page 11, left col. last para.). Shamis teaches dermal fibroblasts derived from hES cells support the normal development of a 3D skin model and repair wounds in this model (Abstract; page 11, left col. last para.). Shamis teaches human pluripotent stem cells may serve as an alternative to adult tissues of more uniform fibroblasts that may provide more predictable tissue outcomes upon their therapeutic use (page 2, left col. para. 3). Flasza teaches the skin graft can be used to treat skin disease, trauma, burns, and scars (page 915, right col. para. 2).
Regarding claim 3, Nazari teaches culturing human dermal fibroblasts with TNF or IL-1β leads to a robust induction of PDPN (Abstract; page 2652, right col. para. 3; page 2654, right col. last para.; page 2655, left col.; page 2656, left col. para. 1; Figure 5A – B; page 2660, right col. last para.; page 2661, left col. para. 1; page 2661, left col. last para.).
Nazari teaches tissue injury triggers the activation and differentiation of multiple cell types to minimize damage and initiate repair processes and in systemic sclerosis (SSc), these repair processes appear to run unchecked leading to aberrant remodeling and fibrosis of the skin and multiple organs, yet the fundamental pathological defect remains unknown (Abstract; page 2650, left col.). Nazari teaches SSc includes a triad of autoimmunity, vasculopathy, and fibrosis, with the fibrotic component dominating the life-threatening pathology (page 2662, left col. para. 2). Nazari teaches in the skin of SSc patients, a transition occurs wherein dermal fibroblasts disappear and podoplanin (PDPN)+ fibroblasts appear (Abstract; page 2651, left col. para. 1; page 2660, right col.). Nazari teaches uncovering these fibroblast changes may provide new tools to study earlier events in SSc before the major fibrotic process (page 2662, left col. para. 2). Nazari teaches by defining the transition, it is possible that disease heterogeneity within SSc can be probed and patients more accurately staged and could provide a new metric for interventional clinical studies (page 2662, left col. para. 2). Nazari teaches human skin undergoing repair and scar formation after an excisional biopsy exhibited the transition with increased PDPN expression and introduction of an incisional wound in mouse skin led to the appearance of PDPN+ cells (page 2654, right col. para. 2; Figure 4). Nazari teaches the rapid observation of PDPN+ cells in these wounds is consistent with fibroblast differentiation and less likely to result from replacement by new cells (page 2654, right col. para. 2). Nazari teaches the transition to increased fibroblast expression of PDPN occurs in inflamed skin from patients with psoriasis, subacute and chronic spongiotic dermatitis, and cutaneous lupus erythematous and the transition was localized to the inflamed papillary dermal layer (page 2656, left col. last para. and right col. para. 1; Figure 6). Nazari teaches recapitulation of the entire transition in vitro would help solidify the hypothesis of transitioning of dermal fibroblasts and this awaits further advances in dermal fibroblast culturing (page 2661, left col. para. 1).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Shamis regarding a method of differentiating human pluripotent stem cells to dermal fibroblasts in 2D adherent culture with the teachings of Nagaoka regarding a defined vitronectin-variant surface for adherent culture and differentiation of human pluripotent stem cells with the teachings of Flasza regarding a GMP method of obtaining, culturing, and producing a skin graft of human dermal fibroblasts with the teachings of Nazari regarding culturing human dermal fibroblasts with TNF or IL-1β leads to a robust induction of PDPN to arrive at the claimed method wherein the fibroblasts are at least 90% papillary fibroblasts. One would have been motivated to combine the teachings of Shamis, Nagaoka, Flasza, and Nazari in a method to produce a 3D tissue model comprising human dermal fibroblasts and papillary fibroblasts from human pluripotent stem cells to study the transition to PDPN+ papillary fibroblasts in inflammatory skin diseases such as SSc and to develop therapies for such diseases as Shamis teaches it is critical to fully characterize the properties of differentiated cells derived from hES cells by developing engineered, pre-clinical tissue models that will better predict their behavior following future therapeutic transplantation to humans and Nazari teaches the fundamental pathological defect of SSc remains unknown and Nazari teaches uncovering these observed fibroblast changes may provide new tools to study earlier events in SSc before the major fibrotic process and Nazari teaches recapitulation of the entire transition in vitro would help solidify the hypothesis of transitioning of dermal fibroblasts and this awaits further advances in dermal fibroblast culturing. One would have a reasonable expectation of success in combining the teachings as Shamis teaches dermal fibroblasts derived from hES cells support the normal development of HSEs and wound healing in this 3D tissue model and Nazari teaches the rapid observation of PDPN+ cells in wounds is consistent with fibroblast differentiation and less likely to result from replacement by new cells.
15. Claim(s) 4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Shamis (Shamis, Yulia, et al. Stem cell research & therapy 2.1 (2011): 10.), hereinafter Shamis which is cited on the IDS filed 09/02/2022 in view of Nagaoka (Nagaoka, Masato, et al. PLoS One 10.8 (2015): e0136350.), hereinafter Nagaoka in view of Flasza (Flasza, Marzena, et al. Regenerative medicine 2.6 (2007): 903-918.), hereinafter Flasza as applied to claims 1, 2, and 5 above, and further in view of in view of Hewitt (Hewitt, Kyle J., et al. Tissue Engineering Part A 15.11 (2009): 3417-3426.), hereinafter Hewitt which is cited on the IDS filed 09/02/2022 in view of Abdian (Abdian N, et. al. Cell Tissue Bank. 2015 Dec;16(4):487-95), hereinafter Abdian.
The limitations of claim 1 are obvious in view of the combined teachings of Shamis, Nagaoka, and Flasza as set forth above. Shamis teaches they expect their findings will help address a central challenge facing clinical application of cells with properties of dermal fibroblasts for regenerative medicine by developing an efficient approach to reproducibly procure these cells from pluripotent stem cells in a way that offers predictable and effective tissue outcomes upon their therapeutic use (page 9, right col. para. 2).
Regarding “insulin, hydrocortisone, epidermal growth factor”, Shamis teaches the method of Hewitt was used to prepare EDK cells (page 2, left col. para. 3 and right col. para. 2; page 9, right col. para. 1) but does not teach the composition of the media. Hewitt teaches the medium contained hydrocortisone, EGF, and insulin (page 3418, right col. para. 2). Hewitt does not teach “fibroblast growth factor (FGF)”. However, Hewitt teaches the integrated events that occur during tissue morphogenesis need to be studied in biological systems in which a high degree of tissue complexity can be achieved (page 3424, right col. last para.). Hewitt teaches the application of 3D tissues will play a pioneering role in moving tissue engineering and discovery science into to translational and clinical relevance (page 3424, right col. last para.). Hewitt teaches the combinatorial model using both 2D and 3D techniques to first commit hES cells to multiple lineages and then to incorporate these cells into bioengineered 3D tissues, provides a novel paradigm to study ectodermal-mesodermal crosstalk during epithelial tissue fabrication (page 3424, right col. last para.; page 3425, left col. para. 1).
Regarding “fibroblast growth factor”, Abdian teaches bFGF is a member of the FGF family, is secreted by human dermal fibroblasts (HDFs), and is an important nutritional factor for cell growth and differentiation and supplementation of culture media with bFGF enhances cell proliferation and improves growth rate compared to media without bFGF (Abstract; page 488, right col. para. 2; page 490, right col. para. 1 – 2; Figure 2; page 492, right col.; page 494, right col. para. 2). Abdian teaches bFGF is important in cell-cycle regulation and progression and fibroblast division stimulation (Abstract). Abdian teaches HDFs can easily be cultured in vitro, and play a critical role in wound healing and synthesis and secretion of extracellular matrix proteins (page 488, left col. para. 1).
It would have been obvious prior to the effective filing date of the invention as clamed for the person of ordinary skill in the art to combine the teachings of Shamis regarding a method of differentiating human pluripotent stem cells to dermal fibroblasts in 2D adherent culture using the method of Hewitt with the teachings of Nagaoka regarding a defined vitronectin-variant surface for adherent culture and differentiation of human pluripotent stem cells with the teachings of Flasza regarding a GMP method of obtaining, culturing, and producing a skin graft of human dermal fibroblasts with the teachings of Hewitt regarding a culture media containing hydrocortisone, EGF, and insulin with the teachings of Abdian regarding adding FGF to culture media increases cell proliferation and growth rate to arrive at the claimed method wherein the medium that is suitable for culturing fibroblasts comprises insulin, hydrocortisone, epidermal growth factor (EGF) and fibroblast growth factor (FGF). One would have been motivated to combine the teachings of Shamis, Nagaoka, Flasza, Hewitt, and Abdian in a method of producing sufficient quantities of clinical-grade human dermal fibroblasts from hPSCs instead of skin biopsies for treating skin wounds as Shamis teaches they expect their findings will help address a central challenge facing clinical application of cells with properties of dermal fibroblasts for regenerative medicine by developing an efficient approach to reproducibly procure these cells from pluripotent stem cells in a way that offers predictable and effective tissue outcomes upon their therapeutic use and Abdian teaches bFGF is an important nutritional factor for cell growth and is important in cell-cycle regulation and progression and fibroblast division stimulation. One would have a reasonable expectation of success in combining the teachings as Abdian teaches culture media containing bFGF produced more HDFs that culture media without bFGF.
16. Claim(s) 6 – 9, 13, and 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Shamis (Shamis, Yulia, et al. Stem cell research & therapy 2.1 (2011): 10.), hereinafter Shamis which is cited on the IDS filed 09/02/2022 in view of Nagaoka (Nagaoka, Masato, et al. PLoS One 10.8 (2015): e0136350.), hereinafter Nagaoka in view of Flasza (Flasza, Marzena, et al. Regenerative medicine 2.6 (2007): 903-918.), hereinafter Flasza as applied to claims 1, 2, and 5 above, and further in view of in view of Hewitt (Hewitt, Kyle J., et al. Tissue Engineering Part A 15.11 (2009): 3417-3426.), hereinafter Hewitt which is cited on the IDS filed 09/02/2022 in view of Zhang (Zhang, Kejing, et al. Development 137.13 (2010): 2095-2105.), hereinafter Zhang as evidenced by Stemgent (Stemgent BMP4 Product Specification Sheet; https://store.reprocell.com/downloads/1536678457S030007.pdf; Accessed on 08/27/2025; previously cited).
The limitations of claim 1 are obvious in view of the combined teachings of Shamis, Nagaoka, and Flasza as set forth above.
Regarding step (a) of claim 6, Nagaoka teaches forming and culturing clusters of hPSCs in 2D on adherent vitronectin-variant and Matrigel surfaces in a medium in mTeSR1 medium (page 2, last para.; page 6, last para.; page 8, para. 1; Figure 2).
Regarding step (b) and step (c) of claim 6 and claims 9, 13 and 14, Nagaoka teaches culturing the hPSC clusters on Matrigel in a medium containing BMP4 (page 2, last para.; page 6, last para.; page 8, para. 1; Figure 2; page 4, para. 1) and Shamis teaches culturing hPSCs in a differentiation medium containing 0.5 nM BMP4 (claim 9) to form EDK cells which display characteristics of dermal fibroblasts (Abstract; page 2, left col. last para. and right col. para. 1 – 2; Figure 1a and b; page 5, left col. para. 4 and right col. last para.; page 6, left col. and right col.; Figure 3). Nagaoka teaches the clusters were observed at 3 days after seeding in the legend for Figure 2. Shamis teaches the method of Hewitt was used to prepare EDK cells and BMP4 was included from day four to day seven (page 2, right col. para. 2). Hewitt teaches the method for forming EDK cells was 14 days (claims 13 and 14) (Figure 1).
Regarding step (d) of claim 6, Shamis teaches at day 14, the derived EDK cells were then propagated on tissue culture plastic until day 21, and then expanded on type I collagen-coated plates (page 2, right col. para. 2).
Regarding claim 7, Nagaoka teaches passaging the hPSCs with enzyme-free Cell Dissociation Buffer (page 2, last para.).
Regarding claim 8, Nagaoka teaches passaging the hPSCs with Accutase (page 2, last para.).
Regarding claims 15 and 16, Shamis and Hewitt teach adding BMP4 at 0.5 nM on day 4 (“day 4” of claims 15 and 16 and “about 0.27 nM” of claim 16) (Shamis: page 2, right col. para. 2; Hewitt: page 3418, right col. para. 2; Figure 1). Shamis and Hewitt do not teach adding BMP4 on day 1 of claims 15 and 16. However, Hewitt teaches BMP4 was added at day 4 to block outgrowth of neural precursors (page 3420, left col. para. 3; page 3424, left col. para. 2). Hewitt teaches the application of 3D tissues will play a pioneering role in moving tissue engineering and discovery science into to translational and clinical relevance (page 3424, right col. last para.).
Regarding “day 1” of claims 15 and 16 and “between 0.2 and 0.4 nM” of claim 15, Zhang teaches addition of 10 ng/mL BMP4 to ESCs at day 0 – 1 and 1 – 2 to suppress neural differentiation (page 2097, left col. last para. and right col.; Figure 2A – B). 10 ng/mL BMP4 is about 0.29 nM based on a BMP4 dimer at 34 kDa as evidenced by Stemgent (page 1, para. 1) [10 ng/mL = 0.00001 g/L x mol/34,000 g = 0.29 nM]. Zhang teaches before day 2, the addition of BMP4 maintained the OCT4+/SOX+ pluripotent cells (page 2097, right col. last para.). Zhang teaches once neural differentiation is initiated, BMP4 cannot inhibit it (page 2098, left col.). Zhang teaches the BMP4-sensitive window appears to be a crucial time during which BMP4 switches its function from maintaining ESC pluripotency to promoting non-neural lineage differentiation (page 2098, right col. para. 1). Zhang teaches seeding ESC cells in chemically defined medium on a cell culture dish coated with FBS and after 5 – 6 days, the cells grew and formed large compact colonies and the colonies were picked, fragmented into smaller clumps using collagenase and mechanical dissociation and passaged at 3-day intervals (page 2096, left col. para. 4). Zhang teaches using knockout serum replacement (KSR) medium to avoid influences from feeder cells and BMP4 was added to this KSR medium (page 2096, right col. para. 4; page 2097, left col. para. 2).
It would have been obvious prior to the effective filing date of the invention as clamed for the person of ordinary skill in the art to combine the teachings of Shamis regarding a method of differentiating human pluripotent stem cells to dermal fibroblasts in 2D adherent culture using the method of Hewitt with the teachings of Nagaoka regarding a defined vitronectin-variant surface for adherent culture and differentiation of human pluripotent stem cells with the teachings of Flasza regarding a GMP method of obtaining, culturing, and producing a skin graft of human dermal fibroblasts with the teachings of Hewitt regarding a 14-day method of producing human dermal fibroblasts from hPSCs using the 14-day method of Hewitt where BMP4 was added at 0.5 nM on days 4 – 7 to block outgrowth of neural precursors followed by 7 days of selecting dermal fibroblasts by culturing on plastic and then collagen with the teachings of Zhang regarding adding BMP4 at about 0.29 nM to ESCs at day 0 – 1 and 1 – 2 to suppress neural differentiation to arrive at the claimed method for differentiating human pluripotent stem cells into fibroblasts according to claim 1, comprising: (a) forming and culturing aggregates or clusters of said human pluripotent stem cells in 2D on an adherent system to support cell attachment and growth in the presence of a medium that is suitable for culturing human pluripotent stem cells; (b) culturing the human pluripotent stem cells or adherent aggregates or clusters of said human pluripotent stem cells in 2D on an adherent system comprising a cell culture surface coated with a defined protein matrix coating in the presence of a medium that is suitable for culturing fibroblasts; (c) differentiating the human pluripotent stem cells obtained in step (b) into fibroblasts by culturing on a protein matrix in the presence of a medium that is suitable for culturing fibroblasts, for 12 to 16 days; and (d) sorting the cells obtained in step (c) to obtain a homogeneous population of differentiated fibroblasts. One would have been motivated to combine the teachings of Shamis, Nagaoka, Flasza, Hewitt, and Zhang in a method to produce clinical-grade human dermal fibroblasts from hPSCs without contaminating neural cells for treating skin wounds as Hewitt teaches the application of 3D tissues will play a pioneering role in moving tissue engineering and discovery science into to translational and clinical relevance and Zhang teaches the BMP4-sensitive window appears to be a crucial time during which BMP4 switches its function from maintaining ESC pluripotency to promoting non-neural lineage differentiation and Zhang teaches once neural differentiation is initiated, BMP4 cannot inhibit it. One would have a reasonable expectation of success in combining the teachings as Shamis teaches EDK cells could support tissue development and enable re-epithelization of wounded human skin equivalents and Nagaoka teaches the vitronectin variant is sufficient to maintain the pluripotency of hPSCs and facilitates the differentiation of the cells towards mesoderm and Flasza teaches the human dermal fibroblast skin graft could completely heal all wounds by 28 days and no scarring or blistering was observed and the human dermal fibroblasts were detected at 28 days after graft application, suggesting persistence of applied cells in the healed wound and Hewitt and Zhang teach the addition of BMP4 to block neural differentiation from PSCs.
Applicant’s Arguments/ Response to Arguments
17. Applicant Argues: Applicant asserts that the prior art does not teach culturing in 2D on an adherent system and using GMP standards as recited in amended claim 1 and 6.
Response to Argument: Applicant's arguments filed 03/18/2026 have been fully considered but they are not persuasive. Nagaoka teaches culturing hPSCs in 2D on an adherent system of Matrigel or vitronectin-derived peptides, the hPSCs form clusters on these surfaces, and differentiating these clusters in a culture media containing BMP4. Nagaoka teaches the vitronectin variant is sufficient to maintain the pluripotency of hPSCs and facilitates the differentiation of the cells towards mesoderm and towards hepatocytes under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs. Flasza teaches a GMP method for obtaining human dermal fibroblasts from neonatal foreskin and culturing human dermal fibroblasts to prepare a skin graft replacement and that all products used were manufactured in accordance with GMP standards in a licensed GMP manufacturing facility. Therefore, the prior art cited in the claim rejections set forth above make obvious the claimed method of culturing in 2D on an adherent system under GMP standards.
Conclusion
No claims allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZANNA M BEHARRY whose telephone number is (571)270-0411. The examiner can normally be reached Monday - Friday 8:45 am - 5:45 pm.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632
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