DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
It is noted that the examiner of record has changed.
Applicant’s election without traverse of Group 1, claims 1 and 3-10 in the reply filed on October 20, 2025 is acknowledged. Based on review of the claims, Group 4, claim 16, drawn to a method of producing a culture medium comprising exosomes from umbilical cord blood stem cells by culturing these cells in media supplemented with GDF-11 has been rejoined.
Claims 11-15 and 21-24 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on October 20, 2025.
Applicant has elected the following species:
Claim 1: Applicant elects growth factors of GDF-11, EGF, FGF2, TGF-β1 and VEGF, i.e., “5GF combination”
Claims 4/5 related to the effect/result: Applicant elects Claim 5
Claim 5 sub-items: Applicant elects a higher ability to regenerate the skin
Claim 7 related to cell culture medium: Applicant elects DMEM
Claim 8 related to product type: Applicant elects cell culture medium containing exosomes released by the umbilical cord blood stem cells
Claim 9 related to formulation: Applicant elects culture medium frozen to powder
Claim 10 related to cell type and effect: Applicant elects fibroblasts and keratinocytes and migration and proliferation.
Based on Applicant’s amended claims dated August 11, 2025 removing “and/or to inhibit inflammation”, all species of claim 5 have been rejoined.
Claim 4 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species.
Claims 2 and 17-20 have been canceled. Claims 4, 11-15, and 21-24 are withdrawn. Claims 1, 3,
5-10, and 16 are examined on the merits.
Priority
The instant application is a 35 U.S.C 371 national stage filing of the International Application No. PCT/KR2021/004088 filed on April 1, 2021. The instant application claims foreign priority under 35 U.S.C 119(a)-(d) to Korean Patent Application KR10-2020-0039848, filed on April 1, 2020. Receipt is acknowledged of a certified copy of the foreign patent application in the original language as required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on January 6, 2023 and December 12, 2024 are in compliance with the provisions of 37 CFR 1.97 and are being considered by the examiner.
Applicant is reminded that the listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
FIG 5. Is objected to as failing to comply with 37 CFR 1.84(p)(5) because they do not include the following reference sign(s) mentioned in the description: Based on the amended specification filed on August 11, 2025, the corresponding description for FIG. 5 on Pgs. 7-8 references Experimental Example 5, which has been removed from the specification on Pgs. 24-25.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Objections to the Specification
The use of the term CAPTUREM and AMICON in Example 1 on Pg. 20 of the instant specification, which is are trade names or a marks used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 1 is objected to because of the following informalities: Claim 1 recites a culture medium containing exosomes with a “released concentration of 0.5x 109/ml ~ 5x109/ml…”. For the purposes of examination, it is interpreted as the released concentration is intended to refer to a released concentration of exosomes, however, as claimed it is not specified that the released concentration is related to the concentration of exosomes or exosome particles/ml. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 5 and 7 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 5 recites “a higher concentration of active ingredients” in line 4. The term “active ingredients” renders the claim indefinite. The phrase “active ingredients” is not defined by the claim, the specification does not provide a standard for measuring the scope of the term, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. As claimed it is unclear what is to be considered an “active ingredient”. Appropriate correction is required.
Claim 5, which depends from claim 1, recites the limitation "the growth factor" in line 7. There is insufficient antecedent basis for this limitation in the claim as claim 1 recites “growth factors” and there is no prior recitation of a growth factor. As claimed it is unclear if growth factor is intended to refer to the previously recited growth factors, one of the previously recited growth factors, or an additional growth factor. Appropriate correction is required.
Claim 7 contains the trademark/trade name KnockOut DMEM in line 7. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a type of media and, accordingly, the identification/description is indefinite.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
Claims 1, 6-7, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Wolvetang et al. (WO 2009055868, hereafter “Wolvetang”) in view of Kern et al. (2006, Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells, 24(5), 1294-1301, hereafter “Kern”) and Kim et al. (2017, Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin. Biochem. and Biophys. Res. Comm., 493(2), 1102-1108., hereafter “Kim”).
With regard to claims 1 and 16, in view of the prior art of Wolvetang, the species of growth factors comprising GDF-11 and FGF2 has been rejoined. Wolvetang teaches a method of maintaining stem cells in an undifferentiated state and a culture media for maintenance of stem cells in an undifferentiated state (Pg. 1, lines 10-15). Wolvetang teaches that stem cells can be derived from umbilical cord blood (Pg. 2, lines 5-6) and that the culture media for culturing a stem cell in an undifferentiated state comprises GDF (Pg. 5, lines 1-2). Wolvetang teaches an exemplary embodiment where human embryonic stem cells were cultured in serum-free media supplemented with GDF-11 (also known as BMP-11) at 20 ng/ml and bFGF, which is considered to reasonably read on FGF2, at 20 ng/ml (Pg. 44, lines 29-31) and Pg. 49, lines 24-29) while maintaining their undifferentiated state (Pg. 49, lines 24-29). With regard to claim 16, Wolvetang is considered to teach culture of stem cells in serum-free media supplemented with at least GDF-11 at a concentration of 20 ng/ml.
While Wolvetang does teach that stem cells can be derived from umbilical cord blood, all exemplary embodiments are drawn toward use of embryonic stem cells.
Kern teaches that mesenchymal stem cells (MSCs) derived from umbilical cord blood can be cultured longer and show higher proliferative ability compared to MSCs from other sources (Abstract) thus exhibiting the highest expansion potential amongst the MSCs investigated by Kern (Pg. 1300, right col., 2nd full para., lines 10-11). Further, Kern teaches that MSCs from umbilical cord blood can be obtained less invasively than MSCs derived from other sources (Pg. 1294, left col., 2nd para.).
Therefore it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to choose stem cells derived from umbilical cord blood as taught by Kern for use in the method of culturing stem cells such that stem cells are maintained in an undifferentiated state as taught by Wolvetang with a reasonable expectation of success. Since Kern teaches that stem cells from umbilical cord blood can be obtained relatively easily and exhibit increased potential for expansion, a skilled artisan would be motivated to choose umbilical cord blood stem cells in order to generate the largest number of undifferentiated stem cells, which Wolvetang teaches can be used for clinical applications such as generation of disease models, testing of pharmaceuticals and/or therapeutics, and tissue repair and/or regeneration (Pg. 2, lines 25-28). A skilled artisan would have had a reasonable expectation of success as both Wolvetang and Kern teach culture of stem cells and would have every reason to expect that Wolvetang’s culture method would achieve similar results when used to culture umbilical cord blood stem cells.
While the combined teachings of Wolvetang and Kern teach culture of umbilical cord blood stem cells in serum free culture media comprising 20 ng/ml of GDF-11 at and 20 ng/ml of FGF2, Wolvetang and Kern are silent as to the secretion of exosomes from umbilical cord blood stem cells and at the specific concentration of released exosomes.
Kim teaches that umbilical cord blood derived mesenchymal stem cells (UBC-MSCs) secrete exosomes that can promote cell migration in fibroblasts (Abstract). Kim teaches that UBC-MSCs were seeded at 1.89 x 105 cells per T25 flask and “conditioned” culture media was collected after 96 hours (Pg. 1103, left col., 3rd full para.) prior to exosome isolation and exosome particle analysis (i.e., diameter and concentration). Kim teaches that exosome concentration from UBC-MSCs seeded at 1.89 x 105 cells per T25 flask and maintained in culture for 96 hours was measured as 1.23 x 1011 particles/ml (Table 2). However, for subsequent investigation of effects of UBC-MSC exosome application on fibroblasts, Kim teaches that an optimal exosome concentration of 1 x 109 particles/ml (Pg. 1105, left col. 3rd para.) was used to examine the therapeutic effects of UCB-MSC exosomes on growth of fibroblasts.
As Kim teaches that media from cultured UCB-MSCs comprise exosomes derived from the UCB-MSCs (Abstract), one having ordinary skill in the art would have every reason to believe that the method of culturing stem cells derived from umbilical cord blood as taught by Wolvetang and Kern would generate culture media comprising exosomes which had been released by umbilical cord blood stem cells. Additionally, the concentration of exosomes released into media during the culture of stem cells would be recognized by one having ordinary skill in the art as dependent on the number of stem cells in culture, the size of the culture vesicle, the amount of media, time in culture, etc., therefore, it would be obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to optimize conditions such as plating density, culture vesicle size, amount of media, and/or time in culture in the method of culturing umbilical cord blood derived stem cells as taught by the combination of Wolvetang and Kern to arrive at the optimal concentration of UCB-MCS exosomes for promoting cell growth of 1 x 109 particles/ml as taught by Kim with a reasonable expectation of success. A skilled artisan would have readily recognized that the concentration of exosomes is a results-effective variable which could be optimized (See MPEP 2144.05(II)). Thus, a skilled artisan would be motivated to alter the plating density as taught by Kim in order to generate a culture media comprising UCB-MSC exosomes at the optimal concentration for therapeutic effects in order to eliminate the step of diluting the conditioned media comprising UCB-MSC exosomes to reach the optimal concentration.
With regard to claim 6, as detailed above in claim 1 and incorporated herein, Kim teaches exosome particle analysis isolated from “conditioned media” generated by culture of UCB-MSCs. Kim teaches that exosomes from UCB-MCS had an average particle size of 120nm (Fig. 1, C-iii and Pg. 1104, right col., 1st para.) Additionally, Kim teaches that exosomes derived from UCB-MSCs were smaller than exosomes from other types of MSCs (Pg. 1104, right col., 1st para.) and that exosomes derived from conditioned media of stem cells are of a small size which makes skin regeneration effects higher due to their ability to penetrate the skin (Pg. 1103, left col., 2nd full para. and Pg. 1108, left col., 1st para.). Further, Kim teaches that exosomes form UCB-MSCs exhibited more standardized/consistent small size, which is considered to reasonably read on “mass-produced”, and that the consistent size provides the benefit of ease of commercialization (Pg. 1108, left col., 1st para.).
With regard to claim 7, based on the prior art of Wolvetang, the species of KnockOut DMEM has been rejoined. As detailed above and incorporated herein, the combined teachings of Wolvetang, Kern, and Kim teach culture of umbilical cord blood stem cells in serum free media supplemented with GDF-11 and FGF, both at 20 ng/ml in order to produce “conditioned” culture medium comprising exosomes at a concentration of 1 x 109 particles/ml. While exemplary embodiments of Wolvetang teach use of KOSR medium (Examples 1 and 4, Tables 2 and 4), Wolvetang also teaches that of serum-free Knockout DMEM and FGF can be used for generation of embryonic cell cultures (Pg. 20, 2nd para.).
Wolvetang is silent as to the use of serum-free DMEM.
Kim teaches culture of umbilical cord blood mesenchymal stem cells (UBC-MBCs) in media comprising DMEM and FGF (Pg. 1103, left col., 3rd full para.), which is considered to reasonably read on serum-free media and subsequent isolation, characterization (Pg. 1104, Characterization of USC-CM exosomes), and investigation of potential use of the UBC-MBC derived exosomes for skin regeneration (Figs 3 and 4 and Pg. 1108, left col.).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention to choose DMEM media as taught by Kim in the method of culturing umbilical cord blood stem cells in serum-free media comprising GDF-11 and FGF as taught by the combination of Wolvetang and Kern with a reasonable expectation of success. One having ordinary skill in the art would have been motivated to choose DMEM as taught by Kim since Kim directly teaches culture of umbilical cord blood mesenchymal stem cells, characterization of exosomes released into the culture media by umbilical cord blood mesenchymal stem cells, and subsequent use of the exosomes for skin rejuvenation (Abstract). Therefore, a skilled artisan would be likely to choose use of DMEM media for culture of UBC-MSCs as taught by Kim in order to replicate the culture conditions which showed successful generation culture medium comprising exosomes from USC-MBCs and generation of exosome exhibiting positive effects on skin as a skilled artisan would recognize these characteristics would be important for therapeutic or commercial use of the exosomes.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Wolvetang, in view of Kern and Kim as applied to claim 1 above and further in view of Kim et al. (US 20190111108, hereafter “US ‘108” for clarity ) and Yeo et al. (2013, Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery. Adv. Drug Deliv. Rev., 65(3), 336-341, hereafter “Yeo”).
With regard to claim 5, which depends from claim 1, MPEP 2111.04 (II) indicates that he broadest reasonable interpretation of a method (or process) claim having contingent limitations requires only those steps that must be performed and does not include steps that are not required to be performed because the condition(s) precedent are not met. Claim 5 recites a contingent limitation wherein the exosomes have higher ability to regenerate the skin. Claim 1 does not recite method steps which include application of exosomes to the skin, therefore the ability to regenerate the skin is considered to be contingent on a method step which is not required by claim 1.
As detailed above and incorporated herein, the combined teachings of Wolvetang, Kern, and Kim teach culture of umbilical cord blood stem cells in serum free media supplemented with GDF-11 and FGF, both at 20 ng/ml in order to produce “conditioned” culture medium comprising exosomes at a concentration of 1 x 109 particles/ml. Wolvetang teaches that the culture medium comprising GDF-11 and FGF at the instantly claimed concentrations maintains stem cells in an undifferentiated state (Pg. 1, 2nd para.) which is important for the use of stem cells for therapeutic, clinical, or research purposes (Pg. 2, 3rd para.) and, further, that in the absence of appropriate culture conditions, stem cells will differentiate, losing pluripotent/multipotent potential, which results in a population of cells which is no longer useful for therapeutic, clinical, or research (Pg. 2, last para and Pg. 3, 1st two paras). Thus, Wolvetang’s culture media can be used to culture stem cells which retain “stemness”.
The combined teachings of Wolvetang, Kern, and Kim are silent as to comparison between exosomes from stem cells cultured in serum comprising growth factors and exosomes from stem cells cultured in serum without growth factors. However, Kim teaches that exosomes from UCB-MSCs and media comprising exosomes from UCB-MSCs, which were cultured in medium comprising EGF and FEG (Pg. 1103, Preparation of USC-CM) contain high concentrations of proteins such as EGF and FGF (Pg. 1104, right col., last para.), which is considered to reasonably read on active ingredients.
Yeo teaches that human mesenchymal stem cells (MSCs) are prolific producers of exosomes (Abstract, Fig. 2), have the potential to mass produce exosomes (Pg. 338, left col., 3rd para.), and can be derived from umbilical cord blood (Pg. 338, left col., 4th para.). Yeo teaches that MSCs differentiate into other cell types (Pg. 338, left col., 4th para.) and that although MSCs can be expanded, expansion is also finite leading to a need to constantly replenish the cell source of exosomes (Pg. 339, left col., 2nd para., lines 1-4).
Thus, based on the teachings of Wolvetang that stem cells can be cultured in media supplemented with GDF-11 and FGF such that they maintain “stemness” instead of differentiating and the teachings of Yeo that mesenchymal stem cells are prolific producers of exosomes for a limited amount of time, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, that the culture medium containing exosomes released by umbilical cord blood stem cells cultured in serum-free media supplemented with GDF-11 and FGF as taught by the combined teachings of Wolvetang, Kern, and Kim would result in a greater number of released exosomes which have higher concentrations of active ingredients. A skilled artisan would have recognized, based on the fact that umbilical cord blood stem cells as cultured by the method taught by Wolvetang, Kern, and Kim, that these cells would maintain their stemness instead of differentiating, and, thus would continue to release greater numbers of exosomes, based on the teachings of Yeo.
Claims 8 and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Wolvetang, in view of Kern and Kim as applied to claim 1 above and further in view of Mathen (WO2018150440, found in IDS dated 12/12/2024).
With regard to claims 8 and 9, as detailed above and incorporated herein, the combined teachings of Wolvetang, Kern, and Kim teach culture of umbilical cord blood stem cells in serum free media supplemented with GDF-11 and FGF, both at 20 ng/ml in order to produce “conditioned” culture medium comprising exosomes at a concentration of 1 x 109 particles/ml. Kim further teaches use of the exosomes derived from umbilical cord blood stem cells for use in rejuvenation of skin (Abstract).
While the combination of Wolvetang, Kern, and Kim all teaches regenerative use of exosomes derived from stem cells for skin, the combination of Wolvetang, Kern, and Kim are silent as to formulations of culture media comprising exosomes.
In view of the prior art, the formulation species of claim 9 of gelled or frozen or dried to powder have been rejoined. Mathen teaches a cell-free conditioned medium comprising factors secreted by human stem cells (Pg. 1, 1st para.) produced by cultivating stem cells in culture medium to allow secretion of factors including exosomes (Pg. 4, step (ii); Pg. 7, 1st & 2nd paras.) and subsequent harvesting of the “conditioned culture medium” (Pg. 4, step (iii)). Mathen teaches that the stem cells are human mesenchymal stem cells which can be derived from umbilical cord blood (Pg. 6, 2nd full para.). Additionally, Mathen teaches that the stem cell culture medium can comprise DMEM (Pg 9, 3rd full para.) and FGF (Pg. 5, 1st full para.; Pg 10, 2nd full para.) Further, Mathen teaches that the harvested conditioned medium (Pg. 11, 3rd para.) can be formulated for cosmetic or therapeutic applications by processes such as lyophilization (Pg. 12, 3rd para.), which is considered to reasonably read on frozen to a power as claimed in claim 9, or as a gel (Pg. 14, 1st full para.) and that conditioned media can be used for skin care and wound healing (Pg. 12, 1st para.) and applied topically (Pg. 12, last para.).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention to apply formulation of the conditioned culture medium comprising exosomes as taught by Mathen to the method of culturing umbilical cord blood stem cells in media supplemented with growth factors in order to produce culture medium comprising exosomes as taught by the combined teachings of Wolvetang, Kern, and Kim with a reasonable expectation of success. A skilled artisan would have been motivated to do this as Mathen teaches that culture media comprising exosomes secreted from stem cells can be lyophilized and used for cosmetic or therapeutic purposes and a skilled artisan would have easily recognized that lyophilization provides long-term storage ability of the therapeutic culture media. Further, a skilled artisan would have been motivated to formulate a composition into a gel in order to generate a composition which could be used topically, which is important for commercialization of a cosmetic or therapeutic product.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Wolvetang in view of Kern and Kim as applied to claim 1 above and further in view of Casado-Díaz et al. (2020, Extracellular vesicles derived from mesenchymal stem cells (MSC) in regenerative medicine: applications in skin wound healing. Fron. in Bioeng. and Biotech., 8, 146., found in IDS dated 12/12/2024, hereafter “Casado-Diaz”).
With regard to claim 10, which depends from claim 1, MPEP 2111.04 (II) indicates that he broadest reasonable interpretation of a method (or process) claim having contingent limitations requires only those steps that must be performed and does not include steps that are not required to be performed because the condition(s) precedent are not met. Claim 10 recites a contingent limitation wherein the exosomes improve the migration and/or proliferation ability of human fibroblasts and/or human keratinocytes. Claim 1 does not recite method steps which include application of exosomes to human fibroblasts and/or keratinocytes, therefore the ability to improve the migration and/or proliferation ability is considered to be contingent on a method step which is not required by claim 1.
As detailed above and incorporated herein, the combined teachings of Wolvetang, Kern, and Kim teach culture of umbilical cord blood stem cells in serum free media supplemented with GDF-11 and FGF, both at 20 ng/ml in order to produce “conditioned” culture medium comprising exosomes at a concentration of 1 x 109 particles/ml.
Kim teaches use of exosomes derived from umbilical cord blood mesenchymal stem cells (UCB-MSCs) for use in rejuvenation of skin (Abstract) Kim further teaches that exosomes from UCB-MSCs and media comprising exosomes from UCB-MSCs contain high concentrations of proteins associated with skin rejuvenation such as EGF and FGF (Fig. 2 and Pg. 1104, right col., last para.) and that UCB-MSC exosomes at 1 x 109 particles/ml increased migration in human fibroblasts (Fig. 3D & 3E and Pg. 1105, right col., 1st para.). Additionally, Kim teaches that UCB-MSC exosomes exhibit skin rejuvenation ability and enhance growth and migration of human fibroblast cells (Pg. 1108, left col., 2nd and 3rd paras.). Thus, Kim teaches that UCB-MSC derived exosomes improve proliferation and migration of human fibroblasts.
Kim is silent as to the effect of UCB-MSC exosomes on keratinocytes.
Casado-Diaz teaches that exosomes derived from MSCs are able to activate proliferation and migration of the main cell types involved in skin regeneration, including fibroblasts and keratinocytes (Abstract). Casado-Diaz additionally teaches that MSCs derived from umbilical cord blood have been used for skin healing (Pg. 7, left col., 2nd para.)
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to use exosomes released during culture of umbilical cord blood stem cells in media comprising GDF-11 and FGF which are able to improve proliferation and migration of human fibroblasts taught by the combination of Wolvetang, Kern, and Kim to the improve the proliferation and migration of keratinocytes as taught by Casado-Diaz with a reasonable expectation of success. Based on the teachings of Casado-Diaz and the combination of Wolvetang, Kern, and Kim, a skilled artisan would have every reason to believe that exosomes from umbilical cord blood stem cells cultured in media supplemented with GDF-11 and FGF would improve the proliferation and migration of human keratinocytes, based on the teachings of Casado-Diaz and would have recognized that use of the exosomes released during culture of umbilical cord blood stem cells in media comprising GDF-11 and FGF could be used to promote healing of the skin.
Claims 1 and 3 are rejected under 35 U.S.C. 103 as being unpatentable over Wolvetang in view of Kern, US ‘108, and Kim.
With regard to claims 1 and 3, Wolvetang teaches a method of maintaining stem cells in an undifferentiated state and a culture media for maintenance of stem cells in an undifferentiated state (Pg. 1, lines 10-15). Wolvetang teaches that stem cells can be derived from umbilical cord blood (Pg. 2, lines 5-6) and that the culture media for culturing a stem cell in an undifferentiated state comprises GDF (Pg. 5, lines 1-2). Wolvetang teaches an exemplary embodiment where human embryonic stem cells were cultured in serum-free media supplemented with GDF-11 (also known as BMP-11) at 20 ng/ml and bFGF, which is considered to reasonably read on FGF2, at 20 ng/ml (Pg. 44, lines 29-31) and Pg. 49, lines 24-29) while maintaining their undifferentiated state (Pg. 49, lines 24-29). With regard to claim 16, Wolvetang is considered to teach culture of stem cells in serum-free media supplemented with at least GDF-11 at a concentration of 20 ng/ml.
While Wolvetang does teach that stem cells can be derived from umbilical cord blood, all exemplary embodiments are drawn toward use of embryonic stem cells.
Kern teaches that mesenchymal stem cells (MSCs) derived from umbilical cord blood can be cultured longer and show higher proliferative ability compared to MSCs from other sources (Abstract) thus exhibiting the highest expansion potential amongst the MSCs investigated by Kern (Pg. 1300, right col., 2nd full para., lines 10-11). Further, Kern teaches that MSCs from umbilical cord blood can be obtained less invasively than MSCs derived from other sources (Pg. 1294, left col., 2nd para.).
Therefore it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to choose stem cells derived from umbilical cord blood as taught by Kern for use in the method of culturing stem cells such that stem cells are maintained in an undifferentiated state as taught by Wolvetang with a reasonable expectation of success. Since Kern teaches that stem cells from umbilical cord blood can be obtained relatively easily and exhibit increased potential for expansion, a skilled artisan would be motivated to choose umbilical cord blood stem cells in order to generate the largest number of undifferentiated stem cells which Wolvetang teaches can be used for clinical applications such as generation of disease models, testing of pharmaceuticals and/or therapeutics, and tissue repair and/or regeneration (Pg. 2, lines 25-28). A skilled artisan would have had a reasonable expectation of success as both Wolvetang and Kern teach culture of stem cells and would have every reason to expect that Wolvetang’s culture method would achieve similar results when used to culture umbilical cord blood stem cells.
While the combined teachings of Wolvetang and Kern teach culture of umbilical cord blood stem cells in serum free culture media supplemented with 20 ng/ml of GDF-11 at and 20 ng/ml of FGF2, they are silent as to use of serum-free media supplemented with GDF-11, EGF, FGF, TGF-β1, and VEGF.
US ‘108 teaches culturing of umbilical cord blood-derived mesenchymal stem cells in media comprising DMEM containing EGF, FGF, VEGF and TGF-β1 (Example 1, Para. [0085]). US ‘108 teaches that GDF-11 is present in culture media obtained by culture of umbilical cord blood derived mesenchymal stem cells, reduction of GDF-11 reduces proliferation of human-derived adult stem cells, and that other growth factors such as EGF, FGF, TGF-β1, and VEGF are involved with GDF-11 expression (Paras. [0037] and [0045]; Figs 7A and 8; Examples 7-1, 7-2). Additionally, US ‘108 teaches that addition of growth factors comprising EGF, FGF, TGF-β1, and VEGF to culture media promotes proliferation of stem cells (Para. [0079]). Further, US ‘108 teaches that when DMEM was supplemented with EGF, FGF, TGF-β1, or VEGF at concentrations of 1 ng/ml or 10 ng/ml, GDF-11 expression from umbilical cord blood derived mesenchymal stem cells is increased (Example 7-2, Fig. 8) and that when all four growth factors were added to media, GDF-11 expression was further increased (Example 7-2).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to apply addition of the growth factors EGF, FGF, TGF-β1, and VEGF at the instantly claimed concentrations which is used for culture of umbilical cord blood derived mesenchymal stem cells as taught by US ‘108 to the method of culturing umbilical cord blood stem cells in culture media comprising GDF-11 and FGF as taught by Wolvetang and Kern with a reasonable expectation of success. Since US ‘108 teaches that addition of EGF, FGF, TGF-β1, and VEGF provides the benefit of promoting stem cell proliferation in culture as well as increases GDF-11 expression in umbilical cord blood derived mesenchymal stem cells and Wolvetang teaches that addition of GDF-11 is beneficial for maintaining stem cells in an undifferentiated state (i.e., maintaining “stemness”), a skilled artisan would have recognized that adding EGF, FGF, TGF-β1, and VEGF to culture media, thereby also increasing GDF-11, would result in both increased proliferation and increased maintenance of stemness of umbilical cord blood derived mesenchymal stem cells thus increasing culture efficiency and generating more stem cells for subsequent therapeutic use.
While the combined teachings of Wolvetang, Kern, and US ‘108 teach culture of umbilical cord blood stem cells in serum free culture media supplemented with supplemented with GDF-11, EGF, FGF, TGF-β1, and VEGF at the instantly claimed concentrations, they are silent as to the secretion of exosomes from umbilical cord blood stem cells and at the specific concentration of released exosomes.
Kim teaches that umbilical cord blood derived mesenchymal stem cells (UBC-MSCs) secrete exosomes that can promote cell migration in fibroblasts (Abstract). Kim teaches that UBC-MSCs were seeded at 1.89 x 105 cells per T25 flask and “conditioned” culture media was collected after 96 hours (Pg. 1103, left col., 3rd full para.) prior to exosome isolation and exosome particle analysis (i.e., diameter and concentration). Kim teaches that exosome concentration from UBC-MSCs seeded at 1.89 x 105 cells per T25 flask and maintained in culture for 96 hours was measured as 1.23 x 1011 particles/ml (Table 2). However, for subsequent investigation of effects of UBC-MSC exosome application on fibroblasts, Kim teaches that an optimal exosome concentration of 1 x 109 particles/ml (Pg. 1105, left col. 3rd para.) was used to examine the therapeutic effects of UCB-MSC exosomes on growth of fibroblasts.
As Kim teaches that media from cultured UCB-MSCs comprise exosomes derived from the UCB-MSCs (Abstract), one having ordinary skill in the art would have every reason to believe that the method of culturing stem cells derived from umbilical cord blood as taught by Wolvetang and Kern would generate culture media comprising exosomes which had been released by umbilical cord blood stem cells. Additionally, the concentration of exosomes released into media during the culture of stem cells would be recognized to one having ordinary skill in the art as dependent on the number of stem cells in culture, the size of the culture vesicle, the amount of media, time in culture, etc., therefore, it would be obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to optimize conditions such as plating density, culture vesicle size, amount of media, and/or time in culture in the method of culturing umbilical cord blood derived stem cells as taught by the combination of Wolvetang and Kern to arrive at the optimal concentration of UCB-MCS exosomes for promoting cell growth of 1 x 109 particles/ml as taught by Kim with a reasonable expectation of success. A skilled artisan would have readily recognized that the concentration of exosomes is a results-effective variable which could be optimized (See MPEP 2144.05(II)). Thus, a skilled artisan would be motivated to alter the plating density as taught by Kim in order to generate a culture media comprising UCB-MSC exosomes at the optimal concentration for therapeutic effects in order to eliminate the method step of diluting the conditioned media comprising UCB-MSC exosomes to reach the optimal concentration.
Conclusion
No claims are allowed.
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/ERIN V PAULUS/Examiner, Art Unit 1631
/ARTHUR S LEONARD/Examiner, Art Unit 1631