The Regents of the University of California

Patent Trials and Appeals Board

Dec 2, 2021

2021001690 (P.T.A.B. Dec. 2, 2021)

UNITED STATES PATENT AND TRADEMARK OFFICE
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
15/195,748 06/28/2016 Sophie Xiaohui Deng 30435.258-US-D1 9472
22462 7590 12/02/2021
GATES & COOPER LLP (General)
HOWARD HUGHES CENTER
6060 CENTER DRIVE
SUITE 830
LOS ANGELES, CA 90045
EXAMINER
FOX, ALLISON M
ART UNIT PAPER NUMBER
1633
NOTIFICATION DATE DELIVERY MODE
12/02/2021 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
docketing-us@gates-cooper.com
gates-cooper@blackhillsip.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte SOPHIE XIAOHUI DENG, HUA MEI,
MARTIN NAKATSU, and SHEYLA GONZALEZ1
Appeal 2021-001690
Application 15/195,748
Technology Center 1600
Before ERIC B. GRIMES, ULRIKE W. JENKS, and
RACHEL H. TOWNSEND, Administrative Patent Judges.
GRIMES, Administrative Patent Judge.
DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134(a) involving claims to a
method of maintaining certain stem cells in an undifferentiated phenotype,
which have been rejected as obvious. We have jurisdiction under
35 U.S.C. § 6(b).
We AFFIRM.

1 Appellant identifies the real party in interest as UCLA. Appeal Br. 2.
“Appellant” refers to “applicant” as defined in 37 C.F.R. § 1.42.
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Application 15/195,748

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STATEMENT OF THE CASE
“Corneal epithelial stem cells [are] also referred to as limbal stem
cells (LSCs).” Spec.2 1:27. “Limbal stem cell deficiency (LSCD) in patients
causes inflammation, vascularization, scarring, pain, and ultimately
blindness.” Id. at 2:3–4. “Transplantation of ex vivo expanded LSCs can
successfully restore vision in many affected individuals.” Id. at 2:7–8.
The Specification describes a method “designed to facilitate human
limbal stem/progenitor cell culture including a novel 3-dimensional (3D)
sandwich method in which limbal stem cells and feeder cells are separately
cultured on opposite sides of a porous membrane.” Id. at 3:23–25. The
porous membrane “allow[s] soluble factors to migrate between the . . . cell
culture compartment[s] while simultaneously preventing cells from
migrating between the . . . cell culture compartment[s].” Id. at 4:10–14.
The Specification states that “[l]imbal stem cells grown under these
culture conditions exhibit a small, compact, cuboidal stem-cell morphology
and other stem cell characteristics that [are] comparable to those observed in
limbal stem cells produced from standard culture methods.” Id. at 3:27–4:1.
Claims 16–25 are on appeal. Claim 16, reproduced below, is
illustrative (emphasis added to disputed limitations):
16. A method of maintaining human limbal stem cells of the
corneal epithelium in an undifferentiated human limbal stem
cell phenotype, wherein the phenotype is characterized by a
small, uniform and compact cellular morpology [sic], the
method comprising culturing the human limbal stem cells in a
system comprising:
a container comprising a culture media for the human
limbal stem cells;

2 Substitute Specification filed Sept. 13, 2016.
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a porous membrane disposed in the culture media so as to
form a first cell culture compartment and a second cell culture
compartment, wherein the porous membrane allows soluble
factors to migrate between the first cell culture compartment
and the second cell culture compartment while simultaneously
preventing cells from migrating between the first cell culture
compartment and the second cell culture compartment, wherein
the porous membrane comprises pores having a size less than
3 μm;
human limbal stem cells in combination with a fibrin gel
disposed in the first cell culture compartment, wherein the
human limbal stem cells in combination with the fibrin gel are
formed in the first cell culture compartment by:
disposing the fibrin gel in the first cell culture
compartment; and
seeding the human limbal cells on the fibrin gel
such that the human limbal cells generate a fibrin cell sheet;
human feeder cells disposed in the second cell culture
compartment, wherein:
the second cell culture compartment consists of the
culture media and the human feeder cells;
the human feeder cells secrete one or more soluble
factors that facilitate the growth of the human limbal stem cells;
the human feeder cells comprise human bone marrow
derived mesenchymal stem cells or human adipose derived
mesenchymal stem cells; and
the human limbal stem cells are disposed in the system at
a location and in an orientation selected to influence the polarity
of the human limbal stem cells.
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Application 15/195,748

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OPINION
Claims 16–25 stand rejected under 35 U.S.C. § 103(a) as obvious
based on Omoto3 and Bizik.4 Final Action5 6. The Examiner finds that
Omoto discloses “ex vivo expansion of human limbal epithelial cells
(HLECs) to create transplantable epithelial sheets.” Id. The Examiner finds
that “[t]he HLECs contain human limbal stem cells.” Id.
The Examiner finds that Omoto teaches the use of human bone marrow
mesenchymal stem cells (MASCs) as feeder cells. Id. “Omoto et al use a
Transwell culture system. Omoto et al seed the MASCs on the bottom of the
paired well, coat the inserts with fibrin, inoculate HLECs onto the fibrin-
coated insert, and culture.” Id. The Examiner finds that the cultured cells
“contained p63α-positive and ABCG2-positive cells, both progenitor (stem)
cell markers,” and “at least some cells have a small, uniform and compact
cellular morphology.” Id.
The Examiner finds that Omoto’s method differs from the claimed
method in that “Omoto et al does not teach the pore size of the Millipore
insert (i.e. the porous membrane) as being less than 3 μm.” Id. at 8. However,
the Examiner finds that Bizik discloses methods “for co-culturing
mammalian cells employing a semi-permeable membrane between the
different cell types,” and teaches that “a semi-permeable membrane having a
pore size of 0.2 to 2 μm is appropriate to achieve this goal.” Id. at 6.

3 Masahiro Omoto et al., “The Use of Human Mesenchymal Stem Cell-
Derived Feeder Cells for the Cultivation of Transplantable Epithelial
Sheets,” Investigative Ophthalmology & Visual Science 50:2109–2015
(2009).
4 Bizik et al., US 2009/0298171 A1, published Dec. 3, 2009.
5 Office Action mailed Feb. 26, 2020.
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The Examiner concludes that it would have been obvious “to have
selected a Transwell insert having a pore size of 0.2–2 μm for use in the set-
up of Omoto et al.” because Bizik “teach[es] that pore sizes of 0.2–2 μm are
appropriate for maintaining physical separation between cell types in a co-
culture, but permitting exchange of nutrients and growth factors, which is
what Omoto et al desire.” Id. at 8.
We agree with the Examiner that the method of claim 16 would have
been obvious to a person of ordinary skill in the art based on Omoto and
Bizik. Omoto describes using “human bone marrow-derived mesenchymal
stem cells as a source of feeder cells for the cultivation of transplantable
corneal epithelial cell sheets.” Omoto 2109, left col. Omoto also refers to
these feeder cells as “marrow adherent stem cells” or “MASCs.” Id.
Omoto states that “[c]ell culture inserts (Transwell, catalog number
3450, Corning, Corning, NY) were coated with 300 μL fibrin . . . as
described previously [citing Higa6], and 5 x 105 feeder cells were seeded in
the bottom of the paired well. The next day, human or rabbit limbal
epithelial cells . . . were inoculated in the culture inserts.” Id. at 2110, right
col. Higa, cited in the above quote, states that “[s]tratified corneal epithelial
sheets were prepared in culture dishes coated with biodegradable fibrin
glue.” Higa 597, left col.
More specifically, a solution containing “fibrinogen and . . . thrombin
was . . . spread rapidly onto the upper chambers of a six-well plate with
culture inserts (Transwell . . . ). Two hours later, the polymerized fibrin-

6 Kazunari Higa et al., “Proliferation and Differentiation of Transplantable
Rabbit Epithelial Sheets Engineered with or without an Amniotic Membrane
Carrier,” Investigative Ophthalmology & Visual Science 48:597–604 (2007).
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coated top chambers were obtained.” Id. at 598, left col. Higa states that
limbal epithelial cells “were seeded onto fibrin- or AM[amniotic membrane]-
coated wells” and provided with aprotinin-containing medium. Id. at 598,
right col.
Higa’s Figure 1A is reproduced (in part) below:

Figure 1A shows “[l]imbal epithelial cells . . . seeded on fibrin- or
AM-coated chambers.” Id. at 598, legend to Fig. 1. More specifically, Figure
1A shows a schematic cross-section of one well of the Transwell plate, with
3T3 feeder cells on the bottom of the well, culture medium (“SHEM +
aprotinin”) in the well, and a culture insert (“Fibrin or AM coated chamber”)
submerged in the medium but suspended above the feeder cells. The culture
insert has a stippled layer on the bottom inside surface indicating the fibrin
or AM coating, with rabbit limbal epithelial cells (hatched circles) on the
coating.
“The cultures were submerged in medium until confluence,” then
“incubated without aprotinin.” Id. at 598, right col. Higa states that “[f]ibrin
remained at the bottom of the cell sheet when cultured with aprotinin . . . and
was dissolved after removal of aprotinin, presumably due to intrinsic
proteolytic activity.” Id. at 601, left col. Higa states that its method
“produce[d] carrier-free sheets . . . as [it] allowed the fibrin to be degraded
by intrinsic proteases.” Id. at 597, right col.
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As the Examiner indicated, Omoto does not disclose the pore size of
the membrane in its Transwell system. Bizik, however, discloses a method of
co-culturing fibroblast or mesenchymal multicell aggregates with a patient
cell sample to regulate growth or status of cells in the cell sample. Bizik
¶¶ 30–32. Bizik specifies that “[w]hile co-culturing, there preferably is a
semi-permeable barrier or membrane between said cell sample and the
multicellular aggregates, said barrier permitting exchange of cytokines and
growth factors but separating physically said spheroids [i.e., aggregates] from
said cell sample.” Id. ¶ 34. Bizik states that “[t]he pore size of said semi-
permeable barrier may preferably be 0.2 to 2 μm.” Id.
Thus, it would have been obvious to use a membrane with a pore size
of less than 3 μm in Omoto’s method, because doing so would allow
cytokines and growth factors produced by Omoto’s feeder cells (bone
marrow-derived mesenchymal stem cells) to reach the limbal epithelial cells
growing in its Transwell cell culture inserts, while physically separating the
limbal cells from the feeder cells.
Appellant argues that the method of claim 16 would not have been
obvious based on the cited references because Omoto does not teach
“maintaining human limbal stem cells of the corneal epithelium in an
undifferentiated human limbal stem cell phenotype characterized by a small,
uniform and compact cellular morpology [sic].” Appeal Br. 3. “In particular,
the Examiner’s belief that FIG. 1 in Omoto shows human limbal stem cells
having a small, uniform and compact cellular morpology [sic] is not
correct.” Id. at 4.
Appellant argues that “a detailed analysis of the Omoto disclosure
confirms that FIG. 1 shows feeder cells growing together with human
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epithelial cells and that the limbal epithelial cells invaded beneath these
feeder cells in the MASC feeders and 3T3 feeders.” Id. at 5. Thus, Appellant
argues, “one looking down on cell cultures where human epithelial cells are
beneath the feeder cells indicates that the small, uniform and compact cells
that the Examiner believes are human epithelial cells are in fact feeder cells
growing on top of the human epithelial cells.” Id.
We agree with Appellant that Omoto does not clearly state that the
limbal epithelial cells grown in Transwell cell culture inserts were
“characterized by a small, uniform and compact cellular” morphology, as
recited in claim 16. But we do not find Appellant’s argument with respect to
Figures 1D–1G persuasive of non-obviousness. It is true, as Appellant
points out, that Omoto states that its Figures 1D–1G show that “[e]pithelial
cells invaded beneath feeder cells.” Omoto 2111, legend to Fig. 1. However,
the cells shown in Omoto’s Figure 1 were not grown in the experiment that
used Transwell cell culture inserts. The Figure 1 cell culture was an
experiment carried out in a “dish” to determine colony-forming efficiency
(CFE). Id. at 2110–11. Omoto states that “MASCs and NIH/3T3 feeder
cells were seeded in a 100-mm dish. . . . The next day, 1000 human limbal
epithelial cells were seeded on the dish. After 2 weeks, . . . CFE was
calculated. . . . CFE was compared with use of the Student’s t-test, and P <
0.05 was considered statistically significant.” Id. at 2110, bridging sentence.
Omoto states that its Figure 1B shows that “[h]uman limbal epithelial
cells formed colonies in MASC and 3T3 feeder cells,” and its Figure 1C
shows that “[n]o statistically significant difference was observed in CFE in
MASC or 3T3 feeder cells.” Id. at 2111, legend to Fig. 1. Thus, Omoto’s
disclosure supports a conclusion that its Figure 1 shows limbal epithelial
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cells cultured directly on a layer of feeder cells, not limbal epithelial cells
grown in the manner recited in claim 16.
Although Omoto does not show or describe the morphology of the
limbal epithelial cells grown in its procedure using Transwell cell culture
inserts, we conclude that a preponderance of the evidence of record supports
the Examiner’s finding that such cells have the morphology recited in claim
16. Omoto’s description of its Transwell culturing procedure is found under
the heading “Engineering of Epithelial Cell Sheets.” Omoto 2110, right col.
When describing “Cultivated Human Epithelial Sheets,” Omoto states that
“[h]uman limbal epithelial cells were cultivated with 3T3 feeders and
MASC feeders on fibrin sealant” (id. at 2111, left col.), which is consistent
with its Transwell experiment.
Omoto states that the “cultivated sheets showed a robust epithelial
sheet of five to six layers with cuboid basal cells and flattened superficial
cells when MASC feeders were used (Fig. 2A). The morphology of MASC
feeder sheets was similar to or more robust than that of 3T3 feeder sheets.”
Id. Omoto also states that “[m]ost, if not all, methods for producing
epithelial sheets have used murine-derived 3T3 cells as feeders.” Id. at 2114,
left col.
In summary, Omoto states that the morphology of human limbal
epithelial cells in cell sheets grown using MASC feeder cells was similar to
that of the same cells in cell sheets grown using 3T3 feeder cells, which is
the standard culturing method. For comparison, Appellant’s Specification
states that the claimed method produces “[l]imbal stem cells . . . [that]
exhibit a small, compact, cuboidal stem-cell morphology and other stem cell
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characteristics that [are] comparable to those observed in limbal stem cells
produced from standard culture methods.” Spec. 3:27–4:1.
Thus, the evidence of record supports a conclusion that both the
claimed method and Omoto’s Transwell culturing method produce cells with
a morphology similar to those of limbal stem cells produced from standard
culturing methods.
Where . . . the claimed and prior art products are identical or
substantially identical . . . the PTO can require an applicant to
prove that the prior art products do not necessarily or inherently
possess the characteristics of his claimed product. Whether the
rejection is based on ‘inherency’ under 35 U.S.C. § 102, on
“prima facie obviousness” under 35 U.S.C. § 103, jointly or
alternatively, the burden of proof is the same, and its fairness is
evidenced by the PTO’s inability to manufacture products or to
obtain and compare prior art products.
In re Best, 562 F.2d 1252, 1255 (CCPA 1977) (citations and footnote
omitted).
Appellant also argues that the claims “require that the second cell
culture compartment consist of the culture media and the human feeder
cells.” Appeal Br. 6. Appellant argues that Omoto’s statement that its
Transwell cell culture inserts “were coated” with fibrin “teaches artisans to
include fibrin coatings on the insert that forms the top of the tissue culture
compartment having the feeder cells.” Id.
Appellant argues that
[a] person of ordinary skill . . . would be led to include fibrin
coatings in the tissue culture compartment having the feeder
cells. This is a direction divergent from the path taken by the
appellant in the invention recited current claims, claims
requiring a fibrin coating to be excluded from the second tissue
compartment containing the feeder cells.
Id.
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The Examiner responds to this argument by noting that Omoto
discloses that “the HLECs are seeded onto the fibrin-coated Transwell
insert.” Ans. 7. The Examiner reasons that,
[a]s cells must be seeded onto a base, then the fibrin-coated
Transwell insert is the base/bottom of the upper cell culture
chamber. . . . [T]he upper chamber is defined by the Transwell
culture insert, and the HLECs are seeded onto the bottom
thereof. As the fibrin-coated Transwell insert, per se, is
considered part of the upper cell chamber . . . there is no fibrin
present in the lower cell culture chamber . . . with the feeder
cells in the set-up of Omoto.
Id.
The issue with respect to this dispute is whether Omoto describes its
Transwell set-up as having a fibrin coating only on the upper surface of the
bottom of the insert (the Examiner’s interpretation) or on both the upper and
lower surfaces of the bottom of the insert (Appellant’s interpretation). We
conclude that a preponderance of the evidence favors the Examiner’s
interpretation.
The evidence supporting the Examiner’s position includes Higa’s
Figure 1A7 (a schematic of which is reproduced at page 6 above), which
shows its “Fibrin or AM coated chamber” as a dark line indicating a cylinder
open at the top with a rim overhanging the edge of the well and a stippled
layer indicated on the top surface of the cylinder’s bottom but no stippled
layer indicated on any other surface of the cylindrical insert. A reasonable
interpretation of the figure is that the stippled layer indicates the fibrin or
AM coating on the Transwell insert.

7 Note that Omoto states that its Transwell cell culture inserts “were coated
with 300 μL fibrin . . . as described previously,” and cites Higa. Omoto
2110, right col.
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In addition, Omoto states that it followed Higa’s procedure for coating
the Transwell inserts with fibrin. Omoto 2110, right col. Higa describes that
procedure as follows:
[A] solution containing 40 mg of human fibrinogen and 0.18 U
of thrombin was diluted with 7.5 mL saline, and 0.3 mL was
spread rapidly onto the upper chambers of a six-well plate with
culture inserts (Transwell . . .). Two hours later, the
polymerized fibrin-coated top chambers were obtained.
Higa 598, left col. (emphasis added).
Higa’s statement that a diluted solution containing fibrinogen and
thrombin was “spread” onto the upper chambers implies that the solution
was spread across a surface and kept in place by gravity, which would apply
only to an upward-facing surface. By contrast, if Higa meant that the cell
culture inserts were coated on all of their surfaces, it would be more
reasonable to describe the inserts as being “dipped into” the solution, or the
solution being “sprayed onto” the inserts.
Finally, Omoto states that “limbal epithelial cells . . . were inoculated
in the culture inserts” and cultured in medium containing aprotinin, a
protease inhibitor. Omoto 2110, right col. After the cells reached confluence,
they were cultured for another three days without aprotinin. Id. Higa, using
the same procedure, states that “[f]ibrin remained at the bottom of the cell
sheet when cultured with aprotinin . . . and was dissolved after removal of
aprotinin, presumably due to intrinsic proteolytic activity.” Higa 601, left
col. Higa thus describes growing the epithelial cells on a layer of fibrin,
which was known to be used as a carrier for cells sheets, then “allow[ing]
the fibrin to be degraded by intrinsic proteases” to produce carrier-free
sheets. See id. at 597, right col.
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In contrast to the evidence discussed above that supports the
Examiner’s interpretation of Omoto, Appellant’s interpretation is supported
only by Omoto’s statement that its Transwell cell culture inserts “were
coated with 300 μL fibrin.” Omoto 2110, right col. This statement is
ambiguous regarding the surface(s) coated with fibrin and, for the reasons
discussed above, we conclude that a preponderance of the evidence of record
supports the Examiner’s interpretation that the fibrin coating was limited to
the inner surface of the bottom of the insert.
In summary, we affirm the rejection of claim 16 under 35 U.S.C.
§ 103(a) based on Omoto and Bizik. Claims 17–25 were not argued
separately and fall with claim 16. 37 C.F.R. § 41.37(c)(1)(iv).
DECISION SUMMARY
In summary:
Claims
Rejected
35 U.S.C. § Reference(s)/Basis Affirmed Reversed
16–25 103(a) Omoto, Bizik 16–25
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a). See 37 C.F.R.
§ 1.136(a)(1)(iv).
AFFIRMED