U.S. patent application number 17/398982 was filed with the patent office on 2022-07-07 for patch graft compositions for cell engraftment.
This patent application is currently assigned to The University of North Carolina at Chapel Hill. The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Lola M. REID, Eliane Wauthier, Wencheng Zhang.
Application Number | 20220211911 17/398982 |
Document ID | / |
Family ID | 1000006213448 |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220211911 |
Kind Code |
A1 |
REID; Lola M. ; et
al. |
July 7, 2022 |
PATCH GRAFT COMPOSITIONS FOR CELL ENGRAFTMENT
Abstract
Compositions and methods of transplanting cells by grafting
strategies into solid organs (especially internal organs) are
provided. These methods and compositions can be used to repair
diseased organs or to establish models of disease states in
experimental hosts. The method involves attachment onto the surface
of a tissue or organ, a patch graft, a "bandaid-like" covering,
containing epithelial cells with supporting early lineage stage
mesenchymal cells. The cells are incorporated into soft gel-forming
biomaterials prepared under serum-free, defined conditions
comprised of nutrients, lipids, vitamins, and regulatory signals
that collectively support stemness of the donor cells. The graft is
covered with a biodegradable, biocompatible, bioresorbable backing
used to affix the graft to the target site. The cells in the graft
migrate into and throughout the tissue such that within a couple of
weeks they are uniformly dispersed within the recipient (host)
tissue. The mechanisms by which engraftment and integration of
donor cells into the organ or tissue involve multiple
membrane-associated and secreted forms of MMPs.
Inventors: |
REID; Lola M.; (Chapel Hill,
NC) ; Zhang; Wencheng; (Chapel Hill, NC) ;
Wauthier; Eliane; (Chapel Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Assignee: |
The University of North Carolina at
Chapel Hill
Chapel Hill
NC
|
Family ID: |
1000006213448 |
Appl. No.: |
17/398982 |
Filed: |
August 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16006464 |
Jun 12, 2018 |
11129923 |
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17398982 |
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62518380 |
Jun 12, 2017 |
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62664694 |
Apr 30, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2506/1346 20130101;
C12N 5/0676 20130101; A61L 27/26 20130101; C12N 5/0668 20130101;
A61L 27/56 20130101; A61L 27/52 20130101; A61L 27/3813 20130101;
A61L 27/3839 20130101; A61L 27/20 20130101; A61L 27/3834 20130101;
A61L 27/58 20130101; A61L 27/227 20130101; A61L 27/3604 20130101;
A61L 27/3886 20130101; C12N 5/0662 20130101; A61L 27/3641 20130101;
C12N 5/067 20130101 |
International
Class: |
A61L 27/38 20060101
A61L027/38; A61L 27/52 20060101 A61L027/52; A61L 27/58 20060101
A61L027/58; A61L 27/56 20060101 A61L027/56; A61L 27/36 20060101
A61L027/36; A61L 27/20 20060101 A61L027/20; A61L 27/26 20060101
A61L027/26; C12N 5/071 20060101 C12N005/071; C12N 5/0775 20060101
C12N005/0775; A61L 27/22 20060101 A61L027/22 |
Claims
1-26. (canceled)
27. A covering or coating for a patch graft or tissue, comprising a
hydrogel or other biomaterial with sufficient viscoelasticity and
resilience to withstand mechanical forces, including such forces
from other tissues and organs.
28. The covering or coating for a patch graft or tissue of claim
27, wherein the hydrogel comprises one or more hyaluronans.
29. The covering or coating for a patch graft or tissue of claim
27, wherein the hydrogel has a viscoelasticity from about 250 to
500 Pa.
30. The covering or coating for a patch graft or tissue of claim
27, wherein the hydrogel has a viscoelasticity from about 250 Pa to
about 350 Pa.
31. The covering or coating for a patch graft or tissue of claim
27, wherein the patch graft or tissue is engrafted to a target
organ, and wherein the mechanical forces are exerted by tissues and
organs adjacent to the target organ.
32. The covering or coating for a patch graft or tissue of claim
31, wherein the target organ is selected from the group consisting
of liver, pancreas, biliary tree, thyroid, thymus thymus,
intestines, lung, prostate, breast, brain, spinal cord, neural
ganglia, skin and underlying dermal tissues, uterus, bone, tendon,
cartilage, kidney, muscle, blood vessels, or heart.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 16/006,464, filed Jun. 12, 2018, which claims priority
under 35 U.S.C. .sctn. 119(e) from U.S. Provisional Patent
Application No. 62/518,380, filed Jun. 12, 2017, and to U.S.
Provisional Patent Application No. 62/664,694, filed Apr. 30, 2018,
the contents of which are hereby incorporated by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 9, 2021, is named 069961-2829_SL.txt and is 6,662 bytes in
size.
FIELD OF THE INVENTION
[0003] The present invention is directed generally to the field of
transplantation of cells or tissue engrafting. More specifically,
from solid organs or tissues into solid organs or tissues,
especially to internal organs. The invention concerns compositions
and methods providing strategies for the rapid transplantation,
engraftment and integration of cells into solid organs and tissues
to treat diseases or conditions of solid organs or tissues, or to
establish model systems of a disease. Representative examples of
this potential are cell therapies for treatment of hepatic or
pancreatic diseases.
BACKGROUND OF THE INVENTION
[0004] There has long been a need for grafting strategies for cells
from solid organs, strategies distinct from those used for
transplantation of hemopoietic cells or for mesenchymal
stem/progenitors. Turner, R., et al. Transplantation 90, 807-810
(2010); Gattinoni, L. et al. Nature Medicine 23, 18-27 (2017);
Trounson A. et al. Cell Stem Cell 17, 11-22 (2015); Sun B. K. et
al. Science 346, 941-945 (2014); Lainas, P. et al. J Hepatol 49,
354-362 (2008). Transplantation of hematopoietic cells and of
mesenchymal cells is done routinely by delivery of cells via a
vascular channel and is dependent on activation of adhesion
molecules in transplanted cells when in relevant target sites
because of micro-environmental signaling, a process referred to as
"homing." Methods used for skin (with similar ones for ocular
targets) employ grafting methods with cells applied directly to
target sites. Sun B. K. et al. Science 346, 941-945 (2014). Many
grafting methods for skin are utilizable for cells from solid
internal organs but require substantial modifications to
accommodate the microenvironment of these internal organs. Grafts
must contend with mechanical forces exerted by interactions of
tissues and organs on each other; examples include the effects of
lungs during breathing, or the compression of the liver against the
diaphragm, or the transient effects of mechanical forces exerted by
the intestinal tract on neighboring tissues during processing of
foods. Grafts, especially those for internal organs, are
challenging to design because of concerns with respect to size,
shape, and complexity in the structure of organs in addition to the
dynamic mechanical forces evident.
[0005] For decades, cell therapies for cells from solid organs
other than skin were attempted using transplantation via a vascular
route or by direct injection into the tissue. Most transplanted
cells, when delivered by either of these strategies, either die or
are transported to ectopic sites, where they can live for months
and create tissue in inappropriate sites, resulting potentially in
adverse effects clinically. Turner, R., et al. Transplantation 90,
807-810 (2010); Lanzoni, G. et al. Stem Cells 31, 2047-2060 (2013).
Engraftment in liver can be improved by coating the cells with
hyaluronans and delivering them vascularly to the liver; the
increased efficiency of engraftment is due to the liver's natural
process of clearance of hyaluronans. Nevi et al. Stem Cell Research
& Therapy 8, 68, 2017. However, this improvement is still less
efficient than that with grafting strategies and, importantly,
still allows for delivery of cells to ectopic sites.
[0006] There remains a need for improved methods of cell
engraftment into solid organs. This disclosure fulfills this need
and provides related advantages.
SUMMARY OF THE INVENTION
[0007] There has long been a need for grafting strategies for cells
from solid organs (Turner, R., et al. Transplantation 90, 807-810
(2010), strategies distinct from those used for transplantation of
hemopoietic cells, mesenchymal stem cells or for skin.
Transplantation of hemopoietic cells and mesenchymal cells is done
routinely via a vascular channel and is dependent on activation of
adhesion molecules in relevant target sites because of
micro-environmental signaling, a process referred to as "homing".
Methods used for skin employ grafting methods with cells applied
directly to target sites.
[0008] Transplantation of cells from solid organs other than skin
have long used vascular delivery. This is not logical, since
adhesion molecules on these cells are always activated and result
in rapid (seconds) cell aggregation that can generate
life-threatening emboli. Even if emboli are managed successfully to
minimize health risks, the efficiency of cell engraftment is low,
only .about.20% for adult cells and even lower (<5%) for
stem/progenitors. Most transplanted cells either die or are
transported to ectopic sites, where they can live for months,
creating tissue in inappropriate sites resulting in possible
adverse effects clinically. The small percentage of cells that
engraft into target sites integrate slowly, requiring weeks to
months to become a significant portion of the tissue. There is
improvement in engraftment in liver if cells are coated with
hyaluronans and delivered vascularly due to the tissue's (e.g.
liver's) clearance of hyaluronans. (Nevi et al. Stem Cell Research
& Therapy 8, 68, 2017).
[0009] Applicants propose a radically different approach, one found
even more successful than coating cells with hyaluronans: placing
grafts directly onto the surface of the target site and using
grafting biomaterials and the unique phenotypic traits of certain
cells when they are in conditions of the graft biomaterials to
enhance transplantation. This parallels some aspects of strategies
of cell therapies for skin but requires substantial, modifications
for internal organs given mechanical effects, abrasion or
compression of organs near to each other, and given the unique
fluid microenvironments around specific organs and the size,
structure, and complexity of organs.
[0010] Described herein are novel patch graft compositions and
methods for transplantation of cells into tissue and solid organs.
In some embodiments, the methods and grafts are adapted for
internal organs, with design features dependent on the level of
maturity of the cells, especially whether cells are stem cells or
mature cells. In some embodiments, the donor cells (optionally
autologous or allogenic) for the patch grafts are disclosed herein
incorporated into the graft biomaterials in optionally as a mixture
of cells or the form of organoids, aggregates of epithelial stem
cells and their native, lineage-stage appropriate mesenchymal cell
partners--e.g. mesenchymal stem/progenitor cells such as early
lineage stage mesenchymal cells (ELSMCs). In some embodiments, the
donor cells are adult cells incorporated into the graft materials
as cell suspensions of adult epithelia and partnered with
mesenchymal stem/progenitor cells, optionally ELSMCs, at ratios
designed to optimize their expression of membrane-associated and/or
secreted matrix metallo-proteinases (MMPs). In some embodiments,
other variables of importance are the grafting biomaterials and the
backing material, both required to be neutral in effects on the
differentiation of the donor cells.
[0011] Aspects of the disclosure relate to a patch graft for
sustaining and maintaining a single cell population or a mixed
population of cells, comprising: (a) a single cell type or a mixed
population having two or more cell types, at least one of which is
at an early lineage stage that is capable of expressing
membrane-associated and/or secreted matrix metalloproteinases
(MMPs), or which has MMPs included from another source (e.g.,
purified or recombinant MMPs), said cell population or mixed
population supported in a medium present in a hydrogel matrix
having a viscoelasticity sufficient to allow for migration of said
mixed population, optionally, within or away from said hydrogel
and/or within or away from the patch graft; (b) a backing
comprising a biocompatible, biodegradable material having a
viscoelasticity sufficient to inhibit a migration of said mixed
population in a direction of said backing; and, optionally, (c) a
hydrogel overlaid on a serosal (i.e. outside) surface of said
backing, which is opposite to that in contact with said mixed
population and, in embodiments where the patch graft is tethered to
a target site, is opposite the side in contact with the target site
(e.g. organ or tissue). In some embodiments, this layer prevents or
inhibits adhesions by or from other tissues or organs. In some
embodiments, the patch graft is configured to sustain and maintain
said mixed population while inhibiting said at least one early
lineage stage cell type from differentiating or further maturing to
a later lineage stage that is no longer capable of expressing
membrane-associated and/or secreted MMPs. The patch graft may be a
single layer plus a backing or multiple layers.
[0012] In some embodiments, said backing is porous or non-porous.
In some embodiments, the backing comprises a porous mesh, scaffold,
or membrane. In some embodiments, the backing comprises silk; a
synthetic textile; or a natural material such as aminion, placenta,
or omentum; or a combination thereof. In some embodiments, said
backing comprises a porous mesh infused with a hydrogel. In further
embodiments, such an infusion prevents cell migration away from the
target organ or tissue. In some embodiments, said backing comprises
a solid material.
[0013] In some embodiments, one or more of said hydrogels comprise
hyaluronans.
[0014] In some embodiments, said medium comprises Kubota's medium
or another medium supportive of stem cells and able to maintain
stemness.
[0015] In some embodiments, said mixed population comprises
mesenchymal cells and epithelial cells. In some embodiments, said
epithelial cells may be ectodermal, endodermal, or mesodermal. In
some embodiments, said mesenchymal cells comprise early lineage
stage mesenchymal cells (ELSMCs). In some embodiments, said ELSMCs
comprise one or more of angioblasts, precursors to endothelia,
precursors to stellate cells, and mesenchymal stem cells (MSCs). In
some embodiments, said epithelial cells comprise epithelial stem
cells. In some embodiments, said epithelial cells comprise biliary
tree stem cells (BTSCs). In some embodiments, said epithelial cells
comprise committed and/or mature epithelial cells. In some
embodiments, said committed and/or mature epithelial cells comprise
mature parenchymal cells. In some embodiments, said mature
parenchymal cells comprise one or more of hepatocytes,
cholangiocytes, and islet cells. In some embodiments, said
mesenchymal cells and epithelial cells both comprise stem
cells.
[0016] In some embodiment said mixed population comprises
autologous and/or allogeneic cells.
[0017] In some embodiments, one or more cell types are genetically
modified.
[0018] Further aspects related to methods employing the disclosed
patch graft compositions. Accordingly, provided herein are methods
of engrafting cells into a target tissue comprising, consisting of,
or consisting essentially of contacting the target tissue with a
patch graft disclosed herein above.
[0019] In some embodiments of the methods, the target tissue is
selected from the group consisting of liver, pancreas, biliary
tree, thyroid, thymus, gastrointestine, lung, prostate, breast,
brain, bladder, spinal cord, skin and underlying dermal tissues,
uterine, kidney, muscle, blood vessel, heart, cartilage, tendons,
and bone tissue. In some embodiments of the methods, the target
tissue is liver tissue. In some embodiments of the methods, the
target tissue is pancreatic tissue. In some embodiments of the
methods, the target tissue is biliary tree tissue. In some
embodiments of the methods, the target tissue is gastrointestinal
tissue. In some embodiments, the tissue is diseased, damaged, or
has a disorder. In some embodiments of the methods, the target
tissue is kidney tissue.
[0020] In some embodiments of the methods, the target tissue is an
organ. In some embodiments of the methods, the organ is an organ of
the musculoskeletal system, the digestive system, the respiratory
system, the urinary system, the female reproductive system, the
male reproductive system, the endocrine system, the circulatory
system, the lymphatic system, the nervous system, or the
integumentary system. In some embodiments of the methods, the organ
is selected from the group consisting of liver, pancreas, biliary
tree, thyroid, thymus, stomach, intestines, lung, prostate, breast,
brain, bladder, spinal cord, skin and underlying dermal tissues,
uterus, kidney, muscle, blood vessel, heart, cartilage, tendon, and
bone. In some embodiments, the organ is diseased, damaged, or has a
disorder.
[0021] Also provided herein are methods of treating a subject with
a liver disease or disorder, the methods comprising, consisting of,
or consisting essentially contacting the subject's liver a patch
graft disclosed herein above. In some embodiments of the methods,
the liver disease or disorder is liver fibrosis, liver cirrhosis,
hemochromatosis, liver cancer, biliary atresia, nonalcoholic fatty
liver disease, hepatitis, viral hepatitis, autoimmune hepatitis,
fascioliasis, alcoholic liver disease, alpha 1-antitrypsin
deficiency, glycogen storage disease type II, transthyretin-related
hereditary amyloidosis, Gilbert's syndrome, primary biliary
cirrhosis, primary sclerosing cholangitis, Budd-Chiari syndrome,
liver trauma, or Wilson disease.
[0022] In other aspects, provided herein are methods of treating a
subject with a disease or disorder of the pancreas, the methods
comprising, consisting of, or consisting essentially of contacting
the subject's pancreas with a patch graft disclosed herein above.
In some embodiments of the methods, the disease or disorder of the
pancreas is diabetes mellitus, exocrine pancreatic insufficiency,
pancreatitis, pancreatic cancer, sphincter of Oddi dysfunction,
cystic fibrosis, pancreas divisum, annular pancreas, pancreatic
trauma, or hemosuccus pancreaticus.
[0023] In other aspects, provided herein are methods of treating a
subject with a gastrointestinal disease or disorder, the method
comprising, consisting of, or consisting essentially of contacting
one or more of the subject's intestines with a patch graft
disclosed herein above. In some embodiments, the gastrointestinal
disease or disorder is gastroenteritis, gastrointestinal cancer,
ileitis, inflammatory bowel disease, Crohn's disease, ulcerative
colitis, irritable bowel syndrome, peptic ulcer disease, celiac
disease, fibrosis, angiodysplasia, Hirschsprung's disease,
pseudomembranous colitis, or gastrointestinal trauma.
[0024] In some aspects, provided herein are methods of treating a
subject with a kidney disease or disorder, the methods comprising,
consisting of, or consisting essentially of contacting one or more
of the subject's kidneys with a patch graft disclosed herein above.
In some embodiments of the methods, the kidney disease or disorder
is nephritis, nephrosis, nephritic syndrome, nephrotic syndrome,
chronic kidney disease, acute kidney injury, kidney trauma, cystic
kidney disease, polycystic kidney disease, glomerulonephritis, IgA
nephropathy, lupus nephritis, kidney cancer, Alport syndrome,
amyloidosis, Goodpasture syndrome, or Wegener's granulomatosis.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIGS. 1A-1D provides information about porcine donor cells
for the patch grafts. FIG. 1A is a schematic of the process and
estimates of the time required for preparing organoids, assembling
patch grafts and doing the surgeries. In FIG. 1B, donor cells for
the stem cell patch grafts were isolated from cell suspensions of
biliary tree tissue from transgenic pigs; the cells were prepared
as organoids in serum-free Kubota's Medium and on low attachment
culture dishes. Organoids of biliary tree stem cells (BTSCs) and of
their early lineage stage mesenchymal cell (ELSMCs) partners,
angioblasts and precursors to endothelia and to stellate cells.
They are shown in a phase micrograph versus one demonstrating
expression of the transgene, green fluorescent protein (GFP). All
of the cells of the aggregate are green, since the transgene is in
both the epithelial cells and the mesenchymal cells. The transgene
was coupled to the histone (H-2B) locus. Histology of the stem cell
organoids that were paraffin embedded, sectioned and stained with
hematoxylin/eosin. (d) Magnified image of an organoid of BTSC and
ELSMCs. FIG. 1C shows immunohistochemistry (IHC) demonstrating
expression of stem cell, hepatic and pancreatic markers indicating
that these cells are precursors to both liver and to pancreas. The
IHC assays indicate outer layers with intermediate stage stem cell
markers such as EpCAM and interior cells expressing very primitive
genes such as pluripotency genes and endodermal transcription
factors (e.g. SOX17, SOX9, PDX1). FIG. 1D is a representative
qRT-PCR assays assessing expression of various genes in the
organoids and indicating that cells are stem cells or early
progenitors. The controls were mature hepatocytes from piglet
livers.
[0026] FIGS. 2A-2F provides information about the major components
of patch grafts. FIG. 2A is a schematic of a patch graft affixed to
the liver of a pig, and on the right, the composition of the
grafts. Early lineage stage cells, both the epithelia and the
mesenchymal cells, are sources for production of matrix
metallo-proteinases (MMPs), key regulators of engraftment. The
matrix components of the graft biomaterials into which donor cells
are placed are soft (.about.100 Pa), without (or with minimal)
sulfation, such as hyaluronan hydrogels. The structure of the graft
consist of layers of biomaterials and cells tethered to the target
site. The medium components are devoid of serum, growth factors and
cytokines influential to differentiation of the donor cells and
should be ones tailored for survival and expansion of early lineage
stage cells such as stem/progenitors. The backing has sufficient
tensile strength to be used in surgical procedures but be neutral
in its effects on the differentiation of the donor cells (e.g. ones
with type I collagen should be avoided). The backing is impregnated
or coated with a more rigid 10.times. hydrogel (.about.700 Pa) to
serve as a barrier to orient the migration of donor cells towards
the target tissue and to minimize adhesions. After attachment to
the target site, a 2.times. HA hydrogel, one that is sufficiently
fluid to be coated or painted onto the serosal surface, is added
and used to further minimize adhesions. FIG. 2B depicts the graft
affixed to the liver or the pancreas of a host. FIG. 2C is a
schematic of the graft demonstrating the layers constituting the
graft composition. FIG. 2D depicts the results of assays
empirically assessing the rheological or viscoelastic properties
(shear and compressive mechanical forces) of the specific hydrogel
layers. FIG. 2E provides a formulation of the viscoelastic
properties of the 3 layers of hydrogels. FIG. 2F is a close up
image of a patch graft sutured to the surface of the liver of a
pig.
[0027] FIGS. 3A-3D depicts the result of immunohistochemistry (IHC)
and histology of the liver patch grafts. FIG. 3A (Panels (a)-(d))
shows the results of Trichrome staining of the patch graft at one
week. Trichrome identifies collagens (blue), cytoplasm (red) and
nuclei (black), and it was used to identify Glisson's capsule
(normally adjacent to the surface of liver lobules) and adhesions
(on the serosal surface of the grafts). There is a high level of
blue staining in the layers at the serosal surface and implicate
adhesions to the graft. Also, the graft has separated from the host
tissue at the interface between the backing and the host; this was
found frequently due to the wealth of MMPs produced at this
interface. The remodeling regions provide evidence of the loss of
classic lobule structure of the liver; they result in a region in
which the donor cells are migrating into the tissue and, in
parallel, altering the host tissue structure. In low magnification
images (a), Trichrome staining of grafts placed on to the liver
validated that extensive remodeling of the Glisson capsule was
occurring and resulted often in a separation between the graft and
the host liver. In higher magnification images (b) the remodeling
region is remarkably broad and consisting of areas (c) near to the
graft where liver lobule structure is missing altogether and (d)
regions within the remaining liver lobules that are undergoing
breakdown in the remodeling process. FIG. 3B (Panels (a)-(b)) shows
the results of Trichrome staining of the patch graft at three
weeks. Hyaluronans in the graft have been resorbed leaving only the
backing (a). With resorption of HA, the Glisson capsule reappears
(b) and the liver lobules near to the graft have stabilized again
into their typical histological patterns, such as lobule and acini
for liver. The arrow in (b) indicates the reappearance of collagens
in the reformation of the Glisson capsule. FIG. 3C (Panels (a)-(c))
and FIG. 3D (Panels (a)-(c)) shows the results of hematoxylin/eosin
staining of a section from the grafts at one week post grafting (C)
and two weeks post grafting (D). The figures at the top are
40.times.. At sites within the figure (a,b,c) are enlargements that
are magnified at 100.times.; the rectangular image below each of
these is magnified at 200.times.. Shown are 3 sites of the graft:
(a) a site within the backing and associated graft biomaterials;
(b) a site at the interface between graft and host tissue; and (c)
a site within the liver lobules. The hematoxylin/eosin staining
yields images that contribute to an appreciation of the engraftment
and migration process that incorporates features of inflammatory
processes.
[0028] FIGS. 4A-4D shows engraftment, migration and rapid
maturation to adult fates within a week. FIG. 4A is a low
magnification image of the patch graft on the surface of a pig
liver after one week. The dashed line indicates the interface of
the graft and host liver. Donor GFP+ cells (with pink nuclei; white
arrows indicate areas with large numbers of the donor GFP+ stem
cells) were visualized by labeling with an antibody to GFP and
secondarily with one coupled to Novo Red, a red fluoroprobe. Nuclei
were stained blue with 4,6-Diamidino-2-phenylindole (DAPI) enabling
recognition of host cells having only blue nuclei and donor ones
having pink nuclei (merge of DAPI and the Novo Red). FIG. 4B
(Sections (a)-(b)) show Host tissue (a) extending into the
hyaluronans (HA, the black background) of the graft; tissue by the
backing contains occasional organoids (inset) but with most donor
cells dispersed into single cells; large numbers of dispersed donor
GFP+ stem cells (b) are seen throughout the host tissue. There is
no evidence for the Glisson capsule in this area that constitutes
the region of remodeling. FIG. 4C demonstrates that engraftment and
migration of donor cells was rapid; within a week, all donor cells
were within the host liver; there were donor cells both near the
graft site and also on the opposite side of the liver lobe
(estimate of the distance is at least 1.5 cm from the graft).
Ongoing studies are analyzing regions of the piglet livers at
greater distances (i.e. other lobes of the liver) to define more
precisely how far the migration can go by the donor cells within a
defined period of time. Shown are donor cells (pink nuclei) near
lobules of host mature hepatocytes (forest green color from
auto-fluorescence of lipofuscins) on the distant side of the liver
lobe from that of the graft site. FIG. 4D (Panels (a)-(b)) shows
that maturation of donor cells to adult fates occurred in parallel
with HAs being resorbed. Enlargement of a region containing donor
GFP+ cells (single cells with pink nuclei) near to host hepatocytes
(a), forest green in color (autofluorescence of lipofuscins), and
readily distinguished from mature donor-derived (b) hepatocytes
that are lavender in color (merge of the pink--GFP, blue--DAPI, and
the green--lipofuscins), that is they were lineage restricted from
donor GFP+ stem cells. With other IHC assays (data not shown), the
bright, spring green color of cells amidst the plates of both host
and donor hepatocytes proved to be endothelia and stellate
cells.
[0029] FIGS. 5A-5C compares engraftment and maturation of cells in
the liver patch grafts after one week and two weeks
post-transplantation. FIG. 5A is an examination of porcine liver at
1 week after patch grafting. Sirius red stain, an azo dye staining
collagens was used and immunohistochemistry for pan-cytokeratin
(pCK) and Sox9; and immunofluorescence (IF) stains were performed
on serial 3-.mu.m sections. At the patch graft site, grafted donor
cells merged with liver lobules. In the upper panels (original
magnification=5.times.), patch grafts are composed of mesenchymal
and epithelial pCK.sup.+ cells (arrows). In middle panels, a higher
magnification is provided (20.times.). Epithelial cells show an
immunophenotype that is typical of biliary tree stem cells (BTSCs)
expressing biliary cytokeratins (pCK) and the endodermal stem cell
marker Sox9. BTSCs within the patch graft are arranged in cell
strings reassembling bile ductules (arrows) and are in direct
continuity with hepatocyte plates of the adjacent liver lobule
(arrowheads). Host hepatocytes in lobules are pCK and Sox9
negative. In lower panels (Original magnification=20.times.), the
immunofluorescence for GFP allows one to identify individual
grafted cells and their progeny. Hepatocytes in lobules adjacent to
the patch graft were GFP positive indicating that these were donor
cells derived that had merged with host liver parenchyma. At the
interface between patch graft and liver lobules,
pCK.sup.+/GFP.sup.+ ductules (that is donor derived cholangiocytes)
were in direct continuity with GFP.sup.+/pCK.sup.+ cells
(donor-derived hepatocytes) within the lobules (arrowheads)
suggesting a maturation of grafting cells towards an hepatocyte
fate. FIG. 5B is an examination of porcine livers 2 weeks after
patch grafting. IF stains reveal that GFP.sup.+ cells are present
within lobules distant to the graft site. They are dispersed
uniformly and so are in a mix of host cells (ones with blue nuclei
from DAPI) and of donor cells (pink/purple nuclei from merge of the
blue from DAPI and the red of the GFP label). They co-express
mature hepatocyte markers such as Hepatocyte Nuclear Factor (HNF)
4.alpha. (a mix of green and pink/purple nuclei) and albumin (green
cytoplasm and with pink/purple nuclei). Separate or merged channels
were included. Nuclei were displayed in blue (DAPI). Original
Magnification: 40.times.. FIG. 5C (Panels 1-3) is an evaluation of
porcine livers a week after patch grafting and demonstrating the
broad region of remodeling that occurs at the interface between the
patch graft and the host tissue. The section in the low
magnification image and in the enlarged image of 1 is
hematoxylin/eosin (lightly stained); that in 2 is stained with
Vector-SG providing a blue/gray color; that in 3 is stained for
alpha-fetoprotein with hematoxylin/eosin background. Specific sites
within 5C are numbered and correlate with enlargements that
indicate the changes occurring within the lobules. The host liver
lobules and acini are breaking down due to the wealth of MMPs
flooding into the area along with the donor cells. The donor cells
are observed at the boundary regions of the lobules, sites also
demonstrating liver-specific markers such as HNF4-a and
.alpha.-fetoprotein, meaning that the cells are maturing to a liver
fate. These traits were not expressed by the BTSCs and so these are
indications that the donor cells are undergoing maturation to an
hepatic fate.
[0030] FIGS. 6A-6D provides information about patch grafts of stem
cell organoids tethered to pancreas. FIG. 6A is a low magnification
(panoramic scan) image of GFP+ donor cells that have engrafted into
much of the pancreas and into the submucosa of the duodenum (a
region containing Brunner's Glands). Immunofluorescent staining of
pig pancreas, liver, and duodenum in the site of the patch graft.
GFP (green), Insulin (red), DAPI (blue). Donor-derived GFP+ cells
occur in the proximity of the site where the patch graft was
positioned, and appear integrated in the pancreas parenchyma. The
silk mesh of the SERI surgical scaffold is observed interposed
among pancreas, liver, and duodenum. FIG. 6B shows that donor cells
mature to functional islets. At higher magnification, donor-derived
GFP+/Insulin+ beta cells (yellow--from merge of the GFP and of the
red from staining for insulin) are observed intermingled with host
GFP-/Insulin+ (red) beta cells in the pancreas parenchyma.
Surrounding the islet cells are a large number of GFP+ cells
displaying a morphology consistent with that of pancreatic exocrine
cells, including acinar and ductal cells. Supporting this
interpretation are the findings in C and D that, indeed, these
cells are producing amylase, a classic acinar marker. FIG. 6C and
FIG. 6D show evidence of functional acinar cells derived from donor
stem cells. Immunofluorescent staining of a serial section from the
same tissue block in the site of the patch graft and with focus on
the region of engrafted GFP+ donor cells. Amylase (green), Insulin
(red), Glucagon (white--not visible in the panoramic scan in C, but
visible at the higher magnification in D), DAPI (blue). Amylase+
acinar cells are the vast majority of the exocrine tissue of the
pancreas. By comparing the staining presented in the serial
sections at low and high magnifications, it is deduced that most of
the donor-derived GFP+ cells in the pancreas have acquired an
amylase+ acinar fate.
[0031] FIGS. 7A-7H offers a characterization of
matrix-metallo-proteinases (MMPs). MMPs are comprised of a large
gene family of calcium-dependent, zinc-containing enzymes that
dissolve extracellular matrix components. There are at least 24
isoforms known in pigs of which a subset are secreted factors (e.g.
MMP1, MMP2, MMP7, MMP9) and a subset are membrane-associated (e.g.
MMP14, MMP15). MMP1 was identified by IHC, especially in the areas
of remodeling, but not by RNA-seq, since there has not yet been an
annotated form of porcine MMP1 available for RNA seq analyses. FIG.
7A, FIG. 7B, FIG. 7C, and FIG. 7D show isoforms of secreted and
membrane-associated categories were expressed by both
stem/progenitors and mature cells. Quantitation of the expression
levels indicated that the membrane-associated forms were similar
for both stem/progenitors and mature cells (note the comparisons in
FIG. 7D). By contrast, secreted forms were expressed at very high
levels in stem/progenitors and at low or negligible levels in
mature cell types. The cell populations of adult cells analyzed
were isolated from suspensions of piglet livers and biliary tree
tissue and comprised of CD45+ cells (hemopoietic cells), CD146+
cells (stellate cells), CD31+ cells (endothelia), EpCAM+/CD45-
cells (adult diploid hepatocytes and cholangiocytes. These
EpCAM+/CD45- cells are the mature parenchymal cells found in piglet
livers. The BTSCs were isolated from the biliary tree by the
protocols given in the examples. FIG. 7E shows representative MMP
expression in regions of remodeling with a BTSC/ELSMCs graft. In a
section adjacent to the patch graft of BTSCs/ELSMCs were stained
with Trichrome indicating the region (bracket) of remodeling. The
region appears as linear stripes of red and blue being cells and
matrix components undergoing dissolution by the "sea" of MMPs. The
stripes end at the edges of lobules that are still mostly intact
but beginning to "fray" at their boundaries from the effects of the
MMPs derived from the invading cells. FIG. 7F shows representative
images of IHC assays for MMP1 (Novo-red+). Methyl green is the
background stain. The liver's lobular/acinar structure has
dissolved into the undulating swirls of cells and marked by the
strong expression of MMP1, a secreted isoform of MMPs. FIG. 7G
shows a section stained for MMP2 (Novo-red+). Hematoxylin is the
background stain. The liver's lobular/acinar structure has
disappeared and has been replaced by a mix of cells with strong
staining for MMP2 (rust brown color). FIG. 7H shows the remodeling
process ongoing within the liver lobules. The liver lobules have
become strips of cells interspersed by invading cells; MMP2+
expression (rust colored) is very high and contributing to the loss
of lobular/acinar structures. With clearance of hyaluronans (by 2-3
weeks), the lobular structures reappeared.
[0032] FIG. 8 is a schematic demonstration of the engraftment and
integration phenomena in liver and on pancreas.
[0033] FIGS. 9A-9E provides information about patch grafts of
mature (adult) hepatocytes partnered with mature mesenchymal cells
(MMCs), such as endothelia or stellate cells. These patch grafts
were unable to engraft. Engraftment was achievable if the
hepatocytes were partnered with early lineage stage mesenchymal
cells (ELSMCs), here being porcine mesenchymal stem cells (MSCs).
If presented with ELSMCs, then engraftment occurred but with
restriction to regions near to the graft. FIG. 9A (Panels
(a)-(b))_shows Trichrome staining of normal pig liver. Bar is 200
.mu.m for low magnification image (a) and 50 .mu.m for the higher
magnification image (b). Note the collagens in the Glisson capsule
and the boundaries between hepatic acini. FIG. 9B (Panels (a)-(b))
shows Trichrome staining of patch graft of normal, adult
hepatocytes partnered with mature mesenchymal cells (MMCs),
endothelia and stellate cells, did not engraft. In the low
magnification image (a) note that the Glisson capsule is intact,
and cells remain atop the capsule. (b) at the higher magnification,
there is evidence of some remodeling (plasticity phenomena) of
cells in the lobule next to the graft (the mottled red color within
the hepatocytes). This plasticity is assumed due to the
membrane-associated MMPs known to be present on both stem cells and
adult cells. FIG. 9C (Panels (a)-(c)) shows IHC assays on patch
graft of normal, adult hepatocytes partnered with mature
mesenchymal cells (MMCs). At the higher magnification (a), it is
evident that engraftment has not occurred. This section was stained
with antibody to RBMY-1 and with hematoxylin as the counterstain
(b). The Glisson capsule is intact and so are the boundary zones
between lobules, and (c) negative control (staining without primary
antibody) to indicate non-specific staining. FIG. 9D (Panels
(a)-(c)) shows Trichrome staining of patch graft of normal, adult
hepatocytes partnered with ELSMCs that here were porcine
mesenchymal stem cells (MSCs) played the role of a cellular source
of MMPs. The graft is separating at the interface between the graft
and the host tissue. The bracket indicates the region of
remodeling. Note that the liver lobules have lost the matrix that
normally constitutes boundary zones between them and appear frayed
at the edges. In the higher magnification (a) are seen donor cells
(pale red compared with the dark red ones in the centers of the
lobules) throughout the image; in (b) is an enlargement of a region
showing that the Glisson capsule is considerably thinner under the
patch (compare with region to the left of the box) and in (c).
Extensive remodeling was evident in the cells adjacent to the graft
(c). FIG. 9E (Panels (a)-(d)) shows a patch graft of hepatocytes
partnered with ELSMCs (porcine MSCs) after one week. The section
(a) was stained with antibody to RBMY-1 (brown) and with methyl
green as the counter stain. The donor cells engrafted (regions of
rust red color) and matured into adult parenchymal cells in the
acini near to the graft. The section (b) shows an enlargement of
the image near to the remains of the thinned Glisson capsule showed
that donor cells (dark brown nuclei) were interspersed uniformly
with host cells (nuclei were methyl green color). The section (c)
is the negative control for (b). The section (d) was stained with
antibody to GFP (coupled with Novus red and yielding a rust brown
color) and with methyl green as the counter stain. Most of the
cells have engrafted and formed a band of dark red, donor (mature)
hepatocytes within the host liver acini. The Glisson capsule
remained but was diminished in thickness. Migration much beyond the
region of the liver near to the graft was not observed within the
three-week time-frame of the experiments.
[0034] FIG. 10 is a schematic comparing engraftment of stem cells
versus adult cells.
[0035] FIG. 11 shows evidence that the engraftment process involves
migration of cells to considerable distances within the host
tissue. Here is demonstrated that for grafts of BTSCs/ELSMCs
organoids at one week post-transplantation. The schematic of the
liver divided into 8 different zones is used to indicate the
regions evaluated for the presence of donor cells. Sections are
prepared from the regions 1-8 and then stained to enable
identification of donor cells. In the table are summarized the
findings showing the distances between the graft and each region
and the proportion of GFP+ cells found. The images to the left of
the table are scans of a representative section from each zone. The
dark brown staining is strongest in 6 near to the graft and is
fainter with increasing distance from the graft, the palest being
zone 1.
[0036] FIGS. 12A-12E provides evidence for migration of donor cells
throughout the host liver. GFP+ cells stained with Novo-red (rust
brown color); host cells are stained with methyl green. FIG. 12A
(Panels (a)-(b)) is a low magnification image of interface of graft
and the host liver. The separation of the graft from the host liver
was often seen (note this also in FIG. 3) and was shown correlated
with exceptionally high levels of secreted MMPs. Enlargement of the
regions (a) and (b) are given below. Note the areas in the low
magnification image and in the enlargement in (b) in which staining
is mottled and with areas showing a washed out appearance and that
proved due to hyaluronan levels in the tissue. FIG. 12B depicts the
intermediate zones to which the cells migrated. Donor cells are
throughout the tissue, both in bile ducts and in the parenchyma of
the acini. FIG. 12C shows the distance zones to which the cells
migrated. Note that only the bile ducts are stained. FIG. 12D
provides enlargements showing donor cells in bile ducts. FIG. 12E
provides enlargements within the parenchyma to show that the donor
cells have GFP labeling in the nuclei.
[0037] FIGS. 13A-13D shows the adverse conditions obtained for
patch grafts with certain backings (see also Tables 1 and 2). These
included necrosis, adhesions, and sites of cholestasis found to
occur when grafts were placed too close to some ducts such that the
swelling caused occlusion of the ducts.
[0038] FIG. 14 shows a chart of both lineage stages for epithelial
cells (FIG. 14A) and mesenchymal cells (FIG. 14B) and the
corresponding biomarker profiles.
[0039] FIG. 15 (Panels A-E) shows organoids of H2B-GFP+
BTSCs/ELSMCs patch grafted onto the Kidney. Evaluation was done at
1-week-post-grafting. Panel A shows Trichrome staining of grafted
kidney. The kidney was prepared in cross-section to expose the
deeper layer that with the graft as a "V" shape. The lower half "V"
with bright blue staining is the graft side on the kidney; the
upper "V" in the figure is a deeper layer to the grafted layer.
Panel B shows H&E staining for the same section of the grafted
kidney. Panel C is the higher magnification of the patch grafted
kidney. The capsule of the kidney under the graft was loosened
(from dissolution by MMPs) in a fashion similar to that in the
liver. Panel D shows IHC staining of GFP+ cells (dark red) that
have engrafted into the kidney at a layer under the patch. Panel E
shows engraftment of the GFP+ cells (dark red) at deeper layers of
the kidney. Necropsy reports indicated that there was no necrosis
found in the grafted kidney or elsewhere in the animals that were
subjected to patch grafts.
BRIEF DESCRIPTION OF THE TABLES (APPENDIX)
[0040] TABLE 1 provides a summary of surgical or other approaches
for patch grafting.
[0041] TABLE 2 provides a comparison of backings tested for the
exemplary patch grafts.
[0042] TABLE 3 provides a summary of the antibodies used for IHC
and IF in the examples.
[0043] TABLE 4 provides a summary of the primers (SEQ ID NOS 1-28,
respectively, in order of appearance) used for qRT-PCR assays.
DETAILED DESCRIPTION
[0044] Embodiments according to the present disclosure will be
described more fully hereinafter. Aspects of the disclosure may,
however, be embodied in different forms and should not be construed
as limited to the embodiments set forth herein. Rather, these
embodiments are provided so that this disclosure will be thorough
and complete, and will fully convey the scope of the invention to
those skilled in the art. The terminology used in the description
herein is for the purpose of describing particular embodiments only
and is not intended to be limiting of the invention. All
publications, patent applications, patents and other references
mentioned herein are incorporated by reference in their
entirety.
[0045] Unless otherwise defined, all terms (including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. It will be further understood that terms, such
as those defined in commonly used dictionaries, should be
interpreted as having a meaning that is consistent with their
meaning in the context of the present application and relevant art
and should not be interpreted in an idealized or overly formal
sense unless expressly so defined herein. While not explicitly
defined below, such terms should be interpreted according to their
common meaning.
[0046] The practice of the present technology will employ, unless
otherwise indicated, conventional techniques of tissue culture,
immunology, molecular biology, microbiology, cell biology, and
recombinant DNA, which are within the skill of the art. See, e.g.,
Sambrook and Russell eds. (2012) Molecular Cloning: A Laboratory
Manual, 4rd edition; the series Ausubel et al. eds. (2012) Current
Protocols in Molecular Biology; the series Methods in Enzymology
(Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A
Practical Approach (IRL Press at Oxford University Press);
MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and
Lane eds. (2014) Antibodies, A Laboratory Manual, 2d edition;
Freshney (2011) Culture of Animal Cells: A Manual of Basic
Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis;
U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1985) Nucleic Acid
Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames
and Higgins eds. (1984) Transcription and Translation; Immobilized
Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical
Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene
Transfer Vectors for Mammalian Cells (Cold Spring Harbor
Laboratory); Makrides ed. (2003) Gene Transfer and Expression in
Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical
Methods in Cell and Molecular Biology (Academic Press, London); and
Herzenberg et al. eds (1996) Weir's Handbook of Experimental
Immunology.
[0047] Unless the context indicates otherwise, it is specifically
intended that the various features of the invention described
herein can be used in any combination. Moreover, the disclosure
also contemplates that in some embodiments, any feature or
combination of features set forth herein can be excluded or
omitted. To illustrate, if the specification states that a complex
comprises components A, B and C, it is specifically intended that
any of A, B or C, or a combination thereof, can be omitted and
disclaimed singularly or in any combination.
[0048] All numerical designations, e.g., pH, temperature, time,
concentration, and molecular weight, including ranges, are
approximations which are varied (+) or (-) by increments of 1.0 or
0.1, as appropriate, or alternatively by a variation of +/-15%, or
alternatively 10%, or alternatively 5%, or alternatively 2%. It is
to be understood, although not always explicitly stated, that all
numerical designations are preceded by the term "about." It also is
to be understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
Definitions
[0049] As used in the description of the invention and the appended
claims, the singular forms "a," "an" and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise.
[0050] The term "about," as used herein when referring to a
measurable value such as an amount or concentration (e.g., the
percentage of collagen in the total proteins in the biomatrix
scaffold) and the like, is meant to encompass variations of 20%,
10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.
[0051] The terms or "acceptable," "effective," or "sufficient" when
used to describe the selection of any components, ranges, dose
forms, etc. disclosed herein intend that said component, range,
dose form, etc. is suitable for the disclosed purpose.
[0052] Also as used herein, "and/or" refers to and encompasses any
and all possible combinations of one or more of the associated
listed items, as well as the lack of combinations when interpreted
in the alternative ("or").
[0053] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
do not exclude others. As used herein, the transitional phrase
"consisting essentially of" (and grammatical variants) is to be
interpreted as encompassing the recited materials or steps "and
those that do not materially affect the basic and novel
characteristic(s)" of the recited embodiment. See, In re Herz, 537
F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in
the original); see also MPEP .sctn. 2111.03. Thus, the term
"consisting essentially of" as used herein should not be
interpreted as equivalent to "comprising." "Consisting of" shall
mean excluding more than trace elements of other ingredients and
substantial method steps for administering the compositions
disclosed herein. Aspects defined by each of these transition terms
are within the scope of the present disclosure.
[0054] As used herein, the term "patch graft" refers to a
composition of cells embedded or comprised in an appropriate
biomaterial that allows for transplanting donor cells (allogeneic
or autologous) to the host. In some embodiments, the term refers to
a composition of cells embedded or comprised in an appropriate
biomaterial that allows for transplanting donor cells to the host.
Biomaterials are ones that can be prepared under defined conditions
(e.g., a basal medium optionally supplemented and/or a medium of
nutritional factors, vitamins, amino acids, carbohydrates,
minerals, insulin, transferrin/Fe, and/or lipids (purified free
fatty acids complexed with purified albumin plus a lipoprotein
carrier molecule such as high density lipoprotein)) and comprised,
optionally solidified, into a soft gel (under 200 Pa, optionally
approximately 100 Pa), and covered with a backing that has
sufficient tensile strength to enable surgical attachment or
otherwise tethered to a tissue or organ of the host and yet be of a
chemistry with minimal effects on the differentiation of the donor
cells. To be avoided are supplements with factors that might drive
differentiation of the cells, especially the early lineage stage
mesenchymal cells (ELSMCs); these include serum, growth factors and
cytokines affecting ELSMCs, and mature matrix components (e.g. type
I collagen).
[0055] The term "backing," as used herein, refers to a material
that serves as a backing or barrier on the surface of the patch
graft capable of tethering the graft to a target site and/or
facilitating migration of the cells therein to the target site
and/or preventing or inhibiting migration of the cells toward the
backing. The backing is or comprises a "biodegradable,
biocompatible material," "biocompatible, biodegradable material,"
or any variation thereof referring to a material which (i) is
biocompatible with the subject into which it is being transplanted,
(ii) exhibits mechanical resilience to withstand the compressive
and shear forces that occur on organs and tissues (especially
internal ones), which in turn enables this material to function as
a surgical tissue, and (iii) has a neutral or minimal effect on the
differentiation status of cells that come in contact with the
material. In some embodiments, the backing of the patch graft
comprises such a material. In such embodiments, the mechanical
resilience of (ii) should be such that the backing can be tethered
the graft to the target site. In further such embodiments, backing
directs cell migration toward the target site--e.g. by affecting
the differentiation of those cells migrating in directions away
from the target site or by physically blocking said migration. In
this regard, suitable materials include but are not limited to
Seri-silk, optionally contour Seri-Silk, or derivatives thereof,
aminions or extracts thereof (for example, of the side facing the
fetus and/or a patch or textile comprised of PGA and/or PLLA.
Non-limiting examples of suitable patches of synthetic materials
include a woven patch comprised of 91% PGA-co-9% PLLA, a knit patch
comprised of 91% PGA-co-9% PLLA, or a non-woven patch comprised of
100% PGA. More generally, suitable backings may include forms of
Bombyx moth silk such as Seri.RTM. Surgical Silk Scaffolds
(Sofregen, New York, N.Y.), other derivatives of Bombyx moth silk,
and synthetic textiles, such as forms of Polyglycolic
acid-co-poly-L-lactic acid (PGA/PLLA).
[0056] In some embodiments, the backing is also bioresorbable. As
used herein, "bioresorbable" refers to a material that can be
broken down by the body of a host or recipient of the graft and
does not require mechanical removal. In some embodiments, the
bioresorbable backing is bioresorbable within a span of about 2 to
about 10 weeks, about 2 to about 20 weeks, about 2 to about 52
weeks, about 4 to about 16 weeks, about 4 to about 12 weeks, or
about 4 to about 8 weeks. In some embodiments, the bioresorbable
backing is bioresorbable within a span of about 4 to about 8 weeks;
about 4 to about 12 weeks, about 4 to about 16 weeks, about 4 to
about 20 weeks, and about 4 to about 52 weeks.
[0057] As used herein, the biomaterials of the graft, and
independent of the backing, include ones that can form hydrogels.
The term "gel" refers to a solid jelly-like material that can have
properties ranging from soft and weak to hard and tough. Gels are
defined as a substantially dilute cross-linked system, which
exhibits no flow when in the steady-state. By weight, gels are
mostly liquid, yet they behave like solids due to a
three-dimensional cross-linked network within the liquid. It is the
crosslinking within the fluid that gives a gel its structure
(hardness, stiffness, mechanical, or viscoelasticity properties)
and contributes to its adhesivity. In this way gels are a
dispersion of molecules of a liquid within a solid in which the
solid is the continuous phase and the liquid is the discontinuous
phase. A "hydrogel," also referred to herein as a "hydrogel
matrix," is a non-limiting example of a gel comprised of a
macromolecular polymer gel constructed of a network of polymer
chains. Hydrogels are synthesized from hydrophilic monomers or
hydrophilic dimers (e.g. in the case of hyaluronan) by either chain
or step growth, along with network formation. A net-like structure
along with void imperfections enhance the hydrogel's ability to
absorb large amounts of water via hydrogen bonding. As a result,
hydrogels develop characteristic firm yet elastic mechanical
properties. They are able to undergo spontaneous formation of new
bonds when old bonds are broken within a material. The structure of
the hydrogels along with electrostatic attraction forces drive new
bond formation through non-covalent hydrogen bonding.
[0058] The biomaterials used for the grafts have mechanical
properties, stiffness, that can be more rigorously defined as the
viscoelasticity of the biomaterials. See
https://en.wikipedia.org/wiki/Viscoelasticity. The graft
biomaterials conducive to engraftment must be very soft (for
example, about 100 Pa), conditions permissive for the donor cells
to remain immature (Lozoya et al. Biomaterials 2011; 32 (30):
7389-7402) and so be able to produce membrane-associated and/or
secreted forms of MMPs.
[0059] As used herein, the term "viscoelasticity" refers to the
property of materials that exhibit both viscous and elastic
characteristics when undergoing deformation. Viscous materials,
like honey, resist shear flow and strain linearly with time when a
stress is applied. Elastic materials strain when stretched and
quickly return to their original state once the stress is removed.
Viscoelastic materials have elements of both of these properties
and, as such, exhibit time-dependent strain. Whereas elasticity is
usually the result of bond stretching along crystallographic planes
in an ordered solid, viscosity is the result of the diffusion of
atoms or molecules inside an amorphous material. Though there are
many instruments that test the mechanical and viscoelastic response
of materials, broadband viscoelastic spectroscopy (BVS) and
resonant ultrasound spectroscopy (RUS) are more commonly used to
test viscoelastic behavior because they can be used above and below
ambient temperatures and are more specific to testing
viscoelasticity. These two instruments employ a damping mechanism
at various frequencies and time ranges with no appeal to
time-temperature superposition. Using BVS and RUS to study the
mechanical properties of materials is important to understanding
how a material exhibiting viscoelasticity will perform
[0060] As used herein, the term "hyaluronan," or "hyaluronic acid,"
refers to a polymer of disaccharide units comprised of glucosamine
and glucuronic acid [1-3] linked by .beta.1-4, .beta.1-3 bonds and
salts thereof. Thus, the term hyaluronan refers to both natural and
synthetic forms of hyaluronans. The naturally occurring hyaluronan
(HA), water-soluble polysaccharide comprising disaccharide units of
D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc),
which are alternately linked, forming a linear polymer. High
molecular weight HA may comprise 100 to 10,000 disaccharide units.
HAs often occur naturally as the sodium salt, sodium hyaluronate.
HA; sodium hyaluronate, and preparations of either HA or sodium
hyaluronate are often referred to as "hyaluronan." Non-limiting
examples of acceptable hyaluronate salts, include potassium
hyaluronate, magnesium hyaluronate, and calcium hyaluronate.
[0061] Other glycosaminoglycans (GAGs) can also be used in the
hydrogel. These include forms of chondroitin sulfate (CSs) and
dermatan sulfates (DSs), polymers of glucuronic acid and
galactosamine, and heparan sulfates (HSs) and heparins (HPs),
polymers of glucuronic acid and glucosamine. The extent and pattern
of sulfation of these GAGs are critical, since the sulfation
patterns dictate the formation of complexes with multiple families
of proteins (e.g. coagulation proteins, growth factors, cytokines,
neutrophilic enzymes). See, e.g., Powell A K, Yates E A, Fernig D
G, Turnbull J E. Interactions of heparin/heparan sulfate with
proteins: appraisal of structural factors and experimental
approaches. Glycobiology. 2004 April; 14(4):17R-30R] Those
appropriate for patch grafts that optimize engraftment comprise
hyaluronans, non-sulfated GAGs, and ones with minimal sulfation
such as forms of chondroitin sulfates found in stem cell niches, as
shown in Karumbaiah L, et al. Chondroitin Sulfate Glycosaminoglycan
Hydrogels Create Endogenous Niches for Neural Stem Cells. Bioconjug
Chem. Dec. 16, 2015; 26(12):2336-49 and Hayes A J, et al.
Chondroitin sulfate sulfation motifs as putative biomarkers for
isolation of articular cartilage progenitor cells. J Histochem
Cytochem. 2008 February; 56(2):125-38 (incorporated herein by
reference).
[0062] As used herein, the term "cell" refers to one or more cells
in the graft. The cells of the present disclosure are eukaryotic.
In some embodiments, this cell is of animal origin, optionally from
a human organ, and can be a stem cell, a mature somatic cell,
progenitor cell, or intermediates in the lineage stages from the
stem cells to the mature cells. The term "population of cells" or
"cells" refers to a group of one or more cells of the same or
different cell type with the same or different origin; this term is
used interchangeably herein with the term "donor cells," which
intend cells that may be autologous or allogeneic. In some
embodiments, this population of cells may be derived from a cell
line, from freshly isolated cells, or in some embodiments, this
population of cells may be derived from a portion of an organ or
tissue, optionally from a donor or a recipient.
[0063] The term "stem cell" refers to cell populations that can
self-replicate (produce daughter cells identical to the parent
cell) and that are multipotent, i.e. can give rise to more than one
type of adult cell. The term "progenitor cell" or "precursor" as
used herein, is broadly defined to encompass progeny of stem cells
and their descendants. Progenitors are cell populations that can be
multipotent, bipotent, or unipotent but have minimal (if any)
ability to self-replicate. Committed progenitors are ones that are
unipotent and can differentiate into a particular lineage leading
to only one mature cell type. Non-limiting examples of stem cells
include but are not limited to embryonic stem (ES) cells, induced
pluripotent stem (iPS) cells, germ layer stem cells, determined
stem cells, (ectodermal, mesodermal or endodermal), perinatal stem
cells, amniotic fluid-derived stem cells, mesenchymal stem cells
(MSCs), angioblasts, and those derived from umbilical cord,
Wharton's jelly, and/or placenta. Intermediates between stem cells
and committed progenitors include cell populations such as
hepatoblasts and pancreatic ductal progenitors and other forms of
transit amplifying cells that may be multipotent but have extensive
proliferative potential but more limited (if any) self-replicative
ability.
[0064] The term "mesenchymal cells" refers to cells derived from
the mesenchyme, including but not limited to angioblasts,
precursors to endothelia, precursors to stellate cells, endothelia,
stellate cells, stromal cells, various subpopulations of mature and
progenitor cells, and mesenchymal stem cells (MSCs) which are
multipotent stromal cells and various subpopulations of mature and
progenitor mesenchymal cells. The MSCs are cell populations
prepared by culture selection processes from tissues (Cathery et
al. Stem Cells 2018; PMID:29732653; Graceb et al. Biochimie 2018:
PMID 29698670; Caplan AI. Stem Cells Int. 2015; PMID: 26273305.
There are at least two major categories of mature mesenchymal
cells: (a) Mature mesenchymal cells (stellate/stromal cells) that
produce and are surrounded by forms of extracellular matrix that
comprise fibrillar collagens (e.g. type I, III, V) and associated
matrix components (fibronectins, chondroitin sulfate proteoglycans,
dermatan sulfate proteoglycans) and bound signals (e.g. growth
factors, cytokines) that form a complex and bound signals (e.g.
growth factors/cytokines) that form a complex associated with cells
that are typically linear (string-like) cell populations.
Nonlimiting examples of such cells include stellate cells, tendon,
stroma, and myofibroblasts. (b) Mature mesenchymal cells such as
endothelia that produce and are surrounded by forms of
extracellular matrix that comprise network collagens (e.g. type IV,
type VI, VIII, X) and associated matrix molecules (laminins,
heparan sulfate proteoglycans, heparin proteoglycans) and bound
signals (e.g. growth factors, cytokines) that together are
associated with cells having more squamous or cuboidal or
cobblestone morphologies. Nonlimiting examples of such cells
include endothelia and myoepithelial.
[0065] The precursors to these mesenchymal cell types include but
are not limited to angioblasts which are multipotent and that can
differentiate into lineages of endothelia (the late stages of which
are fenestrated endothelia) or stellate cells (the late stages of
which are myofibroblasts (stroma). The precursors also include
mesenchymal stem cells (MSCs) which are multipotent cells and can
differentiate into fibroblasts (stroma), osteoblasts (bone cells),
chondrocytes (cartilage cells), myocytes (muscle cells) and
adipocytes (fat cells)). The MSCs may optionally be prepared by
culture selection methods (Cathery et al. Stem Cells 2018;
PMID:29732653; Graceb et al. Biochimie 2018: PMID 29698670; Caplan
A L. Stem Cells Int. 2015; PMID: 26273305.
[0066] The term "epithelial cell expansion" is correlated with the
diameter of a colony of epithelial cells that typically form
colonies with cuboidal or cobblestone morphologies and with
estimates of growth being the composite of the diameters of the
cells of the colony. By contrast, estimates of growth of
mesenchymal cell colonies are correlated with the density of the
colony, since the mesenchymal cells are more migratory and motile,
and the colony density is a reflection of the net sum of cells that
remain within the colony boundaries.
[0067] The term "epithelial cells" refers to cells derived from the
epithelium, specialized cells that provide diverse functions for
the tissue and/or the systemic needs of a host. They are recognized
by their ability to migrate as precursors or immature cells; with
maturation, they become stationary and form layers of squamous or
cobblestone-like or columnar polarized cells with apical, basal and
lateral sides, and that are bound to each other by an assortment of
junctions (connexins, tight junctions, adherens). Their expansion
potential is indicated by the diameter of a colony (not by its
density). The mature epithelial cells provide diverse functions
such as secretion of specialized products or contributions to
metabolism (hepatocytes, cholangiocytes), detoxification
(hepatocytes), production of enzymes (acinar cells), production of
endocrine factors (e.g. islets or other endocrine cells)),
electrical activity (neuronal cells), and absorption (intestinal
cells).
[0068] The term "biliary tree stem cells" (BTSCs) refers to
epithelial stem cells found throughout the biliary tree and located
within peribiliary glands (PBGs), Brunner's Glands, both extramural
and intramural, as well as within the crypts of gallbladder villi.
They have the ability to transition into committed hepatic and/or
pancreatic progenitor cells The hepatic descendants enter into the
liver sinusoids via canals of Hering; the pancreatic progenitors
are found within pancreatic duct glands (PDGs), regions of the
biliary tree located within the pancreas.
[0069] Thus far, at least 7 subpopulations of stem cell populations
have been identified with overlapping traits and ranging from
extremely primitive BTSCs to stem cell populations definable as
hepatic or pancreatic stem cells. Description of what is known for
these is given below. The most primitive ones are found in both the
extramural peribiliary glands--ones tethered to the surface of the
bile ducts--and; the intramural peribiliary glands--ones found
within the bile duct walls. The intramural peribiliary glands
(PBGs) near to the fibromuscular layer in the centers of the bile
duct walls can also be considered crypts (with parallels to
intestinal crypts), niches in which are found the most primitive
stem cell populations. The largest numbers of the PBGs within the
biliary tree network are found within the hepato-pancreatic common
duct and within the large intrahepatic bile ducts. No PBGs occur in
the gallbladder, and instead the stem cell niches within the
gallbladder are the bottoms of the gall bladder villi that contain
intermediate to late stage stem cell populations that are
precursors to hepatic stem cells. The BTSCs are precursors to both
liver and to pancreas. They give rise to hepatic stem cells,
precursors to liver, and to pancreatic stem cells, precursors to
pancreas, and these are found throughout the biliary tree but in
numbers influenced by whether near to the liver versus the
pancreas. Thus, small numbers of pancreatic stem cells and large
numbers of hepatic stem cells are located in the PBGs of the large
intrahepatic bile ducts, whereas small numbers of hepatic stem
cells and large numbers of pancreatic stem cells are located in the
PBGs of the hepato-pancreatic common duct.
[0070] Summaries of genetic signatures are presented in the
Figures. In general, all of the BTSCs subpopulations express
generic biomarkers that include endodermal transcription factors
for both liver and pancreas (e.g. SOX9, SOX17, PDX1), pluripotency
genes (e.g. OCT4, SOX2, NANOG, SALL4, KLF4/KLF5, BMI-1); one or
more of the hyaluronan receptor isoforms (standard and/or variant
isoforms) of CD44; CXCR4; and cytokeratins 8 and 18. Stem cell
subpopulations within the biliary tree and PBGs include (1)
Brunner's Glands stem cells in the submucosa of the duodenum and
that express CK7, TRA-160 and 181 and with traits distinguishable
from stem cells in the intestine; (2) early stage intramural
Biliary Tree Stem Cell (BTSCs) that express sodium iodide symporter
(NIS) and CXCR4, OCT4, SOX2, NANOG, but do not express LGR5 or
EpCAM; (3) intermediate stage intramural BTSCs that express less of
NIS but gain expression of LGR5 but not EpCAM; (4) late stage
intramural BTSCs (the only BTSCs found in the gallbladder) and also
found in high numbers in the large intrahepatic bile ducts and in
the hepato-pancreatic common duct. They express both LGR5 and
EpCAM. These are precursors to hepatic stem cells (in the liver and
expressing SOX17 but not PDX1) and to the pancreatic stem cells (in
the hepato-pancreatic common duct and expressing PDX1 but not
SOX17); (5) hepatic stem cells may be found in the canals of
Hering, in PBGs of the large intrahepatic bile ductules, in PBGs in
the extrahepatic biliary tree; and in the PBGs of the
hepato-pancreatic common duct, but the highest numbers are those at
intrahepatic sites. The hepatic stem cells retain the ability to
self-replicate and to be multipotent. The biomarkers for these
cells include SOX9, SOX17, HNF-4 alpha, ITGB1 (CD29), ONECUT 2,
SALL4, LGR5, CD44, epithelial cell adhesion molecule (EpCAM) found
in the cytoplasm and at the plasma membrane, neural cell adhesion
molecule (NCAM), CD133 (prominin), negligible levels (or none) of
albumin, a complete absence of alpha-fetoprotein (AFP), an absence
of P450 A7, and an absence of secretin receptor (SR). Hepatic stem
cells and hepatoblasts express cytokeratins 8, 18 and 19; (6)
pancreatic stem cells are found in small numbers throughout the
biliary tree (even in the PBGs in the large intrahepatic bile
ducts) but are found in high numbers in PBGs of the
hepato-pancreatic common duct. They have the pluripotency genes and
expression for the other genes noted for all of the stem cell
populations, but they differ in no longer having SOX17; the
subpopulations that will lineage restrict to islets express NGN3.
They express EpCAM throughout the cells and at the plasma membrane
and express low (or no) insulin. Maturation of them is correlated
with increasing insulin expression as well as with expression of
other islet hormones (e.g. glucagon). Those maturing into acinar
populations will express MUC6 and amylase.
[0071] It is noted that hepatic and pancreatic stem cells may also
be found in their respective source organs when they are early in
development (e.g. as ESCs or otherwise), and that any of those
cells disclosed herein may be alternatively generated through
induction (i.e. as iPSCs).
[0072] As used herein, the term "supportive" is used to describe
cells which are able to assist in the propagation of cells from
another lineage stage or provide assistance to neighboring cells
through the production of "paracrine signals", factors active in
their effects on neighboring cells in terms of survival, expansion,
migration, differentiation, and maturation. For example, supportive
mesenchymal cells may be defined by their ability to influence
epithelial cells, optionally through the secretion of matrix
metallo-proteinases (MMPs) and/or one or more paracrine signals or
growth factors. Many of these are summarized in recent reviews.
(Cathery et al. Stem Cells 2018; PMID:29732653; Graceb et al.
Biochimie 2018: PMID 29698670; Caplan AI. Stem Cells Int. 2015;
PMID: 26273305.
[0073] The term "lineage stage partners" refers herein to
mesenchymal cells and/or epithelial cells that are lineage stage
appropriate to support engraftment of the cells. For the hepatic or
biliary tree stem cells, these are comprised of angioblasts
(CD117+, CD133+, VEGFr+, CD31-negative) and their immediate
descendants, precursors to endothelia (CD133+, VEGFr+, CD31+, Van
Wildebrand Factor (vWF+)) and precursors to stellate cells (CD146+,
ICAM-1+, alpha-smooth muscle actin+ (ASMA), vitamin A-negative).
They can be mimicked, in part and/or to some extent, by use of
mesenchymal stem cells (MSCs), such as but not limited to ones
derived from bone marrow or fat tissue. Not to be bound by theory,
it is believed that such cells should be used immediately after
isolation from tissue or after minimal passaging ideally under
serum-free conditions. These cells are collectively referred to
herein as early lineage stage mesenchymal cells (ELSMCs).
[0074] Intermediates in the lineage network are referred to as
"transit amplifying cells," which are cells that can be bipotent
(or multipotent), have considerable proliferative potential but
demonstrate little (if any) true self-replication, have low to
moderate (or even no) pluripotency gene expression, and express
traits indicating commitment to an hepatic (e.g. albumin,
alpha-fetoprotein) or a pancreatic (e.g. insulin, MUC6, amylase)
fate. These include hepatoblasts (the network giving rise to liver)
and pancreatic ductal progenitors (the network giving rise to
pancreas).
[0075] As used herein, the term "pancreatic ductal progenitors"
refers to bipotent cells found within pancreatic ductal glands
(PDGs) within the pancreas and giving rise to acinar cells and
islets. In our studies, we find that they express SOX9, PDX1,
PTF1a, HNF1.beta., EpCAM, LGR5, ICAM-1, CD44, and subpopulations
express NGN3 or MUC6 or amylase. They have been extensively
characterized by others. See, e.g., Rezanejad H., Ouziel-Yahalom L,
Keyzer C A, Sullivan B A, Hollister-Lock J, Li W C, Guo L, Deng S,
Lei J, Markmann J, Bonner-Weir S. Heterogeneity of SOX9 and
HNF1.beta. is dynamic. Stem Cell Reports. Mar. 13, 2018;
10(3):725-738.
[0076] As used herein, the term "hepatoblasts" refers to bipotent
hepatic cells that can give rise to hepatocytic and cholangiocytic
lineages and are found in or adjacent to canals of Hering or in
PBGs within the large intrahepatic bile ducts. They have an
extraordinary ability to proliferate (that is expand) but with less
ability (if any) to self-replicate relative to that observed in
hepatic stem cells or BTSCs. These cells are characterized by a
biomarker profile that overlaps with, but is distinct from, hepatic
stem cells or biliary tree stem cells. They express SOX9, low (or
even negligible) levels of SOX17, high levels of LGR5, HNF4-alpha,
and EpCAM, found primarily at the plasma membrane, and expressing
P450A7, cytokeratin 7, secretin receptor, consistent expression of
albumin in all hepatoblasts, high levels of alpha-fetoprotein
(AFP), intercellular adhesion molecule (ICAM-1) but no expression
of NCAM, and negligible or no expression of pluripotency genes
(e.g. SALL4, KL4/KLF5, OCT4, SOX2, NANOG)) and no expression of
mature hepatic parenchymal markers (e.g. P450s such as P4503A).
[0077] As used herein the term "committed progenitor" refers to a
unipotent progenitor cell that gives rise to a single cell type,
e.g. a committed hepatocytic progenitor cell. In some embodiments,
they do not express pluripotency genes. The committed hepatocytic
progenitors are recognized by expression of albumin, AFP, glycogen,
ICAM-1, various enzymes involved with glycogen synthesis, and the
gap junction gene, connexin 28. These give rise to hepatocytes. A
committed biliary (or cholangiocytic) progenitor gives rise to
cholangiocytes and is recognized by expression of EpCAM,
cytokeratins 7 and 19, aquaporins, CFTR (Cystic Fibrosis
Transmembrane Conductance Regulator), and membrane pumps associated
with production of bile. In some embodiments, a committed islet
progenitor expresses insulin, glucagon, and other islet hormones
albeit at low levels; with maturation the expression levels of the
islet hormones increase but with particular cells expressing
preferentially certain hormones.
[0078] As used herein, the term "aggregates" refers to a plurality
of cells that are amassed together. The aggregates may vary in both
size and shape or may be substantially uniform in size and/or
shape. The cell aggregates used herein can be of various shapes,
such as, for example, a sphere, a cylinder (preferably with equal
height and diameter), or rod-like among others. Although other
shaped aggregates may be used, in one embodiment of the disclosure,
it is generally preferable that the cell aggregates be spherical or
cylindrical. The term "non-aggregated" refers to individual, or
single-celled, stem and/or progenitor cells or mature cells. In
some embodiments, the compositions provided herein can comprise
substantially aggregated cells, substantially non-aggregated cells,
or a mixture thereof.
[0079] The term "organoid" refers herein to a particular cellular
aggregate of donor epithelial cells with mesenchymal cells that is
self-assembled by simple panning methods described herein. In some
embodiments, the mesenchymal cells are supportive mesenchymal
cells. In some embodiments, the organoids are formed after
culturing on low attachment dishes and under serum-free, defined
conditions tailored to the lineage stage(s) of the aggregated cells
in suspension. Others prepare organoids utilizing particular matrix
extracts, such as Matrigel. Indeed, this substance is known to be
the industry standard. See Hindley et al. Dev. Biology 2016;
420:251-261. PMID:27364469. The conditions described in which these
organoids are maintained will not work successfully for the use of
these organoids in the patch grafts described in this invention.
The factors, such as those found in Matrigel, will stop or
substantially reduce the MMP production by the cells which is
required for the success of these patch grafts. Moreover, Matrigel
cannot be a components of conditions for cells to be used
clinically in people or for veterinary purposes.
[0080] The term "culture" or "cell culture" means the maintenance
of cells in an artificial, in vitro environment. A "cell culture
system" is used herein to refer to culture conditions in which a
population of cells may be grown ex vivo (outside of the body)
[0081] "Culture medium" is used herein to refer to a nutrient
solution for the culturing, growth, or proliferation of cells.
Culture medium may be characterized by functional properties such
as, but not limited to, the ability to maintain cells in a
particular state (e.g. a pluripotent state, a proliferative state,
quiescent state, etc.), to mature cells--in some instances,
specifically, to promote the differentiation of progenitor cells
into cells of a particular lineage. Non-limiting examples of
culture media are serum supplemented media (SSM) being any basal
medium supplemented with serum at levels that are typically about
10% to about 20%. The serum can be autologous (the same species as
the cells) or, more commonly, serum from animals that are routinely
slaughtered for commercial purposes (e.g. chickens, cows, pigs,
etc.). Notably, the present embodiments involving stem cells employ
media that avoids incorporation of serum and/or serum components
that drive differentiation. Kubota's medium, a serum-free medium
designed for endodermal stem/progenitors and comprised of a basal
medium (nutrients, amino acids, vitamins, salts, carbohydrates)
with no copper, low calcium (<0.5 mM) and supplemented with
selenium, zinc, insulin, transferrin, lipids but no cytokines or
growth factors. Other media found supportive of stem cells might
also be usable, but they must avoid any factors that cause the
cells to differentiate, since the maturational process will result
in muting of production of membrane-associate and/or secreted
MMPs.
[0082] Basal media are buffers used for cell culture and are
comprised of amino acids, sugars, lipids, vitamins, minerals,
salts, trace elements, and various nutrients in compositions that
mimic the chemical constituents of interstitial fluid around cells.
In addition, cell culture media are usually comprised of basal
media supplemented with a small percentage (typically 2-10%) serum.
For the grafting technologies described herein, conditions are used
to maintain the cells as stem cells or early progenitor cells and
so there is an avoidance of serum or any of the typical supplements
that might drive the cells towards a mature cell fate. In addition
to the customary basal media, various nutritional supplements,
lipids (mixture of free fatty acids complexed with albumin and
carrier molecules such as high density lipoprotein). Only two
hormone/growth factors are added: insulin needed for carbohydrate
metabolism, and transferrin, needed as a Fe carrier for the
polymerases. Kubota's medium, a serum-free medium designed for
endodermal stem/progenitors is comprised of a basal medium (with no
copped, low calcium (<0.5 mM) supplemented with zinc, selenium,
insulin, transferrin, lipids but no cytokines or growth factors.
Other growth factors and cytokines and especially serum are to be
avoided since they will induce differentiation of the donor cells
and, thereby, minimize the production of MMPs, which are required
for the engraftment and migration processes.
[0083] "Kubota's Medium" as used herein refers to any medium
containing no copper, calcium (<0.5 mM), selenium, zinc,
insulin, transferrin/Fe, a mix of free fatty acids bound to
purified albumin and, optionally, also high density lipoprotein
(HDL). In some embodiments, Kubota's Medium comprises any medium
(e.g., RPMI 1640 or DMEM-F12) with no copper, low calcium (e.g.,
0.3 mM), .about.10-9 M selenium, .about.0.1% bovine serum albumin
or human serum albumin (highly purified and fatty acid free),
.about.4.5 mM nicotinamide, .about.0.1 nM zinc sulfate
heptahydrate, .about.10-8 M hydrocortisone (optional component used
for hepatic but not pancreatic precursors), .about.5 .mu.g/ml
transferrin/Fe, .about.5 .mu.g/ml insulin, .about.10 .mu.g/ml high
density lipoprotein, and a mixture of purified free fatty acids
that are added after binding them to purified serum albumin. The
free fatty acid mixture consists of .about.100 mM each of palmitic
acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid,
and stearic acid. Non-limiting, exemplary methods for the
preparation of this media have been published elsewhere, e.g.,
Kubota H, Reid L M, Proc. Nat. Acad. Scien. (USA) 2000;
97:12132-12137, the disclosure of which is incorporated herein in
its entirety by reference.
[0084] In some embodiments, the conditions of these patch grafts
are, therefore, counter to the routine use of media supplemented
with a small percentage (typically 2-10%) serum. Serum has long
been added to provide requisite signaling molecules (hormones,
growth factors, cytokines) needed to drive a biological process
(e.g. proliferation, differentiation). In some embodiments, serum
is not included to avoid differentiation of the cells and/or avoid
inactivating or muting production of MMPs, especially the secreted
forms.
[0085] As used herein the term "amount effective" or "effective
amount" refers to an amount that is sufficient to treat disease
states or conditions (e.g. liver or pancreatic diseases). An
effective amount can be administered in one or more
administrations, applications or dosages. Such delivery is
dependent on a number of variables including the time period during
which the individual dosage unit is to be used, the bioavailability
of the composition, the route of administration, etc. It is
understood, however, that specific amounts of the compositions for
any particular patient depends upon a variety of factors including
the activity of the specific agent employed, the age, body weight,
general health, sex, and diet of the patient, the time of
administration, the rate of excretion, the composition combination,
severity of the particular disease (e.g. liver or pancreatic
disease) being treated and form of administration.
[0086] The terms "equivalent" or "biological equivalent" are used
interchangeably when referring to a particular molecule,
biological, or cellular material and intend those having minimal
homology while still maintaining desired structure or
functionality.
[0087] As used herein, the term "expression" refers to the process
by which polynucleotides are transcribed into mRNA and/or the
process by which the transcribed mRNA is subsequently being
translated into peptides, polypeptides, or proteins. If the
polynucleotide is derived from genomic DNA, expression may include
splicing of the mRNA in a eukaryotic cell. The expression level of
a gene may be determined by measuring the amount of mRNA or protein
in a cell or tissue sample; further, the expression level of
multiple genes can be determined to establish an expression profile
for a particular sample.
[0088] As used herein, the term "functional" may be used to modify
any molecule, biological, or cellular material to intend that it
accomplishes a particular, specified effect.
[0089] The term "gene" as used herein is meant to broadly include
any nucleic acid sequence transcribed into an RNA molecule, whether
the RNA is coding (e.g., mRNA) or non-coding (e.g., ncRNA).
[0090] As used herein, the term "generate" and its equivalents
(e.g. generating, generated, etc.) are used interchangeable with
"produce" and its equivalents when referring to the method steps
that bring the organoid of the instant disclosure into
existence.
[0091] The term "isolated" as used herein refers to molecules or
biologicals or cellular materials being substantially free from
other materials.
[0092] The terms "nucleic acid," "polynucleotide," and
"oligonucleotide" are used interchangeably and refer to a polymeric
form of nucleotides of any length, either deoxyribonucleotides or
ribonucleotides or analogs thereof. Polynucleotides can have any
three dimensional (3D) structure and may perform any function,
known or unknown. The following are non-limiting examples of
polynucleotides: a gene or gene fragment (for example, a probe,
primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA),
transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors,
isolated DNA of any sequence, isolated RNA of any sequence, nucleic
acid probes and primers.
[0093] A polynucleotide can comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure can be imparted before or
after assembly of the polynucleotide. The sequence of nucleotides
can be interrupted by non-nucleotide components. A polynucleotide
can be further modified after polymerization, such as by
conjugation with a labeling component. The term also refers to both
double and single stranded molecules. Unless otherwise specified or
required, any aspect of this technology that is a polynucleotide
encompasses both the double stranded form and each of two
complementary single stranded forms known or predicted to make up
the double stranded form.
[0094] The term "protein", "peptide" and "polypeptide" are used
interchangeably and in their broadest sense to refer to a compound
of two or more subunit amino acids, amino acid analogs or
peptidomimetics. The subunits may be linked by peptide bonds. In
another aspect, the subunit may be linked by other bonds, e.g.,
ester, ether, etc. A protein or peptide must contain at least two
amino acids and no limitation is placed on the maximum number of
amino acids which may comprise a protein's or peptide's sequence.
As used herein the term "amino acid" refers to either natural
and/or unnatural or synthetic amino acids, including glycine and
both the D and L optical isomers, amino acid analogs and
peptidomimetics.
[0095] As used herein, the term "subject" and "patient" are used
interchangeably and are intended to mean any animal. In some
embodiments, the subject may be a mammal. In some embodiments, the
mammal is bovine, equine, porcine, canine, feline, simian, murine,
human, or rat. In some embodiments, the subject is a human.
[0096] The term "tissue" is used herein to refer to tissue of a
living or deceased organism or any tissue derived from or designed
to mimic a living or deceased organism. The tissue may be healthy,
diseased, injured by trauma, damaged and/or have genetic mutations.
The term "natural tissue" or "biological tissue" and variations
thereof as used herein refer to the biological tissue as it exists
in its natural state or in a state unmodified from when it was
derived from an organism. A "micro-organ" refers to a segment of
"bioengineered tissue" that mimics "natural tissue."
[0097] The biological tissue may include any single tissue (e.g., a
collection of cells that may be interconnected) or a group of
tissues making up an organ or part or region of the body of an
organism. The tissue may comprise a homogeneous cellular material
or it may be a composite structure such as that found in regions of
the body including the thorax which for instance can include lung
tissue, skeletal tissue, and/or muscle tissue. Exemplary tissues
include, but are not limited to those derived from liver, pancreas,
biliary tree, lung, intestines, thyroid, thymus, bladder, kidneys,
prostate, uterus, breast, skin and underlying dermal tissues,
brain, spinal cord, blood vessels (e.g. aorta, iliac vein), heart,
muscle, including any combination thereof.
[0098] As used herein, "treating" or "treatment" of a disease in a
subject refers to (1) preventing the symptoms or disease from
occurring in a subject that is predisposed or does not yet display
symptoms of the disease; (2) inhibiting the disease or arresting
its development; or (3) ameliorating or causing regression of the
disease or the symptoms of the disease. As understood in the art,
"treatment" is an approach for obtaining beneficial or desired
results, including clinical results. For the purposes of the
present technology, beneficial or desired results can include one
or more, but are not limited to, alleviation or amelioration of one
or more symptoms, diminishment of extent of a condition (including
a disease), stabilized (i.e., not worsening) state of a condition
(including disease), delay or slowing of condition (including
disease), progression, amelioration or palliation of the condition
(including disease), states and remission (whether partial or
total), whether detectable or undetectable.
ABBREVIATIONS
[0099] AFP, .alpha.-fetoprotein; ALB, albumin; BTSCs, biliary tree
stem cells: CD, common determinant: CD44, hyaluronan receptors;
CD133, prominin; CFTR, cystic fibrosis transmembrane conductance
regulator; CK, cytokeratin protein; CXCR4, CXC-chemokine receptor 4
(also called fusin or CD184; also called platelet factor 4; EGF,
epidermal growth factor; ELSMCs, early lineage stage mesenchymal
cells, consisting of angioblasts and their descendants, precursors
to endothelia and to stellate cells; EpCAM, epithelial cell
adhesion molecule: FGF, fibroblast growth factor; HBs,
hepatoblasts; HGF, hepatocyte growth factor; HpSCs, hepatic stem
cells; KM, Kubota's Medium, a serum-free medium designed for
endodermal stem cells; KRT, cytokeratin gene; LGR5, Leucine-rich
repeat-containing G-protein coupled receptor 5 that binds to
R-spondin; MMPs, matrix metallo-proteinases, a large family of
proteinases associated with dissolution of extracellular matrix,
with cell migration and with regenerative responses; NANOG, a
transcription factor critically involved with self-renewal; NCAM,
neural cell adhesion molecule; NIS, sodium/iodide symporter; OCT4,
(octamer-binding transcription factor 4) also known as POU5F1 (POU
domain, class 5, transcription factor 1), a gene expressed by stem
cells; PDX1, pancreatic and duodenal homeobox 1, a transcription
factor critical for pancreatic development; PBGs, peribiliary
glands, stem cell niches for biliary tree stem cells; SALL4.
Sal-like protein 4 found to be important for self-replication of
stem cells; SOX, Sry-related HMG box; SOX2, a transcription factor
that is essential for maintaining self-renewal, or pluripotency in
embryonic and determined stem cells. SOX9, transcription factor
associated with endodermal tissues (liver, gut and pancreas: SOX17,
a transcription factor essential for differentiation of liver;
VEGF, vascular endothelial cell growth factor; vWF, Von Willebrand
Factor.
[0100] Modes of Practicing the Present Disclosure
[0101] In the examples provided herein, Applicants establish patch
grafting, a novel method for transplantation of cells into internal
organs with design features dependent on whether cells are stem
cells or mature cells. Applicants demonstrate these methods herein
with grafts of biliary tree stem cells (BTSCs), precursors to both
liver and pancreas, and transplanted onto liver or pancreas. The
hosts used for developing these methods are breeds of swine, Sus
scrofa domestics. They are major animal species used in
translational research, surgical models, and procedural training
and are used increasingly as alternatives to monkeys in preclinical
studies.
[0102] Exemplary success was achieved with organoids of biliary
tree stem cells (BTSCs), precursors to liver and to pancreas,
partnered with early lineage stage mesenchymal cells (ELSMCs), and
comprised in soft (.about.100 Pa) hyaluronan (HA) hydrogels. HA
hydrogels, containing organoids, were placed onto Seri-silk
backings (a mesh material) impregnated on their serosal sides with
more rigid HA hydrogel (.about.700 Pa), and were surgically or
otherwise tethered to the surface of the liver or pancreas. Within
a week, grafts caused remodeling of organ capsules and adjacent
tissue and, optionally, distant parenchymal tissue followed by a
merger of donor and host cells. By two weeks, donor cells had
matured to functional adult fates such as hepatocytes (albumin) or
islets (.beta.-cells-insulin). By three weeks, with clearance of
HAs, organ capsules and normal tissue histology returned. The
engraftment/migration/integration processes proved dependent on
multiple plasma membrane-associated and secreted
matrix-metallo-proteinases expressed by the cells.
[0103] These results of these examples are in contrast to those
from past efforts to transplant cells from solid organs into
internal organs, in which transplantation was accomplished either
by direct injection or by delivering cells via a vascular route
(see reviews by Bhatia et al., Lanzoni et al., Weber, and others).
The past methods of transplantation result in small numbers of
cells being engrafted, in risks of emboli that can be life
threatening, and in significant levels of ectopic cell
distribution. These problems have caused cell therapies for
internal solid organs to be used minimally or not at all.
[0104] The patch graft strategy offers an alternative method for
cell therapies, ones that can enable the delivery of adequate cell
numbers and of their integration into the tissue to offer
significant restoration of function(s). The examples demonstrate
safety so long as biomaterials and the backing used were supportive
of maintenance of some or all of the donor cells as immature and so
able to produce the relevant repertoire of MMPs. A common source of
failure was any factor(s) resulting in differentiation of the donor
cells. Not to be bound by theory, it is contemplated herein that
purified MMPs may be incorporated into graft biomaterials and/or
cells may be transformed to secrete MMPs using a recombinant
expression system or other genetic modification technique, as an
alternative to providing a cells in the graft which naturally
produce the requisite MMPs. In such embodiments, the combination of
MMPs incorporated or transduced via construct should include those
identified in the expression profiles provided in the examples
below.
[0105] Composition of a Patch Graft
[0106] Aspects disclosed herein relate to a patch graft comprising
a layer comprising a single population or two or more populations
of cells (e.g. donor cells which may be autologous or allogeneic)
and a source of MMPs and a backing comprising a biocompatible,
biodegradable material, which may be used to tether the graft to a
target site. In some embodiments, the population or populations of
cells include a population of epithelial cells and a population of
mesenchymal cells. In some embodiments, the populations of cells
must be maintained in a particular state or "lineage stage" as part
of the graft, meaning that they do not differentiate or mature
further until incorporation into the organ. This can be achieved by
balancing variables relating to the cell source, MMP content,
medium used, and backing qualities. Each of these aspects is
described in greater detail herein below.
[0107] Not to be bound by theory, it is believed that patch grafts
can be successful with (1) an optimal cell population or mixture of
cells--e.g. donor epithelial cells and a supporting mesenchymal
stem/progenitor cell population that generates membrane-associated
and/or secreted MMPs--in a medium and hydrogel that does not lead
to differentiation of the supporting mesenchymal stem/progenitor
cell population or that otherwise contains appropriate MMPs, and
(2) a backing suitable to tether the graft to the target site and
prevent migration of the cells in the graft toward the backing,
away from the target site.
[0108] Exemplary Cells
[0109] Not to be bound by theory, the cells may be at any
maturational lineage stage including embryonic stem (ES) cells,
induced pluripotent stem (iPS) cells, determined stem cells,
committed progenitors, transit amplifying cells, or mature cells.
However, in certain embodiments, a source of MMPs must be present
in the patch graft. Thus, contemplated herein are cellular sources
of the MMPs for use in the patch grafts. Such cellular sources must
be at an early lineage stage that is capable of expressing
membrane-associated and/or secreted matrix metalloproteinases. A
non-limiting example of such an early lineage stage are early
lineage stage mesenchymal stem cells (ESMLCs).
[0110] In some embodiments, the cells to be grafted are epithelial
cells partnered with mesenchymal cells. In some embodiments, the
epithelial cells comprise epithelial stem cells. In some
embodiments, the epithelial cells comprise committed and/or mature
epithelial cells. In some embodiments, the committed and/or mature
epithelial cells comprise mature parenchymal cells. In some
embodiments, the mature parenchymal cells comprise one or more of
hepatocytes, cholangiocytes, or islet cells. In some embodiments,
the mesenchymal cells comprise ELSMCs. In some embodiments, the
ELSMCs comprise one or more of angioblasts, precursors to
endothelia, precursors to stellate cells, and MSCs. In some
embodiments, the epithelial cells and mesenchymal cells are lineage
stage partners of one another. In some embodiments, the epithelial
cells and the mesenchymal cells are not lineage stage partners of
one another, e.g. are not at approximately the same lineage stage
or maturation stage, respectively. In some embodiments, the
epithelial cells are mature cells. In some embodiments, the
mesenchymal cells are ELSMCs.
[0111] In some embodiments, at least one of the epithelial cells
and the mesenchymal cells are derived from a donor. In some
embodiments, the donor is a subject in need of a tissue transplant.
In some embodiments, the donor is the source of healthy cells for a
tissue transplant. In some embodiments, at least one of the
epithelial cells and the mesenchymal cells are autologous to an
intended recipient of the patch graft. In some embodiments, all of
the cells (i.e. epithelial and mesenchymal) are autologous to the
intended recipient of the graft. In some embodiments, the donor of
cells may be one other than the recipient (allograft) or may also
be the subject (autologous) having the internal organ in a diseased
or dysfunctional condition, optionally, wherein are obtained from a
portion of the internal organ that is not diseased or dysfunctional
and/or that the cells have been genetically modified to restore
function.
[0112] In another aspect, the mesenchymal cells are lineage-stage
partners of the donor cells, e.g. at a comparable or corresponding
lineage stage. In another aspect, the mesenchymal cells are not
lineage-stage appropriate partners of the donor cells. The
mesenchymal lineage stage cells may be angioblasts, early lineage
stage precursors to endothelia and/or stellate cells, mesenchymal
stem cells, endothelia or stellate cells, or derivatives of these
cell populations.
[0113] For stem cell transplants, epithelial cells should be
partnered with their native, lineage stage partner mesenchymal
cells (angioblasts and/or precursors to endothelia or to stellate
cells). For adult epithelial cells, appropriate partners include
early lineage stage mesenchymal cells (ELSMCs) that are comprised
of angioblasts and/or precursors to stellate cells and to
endothelial cells. Applicants have shown that one can use
preparations of mesenchymal stem cells (MSCs) in combination with
adult cells to achieve engraftment. In some embodiments, certain
MSCs may be preferable to others. Not to be bound by theory, it is
believed that grafts may be optimized by selecting combinations of
cells which require minimal, if any culturing of the cells and that
will avoid serum and matrix components that might drive
differentiation of the cells. Not to be bound by theory, it is
further understood that the epithelial-mesenchymal relationship is
important, since the paracrine signaling supports the production of
MMPs. However, mature epithelial cells partnered with mature
endothelia will survive in the graft and will be functional cells
but will not engraft. Thus, if the mature epithelial cells are
partnered with mature stroma to form a graft, the resulting grafts
are likely to become fibrotic.
[0114] For treatment of a diseased or dysfunctional organ, cells
may be from a donor other than the recipient (allografts) or may
also be autologous transplants and so from the subject having the
internal organ in a diseased or dysfunctional condition,
optionally, wherein are obtained from a portion of the internal
organ that is not diseased or dysfunctional and/or that the cells
have been genetically modified to restore function.
[0115] For establishing a model system to study a disease, cells
can be ones that have the disease and that are transplanted
onto/into normal tissue in an experimental host.
[0116] In some embodiments, the epithelial cells may be stem cells
combined with supportive mesenchymal cells, optionally ELSMCs, to
form organoids, which optionally self-assemble. These organoids may
be embedded or comprised in a hyaluronan hydrogel. The stem and/or
progenitor cells of the present disclosure can include any stem
and/or progenitor cell known in the art, including for example, an
embryonic stem cell (ESC), an embryonic germ cell (EGC), an induced
pluripotent stem cell (iPSC), a pancreatic stem cell (PSC), hepatic
stem cell (HpSC), biliary tree stem cell (BTSC), an hepatoblast, a
pancreatic ductal progenitor, a committed pancreatic progenitor
cell, or a committed hepatic progenitor cell. In some embodiments,
the cell populations comprise only stem cells such as pancreatic
stem cells, hepatic stem cells, biliary tree stem cells (BTSCs) or
Brunner's Glands stem cells. In other embodiments, the cells
comprise only multipotent progenitor subpopulations such as
hepatoblasts or pancreatic ductal progenitor cells, or the graft
can contain committed, unipotent progenitors (e.g. hepatocytic or
biliary or islet or acinar committed progenitor cells). In other
embodiments, the cells comprise a mixture of stem cells and
progenitors.
[0117] If adult epithelial cells are used, then they may be mixed
at relevant ratios with ELSMCs into the grafting biomaterials. The
ratios of cell mixture may be determined so as to mimic the target
tissue. Alternatively or in addition, the ratios may be determined
through self-assembly of the organoids. The organoids or cell
mixtures are embedded in the soft grafting biomaterials such as the
soft hyaluronan hydrogel. If a stem cell graft, then the stem
and/or progenitor cells of the present disclosure can include any
stem and/or progenitor cell known in the art, including for
example, an embryonic stem cell (ESC), an embryonic germ cell
(EGC), an induced pluripotent stem cell (iPSC), a Brunner's Glands
stem cells (BGSCs), a biliary tree stem cell (BTSC), a pancreatic
stem cell (PSC), an hepatic stem cell (HpSC), transit amplifying
cells (e.g. hepatoblasts or pancreatic ductal progenitors), and
committed, unipotent progenitors (e.g. a committed pancreatic
progenitors or hepatocytic or cholangiocytic progenitor). In some
embodiments, the cell populations comprise only stem cells. In
other embodiments, the cells comprise only progenitor
subpopulations. In other embodiments, the cells comprise a mixture
of stem cells and progenitors or a mixture of stem/progenitor cells
and more mature cells. In yet others, there can be a chimeric mix
of adult cells (e.g. hepatocytes, cholangiocytes, enterocytes,
islets) and ELSMCs.
[0118] The stem cell and/or progenitor cells can be identified by
any method known to one who is skilled in the art. Non-limiting
examples include using a combination of assays defining
self-replicative ability and ones demonstrating multipotency by
morphological analysis, by gene and/or protein expression, cell
surface markers, and the like. In some embodiments, the stem and/or
progenitor cells express at least one marker indicative of early
stage liver cell lineage cell (e.g., SOX 17, HNF-4alpha, HNF6,
HES1, CK19)) and at least one marker indicative of early stage
pancreatic cell lineage (e.g., PDX1, PROX1, NGN3, HNF.beta.1). For
example, stem and/or progenitor cells, in particular BTSCs, can be
identified by expression of SOX9, SOX17, PDX1, CD133, NCAM, sonic
hedgehog (SHH), sodium iodide symporter (NIS), LGR5, LGR6, EpCAM,
various isoforms of CD44, CXCR4, and various pluripotency genes
(e.g. OCT4, SOX2, NANOG, KLF4, KLF5, SALL4, BMi-1) or any
combination thereof.
[0119] In some embodiments, the stem and/or progenitor cells
express at least one marker indicative of early parental stage cell
lineages such as parental lineages for liver and pancreas. Thus
they would express one(s) shared by both hepatic and pancreatic
lineages (e.g. SOX9, LGR5/LGR6, EpCAM, CD133, CK19) and one(s) for
hepatic lineages (e.g., SOX 17, HNF-4-alpha, HNF6, HES1) and one(s)
for early stage pancreatic cell lineages (e.g., PDX1, PROX1, NGN3,
HNF.beta.1). For example, stem and/or progenitor cells, in
particular BTSCs, can be identified by expression of SOX9, SOX17,
PDX1, CD133, NCAM, sonic hedgehog (SHH), sodium iodide symporter
(NIS), LGR5, LGR6, EpCAM, and various pluripotency genes (e.g.
OCT4, SOX2, NANOG, KLF4, KLF5, SALL4, BMi-1) or any combination
thereof.
[0120] Generation of Mature Cell Types
[0121] The stem and/or progenitor cells can also be differentiated
into a more mature cell type, if one is desired. This can be done
in vitro by spontaneous differentiation and/or by directed
differentiation. Directed differentiation can involve use of a
defined media, genetically modifying the stem and/or progenitor
cells to express a gene of interest, or combinations thereof.
[0122] Non-limiting examples of defined media to differentiate
cells include the hormonally-defined media (HDM) used for
differentiation of endodermal stem cells to adult fates.
Supplements can be added to Kubota's Medium to generate a
serum-free, hormonally defined medium (HDM) that will facilitate
differentiation of the normal hepatic or biliary tree stem cells to
specific adult fates. These include supplementation with calcium to
achieve at or above 0.6 mM concentration, 1 nM tri-iodothyronine
(T3), 10.sup.-12 M copper, 10 nM of hydrocortisone and 20 ng/ml of
basic fibroblast growth factor (bFGF). The medium conditions over
and above these needed to selectively yield hepatocytes (HDM-H)
versus cholangiocytes (HDM-C) versus pancreatic islets (HDM-P) are:
[0123] 1) HDM-H: supplementation further with 7 .mu.g/L glucagon, 2
g/L galactose, 10 ng/ml epidermal growth factor (EGF) and 20 ng/ml
hepatocyte growth factor (HGF); [0124] 2) HDM-C: supplementation
further with 20 ng/ml vascular endothelial cell growth factor
(VEGF) and 10 ng/ml HGF; and [0125] 3) HDM-P: prepared without
glucocorticoids and further supplemented with 1% B27, 0.1 mM
ascorbic acid, 0.25 .mu.M cyclopamine, 1 .mu.M retinoic acid, 20
ng/ml of FGF-7 for 4 days, then changed with one supplemented with
50 ng/ml exendin-4 and 20 ng/ml of HGF for 6 more days of
induction.
[0126] The HDM provided herein can be supplemented with additional
growth factors including, but not limited to, Wnt signals,
epidermal growth factors (EGFs), fibroblast growth factors (FGFs),
hepatocyte growth factors (HGFs), insulin-like growth factors
(IGFs), transforming growth factors (TGFs), nerve growth factors
(NGFs), neurotrophic factors, various interleukins, leukemia
inhibitory factors (LIFs), vascular endothelial cell growth factors
(VEGFs), platelet-derived growth factors (PDGFs), stem cell factors
(SCFs), colony stimulating factors (CSFs), GM-CSFs, erythropoietin,
thrombopoietin, heparin binding growth factors, IGF binding
proteins, and/or to placental growth factors.
[0127] The HDM provided herein can be supplemented with cytokines
including, but not limited to interleukins, lymphokines, monokines,
colony stimulating factors, chemokines, interferons and tumor
necrosis factor (TNF).
[0128] Applicants have shown that hyaluronans can influence stem
and/or progenitor cells to express factors that regulate critical
cell adhesion molecules needed for cell attachment and cell-cell
interactions and to prevent the stem and/or progenitor cells from
internalization of those attachment factors following cell
suspension preparations, cryopreservation, or with transplantation.
Non-limiting examples of such attachment factors include integrins.
Integrins are a large family of heterodimeric transmembrane
glycoproteins that function to attach cells to extracellular matrix
proteins of the basement membrane, ligands on other cells, and
soluble ligands. Integrins contain a large and small subunit,
referred to as .alpha. and .beta., respectively. This subunits form
.alpha..beta. heterodimers and at least 18 .alpha. and eight .beta.
subunits are known in humans, generating 24 heterodimers. In some
embodiments, the stem and/or progenitor cells express higher levels
of integrin subunits, for example, ITGa1, ITGa2, ITGa2B, ITGa3,
ITGa4, ITGa5, ITGa6, ITGa7, ITGa8, ITGa9, ITGa10, ITGa11, ITGaD,
ITGaE, ITGaL, ITGaM, IIGaV, IIGaX, ITG.beta.1, ITG.beta.2,
ITG.beta.3, ITG.beta.4, ITG.beta.5, ITG.beta.6, ITG.beta.7 and
IG.beta.8. In one preferred embodiment, the stem and/or progenitor
cells express higher levels of integrin subunit beta 1 (ITG.beta.1)
and/or integrin subunit beta 4 (ITG.beta.4). Takada Y. et al.
(2007) Genome Biol. 8(5): 215.
[0129] In some embodiments, the stem and/or progenitor cells of the
present disclosure differ from naturally occurring stem and/or
progenitor cells at least in that they express an integrin subunit
in an amount that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%
greater than the amount of the integrin subunit in unmodified stem
and/or progenitor cells. It is contemplated that an increase in an
integrin subunit can help the stem and/or progenitor cell to
attach, form cell-cell interactions, and to prevent the stem and/or
progenitor cells from internalization should this be desired.
[0130] MMPs
[0131] The MMPs are one of the key factors facilitating engraftment
and integration. MMPs are comprised of many isoforms (at least 28;
in the pigs, 24 isoforms are known) of which some are secreted
(e.g. MMP1, MMP2, MMP7, MMP9) and some are plasma membrane
associated (e.g. MMP14, MMP15). Not to be bound by theory, it is
believed that a mix of these is required for engraftment,
especially a mix of the secreted forms. All cells examined produce
varying amounts of both secreted and membrane associated forms, but
stem/progenitors produce very high levels of the secreted forms.
Engraftment is dependent on these secreted MMPs (and with some
known synergies with the membrane-associated forms). A cellular
source of these is the practical way to provide the requisite MMPs
to achieve engraftment. As an alternative approach, Applicants
contemplate incorporation of purified/recombinant forms of the MMPs
into the graft biomaterials and/or genetic engineering of cells in
the graft to produce the requisite MMPs.
[0132] The cells can successfully engraft as long as there are
sources, ideally cellular sources, of multiple matrix
metallo-proteinases (MMPs), optionally one or both of secreted and
membrane-associated ones. MMPs are produced by all cell types, both
immature and mature cells, but they vary as to which isoforms are
produced and at what level of expression of particular MMPs.
Representative secreted ones include MMP1, MMP2, MMP7 and MMP9.
Representative membrane-associated ones include MMP14 and MMP15.
Empirically it has been found that the highest production of
secreted MMPs is by early lineage stage cells, stem cells and early
progenitors. The biomaterials of the graft support the ability of
both the epithelial and mesenchymal cells to produce these multiple
forms of matrix metallo-proteinases (MMPs) that dissolve capsules
around organs or tissues and enable migration of cells by means of
dissolution of multiple forms of extracellular matrix
components.
[0133] More generally, matrix metallo-proteinases (MMPS) are a
large family of zinc-dependent proteinases that are involved in
breakdown and modulation of extracellular matrix component and that
are involved in implantation, invasion, angiogenesis,
vascularization, and migration in normal and pathogenic processes.
There are at least 28 isoforms that comprise matrixins,
adamalysins, astacins, serralysins, etc. Their roles have been
characterized in normal processes such as the implantation of the
placenta, as well as in pathogenic ones such as invasion and
metastases of cancers.
[0134] The studies described herein offer evidence for entirely new
roles of MMPs that contribute to engraftment, migration and
integration of transplanted cells. Stem/progenitors, both
epithelial ones and mesenchymal ones, express multiple MMP isoforms
that are especially potent in these roles. Maturation of the cells
results in muting the expression of one or more of the potent
stem/progenitor-cell-associated MMPs and so diminishing the
invasion and migration processes. Adult cells also express MMPs,
primarily ones that are membrane bound (MT-MMPs), said MMPs are
involved in plasticity processes but not the wholesale engraftment
and integration of cells into tissues. However, there are some
synergies between the MT-MMPs and the secreted forms. The net sum
of this realization is that the graft biomaterials, backing and
other conditions must be ones that, among other characteristics,
optimize expression of the various MMPs, such as the secreted MMPs,
enabling the grafting and migration processes to occur. Therefore,
factors driving differentiation of the transplanted cells will, in
parallel, mute the complex MMP responses. This realization means
that factors to be avoided include serum (which drives
differentiation), soluble signals that drive differentiation (e.g.
certain growth factors, cytokines and hormones); extracellular
matrix components that drive differentiation (e.g. collagens,
adhesion molecules, highly sulfated
glycosaminoglycans/proteoglycans); and mechanical forces contribute
to rigidity (the viscoelasticity properties, which drive
differentiation) of the graft.
[0135] In some embodiments, one or more of the cells in the mixture
is a source of secreted and/or membrane-associated MMPs. The
secreted MMPs may optionally be produced naturally by the one or
more of the epithelial or mesenchymal cells or optionally be
produced due to transformation of the one or more of the epithelial
or mesenchymal cells with a recombinant expression vector or
genetic editing for MMP production. In some embodiments, such as
but not limited to those involving stem/progenitor cell populations
that naturally secrete MMPs, variables that mute MMP
expression--optionally membrane-associated and/or secreted MMP
expression--are controlled in the patch graft. Non-limiting
examples of such variables include variables that result in
maturation of stem/progenitor cells, such as but not limited to
serum supplementation to media or to the graft biomaterials,
hormones or other soluble signals that influence differentiation of
the epithelial and/or mesenchymal cells, oxygen levels (as
anaerobic conditions keep the cells immature, whereas higher oxygen
levels promote differentiation), and the rigidity of graft
materials (as rigidity or mechanical forces such as shear force and
compression may drive differentiation).
[0136] For stem cell grafts, both the epithelial cells and their
mesenchymal cell partners are optimally stem cells or progenitors,
since both provide contributions of multiple types of MMPs. To
engraft adult cells, the one of the epithelial or mesenchymal cells
should optimally provide a cellular source of membrane-associated
and/or secreted MMPs, e.g. optionally using ELSMCs as the cellular
source of membrane-associated and/or secreted MMPs. Thus, grafts in
which both the epithelia and the mesenchymal cells are mature cell
types are not successful for engraftment. If mature endothelia,
then the epithelial cells are likely to survive and to proliferate
and function but will not engraft; if mature stroma, then the
grafts are likely to become fibrotic.
[0137] In summary, engraftment will occur if both
epithelial-mesenchymal cell partners are stem/progenitors or if
there at least one of the epithelial or mesenchymal cells is a stem
cells, e.g. optionally using ELSMCs as a source of
matrix-associated and/or secreted isoforms of matrix
metalloproteases (MMPs), or if purified/recombinant forms of those
MMPs are provided in the graft biomaterials. The early lineage
stage mesenchymal cells (ELSMCs) appropriate for patch grafts can
be angioblasts, precursors to endothelia, early lineage stage
endothelia, precursors to stellate cells, early stage stellate
cells, or mesenchymal stem cells (MSCs), or mixtures of these.
[0138] Thus, contemplated herein is a composition for use as a
patch graft comprising at least a population of cells (e.g.
epithelial and mesenchymal cells) and a source of MMPs (i.e. a
population of cells at an early lineage stage that is capable of
expressing membrane-associated and/or secreted matrix
metalloproteinases (MMPs), optionally supported by the conditions
of the medium and/or hydrogel.
[0139] Medium Components
[0140] For use in combination with the cells and source of MMPs
disclosed herein, one can use any medium (comprising nutrients,
vitamins, salts, etc.) plus critical soluble factors such as
insulin, transferrin/Fe and lipids that is found useful for
expansion and/or survival of stem/progenitors. One must avoid all
factors that cause the cells to mature, since maturation will
result in a reduction or muting of expression of MMPs. The factors
to be avoided include serum, soluble signals that drive
differentiation, extracellular matrix components that drive
differentiation, and rigidity or mechanical forces (compression,
abrasion). A non-limiting example of such a media is Kubota's
medium.
[0141] Thus, contemplated herein is a composition for use as a
patch graft comprising at least a population of cells and a source
of MMPs (e.g. a population of cells at an early lineage stage that
is capable of expressing membrane-associated and/or secreted matrix
metalloproteinases (MMPs), supported in a suitable medium, or
purified MMPs). A non-limiting example of a suitable medium is
Kubota's medium. Other stem cell mediums, such as those used for
embryonic stem (ES) cells or induced pluripotent stem (iPS) cells
may likewise be suitable as long as they do not contain soluble
signals or matrix signals that will drive the differentiation of
the cells that are the source of the MMPs or as long as MMPs are
present or included from other sources.
[0142] Hydrogel
[0143] The patch graft comprises one or more hydrogel components.
In some aspects, the biomaterials that can form hydrogels, or a
parallel insoluble complex (e.g. a non-collagenous gelatin),
comprise hyaluronans, thiol-modified hyaluronans, other
glycosaminoglycans (GAGs), or combinations thereof. A trigger for
solidification can be any factor eliciting cross-linking of the
matrix components or gelation of those components that can gel. The
cross-linker may comprise Poly(ethylene glycol) (PEG) or
PEG-diacrylate (PEGDA) hydrogel or a disulfide-containing
derivative thereof. Notably, biomaterials comprised in the hydrogel
should be selected for the ability to support the stemness in the
one or more cell populations disclosed for use in the patch graft,
e.g. ELSMCs.
[0144] Matrix components supportive of maintenance of stemness can
be used but not those components driving differentiation.
Non-limiting examples of supportive components include hyaluronans
or non-sulfated (or minimally-sulfated) glycosaminoglycans. These
are especially useful since thy can be "tuned," that is modified to
having varying levels of rigidity (optionally measured as
viscoelasticity). Accordingly, in some aspects, the population of
cells, optionally isolated cells of an internal organ, may be
solidified ex vivo within the biomaterials prior to introducing the
cells into the hosts, or in the alternative, injected as a fluid
substance and allowed to solidify in vivo.
[0145] The very soft versions (e.g. .about.100 Pa) of hydrogels are
ideal for maintaining the donor cells in an immature state). More
rigid versions (e.g. >500 Pa) can be used to cause the cells to
mature enough to shut off MMP production and so block migration.
More rigid versions can also minimize adhesions from neighboring
tissues. In certain embodiments, the population of cells and the
source of MMPs, optionally another population of cells (i.e.
population of cells at an early lineage stage that is capable of
expressing membrane-associated and/or secreted matrix
metalloproteinases (MMPs)
[0146] Not to be bound by theory, it is believed that that forms of
extracellular matrix found in amnions are able to keep the donor
cells immature. Thus, amnions are contemplated both for use in the
hydrogel and, optionally, as an alternative biocompatible,
biodegradable material.
[0147] Notably materials known to cause maturation include certain
components derived from mature extracellular matrix, such as but
not limited to type I collagen. These materials should be excluded
from all elements of the patch graft, including but not limited to
the cells, the hydrogel, the medium, the backing, and/or any
further components.
[0148] Thus, contemplated herein is a composition for use in the
patch graft comprising at least a population of cells and a source
of MMPs (i.e. a population of cells at an early lineage stage that
is capable of expressing membrane-associated and/or secreted matrix
metalloproteinases (MMPs), supported in a suitable medium and
comprised in a hydrogel.
[0149] As noted above, rigidity can drive the ability of cells to
differentiate. Further rigid hydrogels may have an effect on the
ability of cells to migrate. As the cells must migrate into the
organ, the hydrogel in which the cells are comprised should have a
viscoelasticity sufficient to allow for migration of said cells,
optionally, within or away from the hydrogel and/or the patch
graft. Non-limiting examples of such viscoelasticity include by are
not limited a viscoelasticity ranging from about 50 to about 100 Pa
or about 250 Pa, for example at least about 50 Pa, at least about
100 Pa, at least about 150 Pa, at least about 200 Pa, at most about
250 Pa, at most about 200 Pa, at most about 150 Pa, at most about
100 Pa, and/or any individual value in between such as but not
limited to about 50 Pa, about 100 Pa, about 150 Pa, about 200 Pa,
and about 250 Pa.
[0150] Not to be bound by theory, it is believe that when the cells
migrate from the patch graft into the target organ or tissue, they
migrate with some of the hydrogel associated with them or coating
them. The hydrogel shields the cells from the signals in the tissue
microenvironment which would influence the cells to differentiate
or mature, and enables the cells to remain immature. This
facilitates the cells migrating through the parenchymal tissue. As
the hyaluronans in the hydrogel gradually get degraded and removed,
the cells begin to differentiate or mature and begin adult cell
functions.
[0151] Methods of Generating Organoids
[0152] Not to be bound by theory, it has been determined that early
stage lineage cells may have a high rate of graft success when
incorporated into an organoid or an aggregate. Such organize may
optionally comprise early lineage stages of both epithelial and of
mesenchymal cells.
[0153] Thus, provided herein is a method of forming organoids, the
method comprising, consisting of, or consisting essentially of
culturing a mixture of epithelial cells and mesenchymal cells in a
container suitable for tissue culture and in the presence of a
culture medium, removing mature cells that attach to a surface of
the container by panning, and recovering self-assembled organoids
from the suspension of cells in the culture media. Also disclosed
herein is a composition comprising an organoid generated as
such.
[0154] In some embodiments, the procedure involves panning to
eliminate mature cells by selective, rapid (15-30 minutes)
attachment of them to regular culture dishes under serum-free
conditions and at 37.degree. C., since even under these conditions,
the mature cells express various matrix components that enable cell
attachment. Multiple rounds (e.g. 4-5) of such a panning process
enriches the cell suspension for the earlier lineage stage cells.
Then the cell suspension is transferred to low attachment dishes
and again in serum-free medium, one designed for the early lineage
stage cells, and left overnight in an incubator at 37.degree. C.
The conditions foster self-assembly of the lineage-stage-matched
epithelial and mesenchymal cells into organoids. Organoids can be
obtained from mixing of early stages of epithelia (ES cells, iPS
cells, determined stem cells, transit amplifying cells,
progenitors) with early stages of mesenchymal cells (angioblasts,
precursors to endothelia, precursors to stellate cells).
[0155] Mixtures of adult epithelial cells with mature mesenchymal
cells and chimeric mixtures of mature epithelial cells with early
lineage stage mesenchymal cells (ELSMCs) do not usually generate
organoids but can be used as mixtures of the cells in suspension in
the graft biomaterials. If mature epithelia (e.g. hepatocytes,
cholangiocytes, islets, acinar cells, enterocytes, etc.) are
partnered with mature mesenchymal cells (e.g., endothelia, stellate
cells, stromal cells, myofibroblasts), the mixtures will not result
in successful integration of the grafts into the target site or
organ but rather in ones that persist at the surface of the organs
or tissues. If chimeric mixtures are used comprising adult and
stem/progenitors (e.g. mature hepatocytes with angioblasts), then
engraftment does occur, since there is a source of MMPs that enable
engraftment and migration of the cells.
[0156] In another aspect, the isolated cells of the internal organ
may be solidified ex vivo within the biomaterials prior to
introducing the cells into the hosts, or in the alternative,
injected as a fluid substance and allowed to solidify into a graft
in vivo. Preferably, the cells are introduced at or near the
diseased or dysfunctional tissue, and may be introduced via
injection or grafted onto/into the tissue, or using an appropriate
surgical method.
[0157] In another aspect, the biomaterials that can form hydrogels,
or a parallel insoluble complex, can comprise hyaluronans,
thiol-modified hyaluronans or other glycosaminoglycans (GAGs). A
trigger for solidification can be any factor eliciting
cross-linking of the matrix components or gelation of those
components that can gel. The cross-linker may comprise
Poly(ethylene glycol) (PEG) or PEG-diacrylate (PEGDA) hydrogel or a
disulfide-containing derivative thereof.
[0158] In another aspect, this disclosure provides a methods of
forming organoids by culturing a first type of cells (epithelia)
with one or more second type of cells (mesenchymal cells), wherein
the second type of cells is at a maturational stage to be an
appropriate lineage partner of the first type of cells. In some
embodiments, this can be achieved by removing mature cells that
attach to culture dishes by panning; transferring the cells that
did not attach to low attachment culture dishes and in an
appropriate medium; and recovering organoids that self-assemble
under these conditions. The first type of cells may be epithelial
stem cells, committed progenitors of epithelial cells, or mature
cells (e.g. hepatocytes). The second type of cells may be stem
cells of the mesenchymal lineages (e.g. angioblasts, mesenchymal
stem cells), progenitors of those lineages (e.g. endothelial or
stellate cell progenitors), or a mixture of early lineage stage
mesenchymal cells. Critically, such formation cannot occur under
all conditions. For example, culturing in Matrigel does not
generate suitable organoids for successful patch grafting. Though
Matrigel-prepared organoids might engraft, the extent of
engraftment will be muted relative to that with organoids prepared
in defined conditions. Moreover, Matrigel cannot be a component of
conditions that are to be used for clinical products.
[0159] In another aspect, this disclosure provides a method for
engrafting cells into an organ comprising contacting a patch graft
comprising multiple layers including a biocompatible, biodegradable
backing that is neutral in effects on the differentiation of the
donor cells; a second layer comprising one or more biomaterials,
such as hyaluronans, that can be solidified such as into a
hydrogel; a mixture of epithelial cells and supportive mesenchymal
cells that are incorporated into the solidified biomaterial; and
this Bandaid-like structure attached to a target site by sutures or
surgical glue. On the serosal surface of the backing is added a
layer of the solidified biomaterials prepared to achieve 400 Pa or
higher, a level at least twice that found in the soft biomaterials
into which the donor cells are incorporated. The cells within the
patch graft are able to engraft and migrate into and throughout the
tissue/organ and then to mature to relevant adult fates, dictated
by the microenvironment in which they become located. The higher
Pascal levels of the biomaterials embedded or comprised into a
porous backing blocks the migration of the cells in the wrong
direction and that added to the serosal surface of the graft
minimizes adhesions of cells from other organs and tissues.
[0160] Organoids
[0161] According to one embodiment disclosed herein, organoids,
floating aggregates of biliary tree stem cells (hereinafter
"BTSCs") and early lineage stage mesenchymal cells (hereinafter
"ELMCs") proved the most successful method of incorporating cells
in the grafts. It is disclosed herein that BTSCs and ELMCs can
self-select into organoids by panning to eliminate the mature
stellate/stromal cells, and this a proved more efficient and
effective in establishing lineage-stage appropriate
epithelial-mesenchymal partners for the grafts. In another aspect,
this disclosure provides a methods of forming organoids by
culturing a first type of cells with a second type of cells,
wherein the second type of cells is a stage appropriate lineage
partner of the first type of cells, removing mature cells that
attach to the culture dish by panning, and recovering the
self-assembled organoids from the suspension of the culture. The
first type of cells may be epithelial stem cells or committed
epithelial cells. The second type of cells may be cells of the
mesenchymal lineage, mesenchymal stem cells, or early lineage stage
mesenchymal cells. Further aspects relate to the self-assembled
organoid and uses thereof.
[0162] In some embodiments, either the donor cells and/or the
supporting mesenchymal cells express matrix metallo-proteinases
(hereinafter MMPs). Without being limited by theory, it is believed
that the MMPs allows for merger of donor and host cells, and the
dissolution of Glisson's capsule (or the equivalent capsule around
the tissue or organ). The disclosure herein provides that in some
embodiments, the early stage stem cells or ELMCs express high
levels of MMPs, whereas the mature hepatocytes express low levels
of MMPs. In some embodiments, partnering mature hepatocytes with
mature sinusoidal endothelia (CD31+++, VEGF-receptor+, type IV
collagen+ and negative for CD117) and those for adult
cholangiocytes are associated with mature stellate and stromal
cells (ICAM-1+, ASMA+, Vitamin A++, type I collagen+) results in
cell aggregates that remain at the surface of the organ and cannot
be effectively engrafted. In some embodiments, engraftment of
mature epithelial cells requires that they are partnered with
immature mesenchymal cells that produce the requisite MMPs for
engraftment and migration.
[0163] According to one embodiment disclosed herein, organoids,
floating aggregates of stem/progenitor cells, such as BTSCs and
ELSMCs, proved the most successful presentation of cells for
success at patch grafting. It is disclosed herein that BTSCs and
ELSMCs can self-select into organoids by elimination of the mature
mesenchymal cells by standard panning procedures for cells that
attach to regular dishes under serum-free conditions, followed by
culturing the remaining cells (those that did not attach) in low
attachment dishes and in serum-free, defined medium. Organoids
self-assemble under these conditions.
[0164] In another aspect, this disclosure provides a method of
forming organoids by culturing a first type of cells, epithelia,
with a second type of cells, mesenchymal cells, wherein the second
type of cells is a stage appropriate lineage partner of the first
type of cells, removing mature cells that attach to the regular
culture dishes by panning procedures, and recovering the organoids
that self-assemble from the suspension of the culture on culture
dishes that are low attachment ones. The first type of cells may be
epithelial stem cells, transit amplifying cells committed
epithelial progenitors. The second type of cells may be stem cells
of the mesenchymal cell lineages, transit amplifying cells or
committed mesenchymal progenitors.
[0165] In some embodiments, either the donor cells and/or the
supporting mesenchymal cells express matrix metallo-proteinases
(hereinafter MMPs). Without being limited by theory, it is believed
that the MMPs results in dissolution of the capsules around tissues
or organs and allows for merger of donor and host cells. The
disclosure herein provides that in some embodiments, the early
stage stem cells or ELMCs express high levels of MMPs, whereas the
mature hepatocytes express low levels of MMPs. In some embodiments,
partnering mature hepatocytes with mature sinusoidal endothelia
(CD31+++, VEGF-receptor+, type IV collagen+ and negative for CD117)
and those for adult cholangiocytes are associated with mature
stellate and stromal cells (ICAM-1+, ASMA+, Vitamin A++, type I
collagen+) results in cell aggregates that remain at the surface of
the organ and cannot be effectively engrafted. In some embodiments,
engraftment of mature epithelial cells requires that they are
partnered with immature mesenchymal cells that produce the
requisite MMPs for engraftment and migration.
[0166] According to one embodiment disclosed herein, organoids,
floating aggregates of stem/progenitor cells, such as BTSCs and
ELSMCs, proved the most successful presentation of cells for
success at patch grafting. It is disclosed herein that BTSCs and
ELSMCs can self-select into organoids by elimination of the mature
mesenchymal cells by standard panning procedures for cells that
attach to regular dishes under serum-free conditions, followed by
culturing the remaining cells (those that did not attach) in low
attachment dishes and in serum-free, defined medium. Organoids
self-assemble under these conditions.
[0167] In another aspect, this disclosure provides a method of
forming organoids by culturing a first type of cells, epithelia,
with a second type of cells, mesenchymal cells, wherein the second
type of cells is a stage appropriate lineage partner of the first
type of cells, removing mature cells that attach to the regular
culture dishes by panning procedures, and recovering the organoids
that self-assemble from the suspension of the culture on culture
dishes that are low attachment ones. The first type of cells may be
epithelial stem cells, transit amplifying cells committed
epithelial progenitors. The second type of cells may be stem cells
of the mesenchymal cell lineages, transit amplifying cells or
committed mesenchymal progenitors.
[0168] In some embodiments, for success with patch grafting
strategies, either the donor cells and/or the supporting
mesenchymal cells must express multiple matrix metallo-proteinases
(hereinafter MMPs) and especially secreted forms of MMPs. Without
being limited by theory, it is believed that multiple isoforms of
the MMPs allows for the dissolution of the capsule around the organ
or tissue followed by rapid migration of donor cells into the host
tissue. The disclosure herein provides that the early stage
epithelial stem cells and/or ELSMCs express high levels of
membrane-associated and/or secreted MMPs, whereas the mature cells
(e.g. hepatocytes) express low levels of secreted MMPs even if they
express plasma membrane-associated MMPs. Engraftment of such adult
cells (e.g. hepatocytes, cholangiocytes, islets, enterocytes, etc.)
requires that the mesenchymal partner be a cellular source of MMPs,
particularly the secreted forms of MMPs if engraftment is to occur.
An alternative is to provide the relevant isoforms of MMPs, that is
purified forms of them, in the biomaterials of the graft.
[0169] According to this disclosure, the numbers of cells that can
be engrafted using a patch graft are considerable (>10.sup.8)
and dictated by the dimensions of the graft, the number and size of
the organoids (or the number of cells-if not part of organoids),
whether the donor cells are stem cells or mature cells, and the
expression of secreted and membrane-associated MMPs (whether from
the epithelia and/or from the mesenchymal cells). These findings
are quite distinct from the limited numbers of cells (e.g.
10.sup.5-10.sup.6) feasible with vascular delivery or by injection
grafting.
[0170] It is disclosed herein that the making the grafts comprises
mixing of cells with appropriate biomaterials that can become
insoluble and keep cells localized to the target site. In another
aspect, the isolated cells of the internal organ may be solidified
ex vivo within the biomaterials prior to introducing the cells into
the hosts, or in the alternative, injected as a fluid substance and
allowed to solidify in vivo. In another aspect, the biomaterials
that can form hydrogels, or a parallel insoluble complex, can
comprise hyaluronans or other non-sulfated or minimally sulfated
glycosaminoglycans, a thiol-modified sodium hyaluronate or plant
derived material (e.g. alginates). A trigger for solidification can
be any factor eliciting cross-linking of the matrix components or
gelation of those that can gel. The cross-linker may comprise
polyethylene glycol diacrylate or a disulfide-containing derivative
thereof. Preferably, the insoluble complex of cells and
biomaterials possesses a viscoelasticity ranging from about 0.1 to
200 Pa, preferably about 0.1 to about 1 Pa, about 1 to about 10 Pa,
about 10-100 Pa, or about 100 to about 200.
[0171] Preferably, the cells are introduced at or near the diseased
or dysfunctional tissue, and may be introduced via injection or
surgical delivery. Without being limited by theory, it is an
hypothesis herein that more rigid HA hydrogels, (e.g. >500 Pa),
triggers differentiation of the cells and reduces engraftment due,
in part, to the reduction in expression of MMPs with maturation
and, in parallel reduction in ability to migrate.
[0172] Backing
[0173] There are multiple options for the biocompatible,
biodegradable backing with neutrality to the maturational state of
the donor cells. They include forms of Bombyx moth silk such as
Seri.RTM. Surgical Silk Scaffolds or Contour Seri-Silk (Sofregen,
New York, N.Y.), other derivatives of Bombyx moth silk, amnion
derivatives, omentum, placenta, and synthetic textiles or
materials, such as forms of Polyglycolic acid-co-poly-L-lactic acid
(PGA/PLLA). Critical to the effectiveness of the backing is that it
has minimal effects on the differentiation of the donor cells.
Thus, many forms of backings used clinically are not useful for
patch grafting, since they are comprised of components (e.g. forms
of mature types of extracellular matrix) that induce
differentiation of the donor cells.
[0174] The backing must have sufficient tensile strength to permit
attaching the graft to the target site by sutures or by surgical
glue. It should be comprised of a biocompatible, biodegradable
material that is capable of degrading within a couple of months but
with degradation products that do not alter the maturational state
of the donor cells. Thus, the products should have minimal effects
on the pH or on other facets of the environment. The backing must
also be able to fit to the surface of the target site; so flexible
backing will facilitate using the grafts on sites of significant
curvature. Seri-Silk is a non-limiting example of a suitable
material for the backing. An aminion derived alternative is also
contemplated as a suitable material for the backing, such as but
not limited to the aminion derived material produced by Osiris
Therapeutics, Inc (Columbia, Md.).
[0175] Backing may be sourced from a porous scaffold, such as
Seri-silk, or a non-porous membrane, such as amnion or placental
membrane or omentum, or can be a porous or non-porous synthetic
textile, or a combination thereof. If the backing is porous it
should be infused/impregnated with a biomaterial to seal it and so
inhibit migration of said population of cells in the direction of
the backing, i.e. away from the target site, or through the backing
The critical features of the backing material that it is
biocompatible, biodegradable, neutral as defined above, and has
sufficient tensile strength as described above. Further, the
material may optionally be bioresorbable.
[0176] The backing may be further optimized depending on the use.
For example, in some embodiments, a patch graft is useful for skin
and underlying dermal tissues if it comprises a backing designed to
survive the drying effect of air.
[0177] Hydrogel matrices as disclosed herein above may also be
useful in other parts of the patch graft. For example, should the
biocompatible, biodegradable backing be porous, a hydrogel may be
used to inhibit migration of said population of cells in the
direction of the backing. Such a hydrogel would require a higher
viscoelasticity compared to the hydrogel, e.g., between 1.5 and 15
fold greater, for example 2 fold greater. Non-limiting examples of
a suitable viscoelasticity include by are not limited a
viscoelasticity properties ranging from about 250 to about 600 Pa,
for example at least about 250 Pa, at least about 300 Pa, at least
about 350 Pa, at least about 400 Pa, at least about 450 Pa, at
least about 500 Pa, at least about 550 Pa, at most about 600 Pa, at
most about 550 Pa, at most about 500 Pa, at most about 450 Pa, at
most about 400 Pa, at most about 350 Pa, at most about 200 Pa
and/or any individual value in between such as but not limited to
about 250 Pa, about 300 Pa, about 350 Pa, about 400 Pa, about 450
Pa, about 500 Pa, about 550 Pa, and about 600 Pa. Further
non-limiting examples of suitable viscoelasticity include by are
not limited a viscoelasticity ranging from about 600 to about 800
Pa, for example at least about 600 Pa, at least about 650 Pa, at
least about 700 Pa, at least about 750 Pa, at most about 800 Pa, at
most about 750 Pa, at most about 700 Pa, at most about 650 Pa, at
most about 600 Pa, and/or any individual value in between such as
but not limited to about 600 Pa, about 650 Pa, about 700 Pa, about
750 Pa, and about 800 Pa. Still further non-limiting examples
include the range from about 250 Pa to about 800 Pa.
[0178] Further still, the hydrogels disclosed herein may be useful
as a coating to prevent adhesion on the serosal surface of the
backing, which is opposite to the side of the backing adjacent to
the cells. Such a hydrogel may should have a viscoelasticity
between that suitable for the hydrogel in which the cells are
comprised and that suitable to seal the backing. Non-limiting
examples of a suitable viscoelasticity include by are not limited a
viscoelasticity ranging from about 250 to about 400 Pa or about 500
Pa, for example at least about 250 Pa, at least about 300 Pa, at
least about 350 Pa, at least about 400 Pa, at least about 450 Pa,
at most about 500 Pa, at most about 450 Pa, at most about 400 Pa,
at most about 350 Pa, at most about 200 Pa and/or any individual
value in between such as but not limited to about 250 Pa, about 300
Pa, about 350 Pa, about 400 Pa, about 450 Pa, and about 500 Pa.
[0179] Grafts, In General
[0180] In general, a patch graft may be designed using the
aforementioned methods and components for transplantation of donor
(allogeneic or autologous) cells to a solid organ or tissue and
with conditions sustaining and maintaining donor cells at an early
maturational lineage stage. More particularly, a patch graft is
contemplated, which useful for transplantation of donor cells
(allogeneic or autologous) to a solid organ or tissue, with
conditions sustaining and maintaining some or all of the donor
cells at an early maturational lineage stage. In some embodiments,
the donor cells are a mixture of epithelial and mesenchymal cells.
In some embodiments both donor cell populations are stem/progenitor
cells. In some embodiments, the epithelial cells are mature cells
(e.g. hepatocytes, islets, etc.) and the mesenchymal cells are
stem/progenitor cells. In some embodiments, the conditions of the
graft biomaterials, e.g. the medium and matrix components, enable
both the donor cell populations or at least the mesenchymal cell
population to remain as stem/progenitor cells. In some embodiments
the medium comprises a basal medium and soluble signals. In further
embodiments, this basal medium and soluble signals are supportive
of maintenance of stemness in both donor populations or at least in
the mesenchymal cell population. In some embodiments, the matrix,
optionally comprising extracellular matrix components, and its
level of rigidity are supportive of maintenance of stemness of both
the donor populations or at least the mesenchymal cell population.
In some embodiments, the matric comprises hyaluronans, optionally
prepared as a soft hydrogel having a viscoelasticity of about 50 Pa
to about 150 Pa. In some embodiments, the patch graft comprises a
backing which has sufficient mechanical strength to enable the
graft to be tethered to the target site and consists of a
biocompatible, biodegradable material that does not significantly
alter the maturational lineage stage of the donor cells.
Optionally, without further modifications, the backing should be
adequate on its own to protect the layer containing the donor cells
without significantly affecting the donor cells' maturational
lineage stage. In some embodiments, the backing is a mesh or
scaffold and is further impregnated with a biomaterial such as
hyaluronana with a viscoelasticity sufficiently high as to make any
cells migrating into it mature enough to abrogate the migration of
the donor cells in a direction other than towards the target site.
In some embodiments, this viscoelasticity is about 500 Pa or
greater. In some embodiments, the serological surface of the graft
is coated with a biomaterial to minimize adhesions from adjacent
tissue or organs. In some embodiments, these biomaterials have a
viscoelasticity of about 200 Pa to about 300 Pa.
[0181] The proposed backing is contemplated to have sufficient
resilience to withstand mechanical forces, is able to be tethered
to the target organ or tissue, and has sufficient flexibility to be
tethered to locations with curvature. Also any biomaterial (other
than a hydrogel) can be utilized so long as the biomaterial is
capable of sustaining and maintaining the cell populations and has
viscoelasticity properties sufficient to allow for migration of the
cell population within or away from the patch graft.
[0182] In another embodiment, the patch graft is useful for
sustaining and maintaining a population of cells and comprises: (a)
a population of cells (optionally of a single type), supported in a
medium in a hydrogel or other biomaterial having viscoelasticity
sufficient to allow for migration of the cells within or away from
the patch graft; and (b) a backing comprising a biocompatible,
biodegradable material having a viscoelasticity sufficient to
inhibit (or provide a barrier to) migration of the cell population
in a direction of the backing,
[0183] It is important to note that MMPs can be membrane-associated
and/or secreted MMPs; they can be provided by MMP producing cells,
derived from such cells, or they can be added to the compositions
of interest (e.g., purified or produced recombinantly).
[0184] In another embodiment, a covering or coating for a patch
graft or tissue is provided which comprises a hydrogel or other
biomaterial with sufficient viscoelasticity and resilience to
withstand mechanical forces applied against the covering or
coating, including such forces being applied from or by other
tissues and organs. By use of the covering or coating, a method is
provided for inhibiting or preventing a formation of adhesions
(which may involve or result from mechanical forces or contact from
other organs and tissues), which method comprises covering or
coating a surface with a hydrogel or other comparable
biomaterial.
[0185] In yet another embodiment, a method of engrafting cells into
a target tissue is provided, which comprises contacting a target
tissue with a patch graft, comprising: (a) a population of cells,
including at least one population having an early lineage stage,
comprising a single type or multiple types of cells supported in a
medium in a hydrogel or other biomaterial having rheological
properties (e.g., viscoelasticity) sufficient to allow for
migration of cells of the population within or away from the patch
graft; and (b) a backing comprising a biocompatible, biodegradable
material having rheological properties (e.g., viscoelasticity)
sufficient to inhibit (or provide a barrier to) migration of cells
of the population in a direction of said backing, the patch graft
configured to sustain and maintain said population of cells while
inhibiting said at least one population having an early lineage
stage from differentiating or further maturing to a later lineage
stage. In a further embodiment, a method is provided in which the
one population having an early lineage stage is capable of
expressing membrane-associated and/or secreted matrix
metalloproteinases (MMPs). In another embodiment, the cells do not
have this capability but MMPs are present or included from other
sources (e.g. recombinant).
[0186] Grafts with a Cell Source of MMPs
[0187] Aspects of the disclosure relate to a patch graft for
sustaining and maintaining a mixed population of cells, comprising:
(a) a mixed population having two or more cell types, at least one
of which is at an early lineage stage that is capable of expressing
secreted and/or membrane-associated and/or secreted matrix
metalloproteinases (MMPs), said mixed population supported in a
medium present in a hydrogel matrix having a viscoelasticity
sufficient to allow for migration of said mixed population,
optionally, within or away from said hydrogel and/or within or away
from the patch graft; (b) a backing comprising a biocompatible,
biodegradable material having a viscoelasticity sufficient to
inhibit a migration of said mixed population in a direction of said
backing; and, optionally, ((c) a hydrogel overlaid on a serosal
(i.e. outside) surface of said backing, which is opposite to that
in contact with said mixed population and, in embodiments where the
patch graft is tethered to a target site, is opposite the side in
contact with the target site (e.g. organ or tissue). In some
embodiments, this layer prevents or inhibits adhesions by or from
other tissues or organs. In some embodiments, the patch graft is
configured to sustain and maintain said mixed population while
inhibiting said at least one early lineage stage cell type from
differentiating or further maturing to a later lineage stage that
is no longer capable of expressing membrane-associated and/or
secreted MMPs.
[0188] In some embodiments, the graft might contain only one cell
type such as an embryonic stem (ES) cell or induced pluripotent
stem (iPS) cells. This can be successful as long as this cells are
a cellular source of MMPs or, alternatively, other sources such as
purified (e.g. recombinant) forms of MMPs are added to the
graft.
[0189] In some embodiments, said backing is porous or non-porous.
In some embodiments, the backing comprises a porous and/or
non-porous mesh, scaffold, or membrane. In some embodiments, the
backing comprises silk; a synthetic textile; or a natural material
such as amnion, placenta, or omentum or derivatives thereof; or a
combination thereof. In some embodiments, said backing comprises a
porous mesh infused with a hydrogel or other biomaterial used to
convert it into a barrier. In further embodiments, such an infusion
prevents cell migration away from the target organ or tissue. In
some embodiments, In some embodiments, said backing comprises a
solid material.
[0190] In some embodiments, one or more of said hydrogels comprise
hyaluronans.
[0191] In some embodiments, said medium comprises Kubota's Medium
or another medium supportive of stem cells and able to maintain
stemness.
[0192] In some embodiments, said mixed population comprises
mesenchymal cells and epithelial cells. In some embodiments, said
epithelial cells may be ectodermal, endodermal, or mesodermal. In
some embodiments, said mesenchymal cells comprise early lineage
stage mesenchymal cells (ELSMCs). In some embodiments, said ELSMCs
comprise one or more of angioblasts, precursors to endothelia,
precursors to stellate cells, and mesenchymal stem cells (MSCs). In
some embodiments, said epithelial cells comprise epithelial stem
cells. In some embodiments, said epithelial cells comprise biliary
tree stem cells (BTSCs). In some embodiments, said epithelial cells
comprise committed and/or mature epithelial cells. In some
embodiments, said committed and/or mature epithelial cells comprise
mature parenchymal cells. In some embodiments, said mature
parenchymal cells comprise one or more of hepatocytes,
cholangiocytes, and islet cells. In some embodiments, said
mesenchymal cells and epithelial cells both comprise stem
cells.
[0193] In some embodiment said mixed population comprises
autologous and/or allogeneic cells.
[0194] In some embodiments, one or more cell types are genetically
modified.
[0195] "Layered" Grafts
[0196] In some embodiments, the patch graft is understood as a
multi-layered graft. For example, provided herein are patch grafts
comprising, consisting of, or consisting essentially of multiple
layers including, at least: (a) a soft first layer of hydrogel
comprising donor cells, optionally epithelial cells and/or
mesenchymal cells; (b) a stiff second layer of hydrogel; and (c) a
third layer comprising a biocompatible, biodegradable backing. In
some embodiments, particular those where the third layer is porous,
the second layer is incorporated, impregnated, and/or infused into
the third layer. In some embodiments, the patch grafts further
comprise a fourth layer of hydrogel. In some embodiments of the
patch graft, the fourth layer is coated or painted onto a serosal
surface of the graft. In some embodiments of the patch graft, the
first layer is adapted to directly contact a target tissue or
organ.
[0197] As used herein, "soft" refers to a hydrogel layer that
exhibits a low level of internal pressure as determined
quantitatively by Pascal (Pa) assays. A Pascal is defined as one
newton per square meter. In some embodiments, a soft layer has a
viscosity of about 10 Pa to about 300 Pa, about 50 Pa to about 250
Pa, about 100 Pa to about 250 Pa, about 50 Pa to about 200 Pa,
about 150 Pa to about 200 Pa, or about 100 Pa to about 200 Pa. In a
particular embodiment, a soft hydrogel layer has a viscosity that
is less than or about 200 Pa.
[0198] As used herein, "stiff" refers to a hydrogel layer that
exhibits a high level of internal pressure as determined
quantitatively by Pascal (Pa) assays. In some embodiments, a stiff
layer has a viscosity of about 300 Pa to about 3000 Pa, about 300
Pa to about 1000 Pa, about 400 Pa to about 750 Pa, about 400 Pa to
about 550 Pa, about 450 Pa to about 600 Pa, or about 500 Pa to
about 600 Pa. In a particular embodiment, a stiff hydrogel layer
has a viscosity that is greater than or about 500 Pa.
[0199] Preferably, for the first layer of the layered graft, the
insoluble complex of cells and biomaterials possesses a viscosity
or viscoelasticity ranging from about 0.1 to 200 Pa, preferably
about 0.1 to about 1 Pa, about 1 to about 10 Pa, about 10 to 100
Pa, or about 100 to about 200, or about 50 to about 250 Pa, or
about 200 Pa. Preferably, for the first layer of the layered graft,
the insoluble complex of cells and biomaterials possesses a
viscoelasticity ranging from about 0.1 to 200 Pa, preferably about
0.1 to about 1 Pa, about 1 to about 10 Pa, about 10-100 Pa, or
about 100 to about 200.
[0200] In some embodiments, one or more of the cells in the mixture
is a source of secreted and/or membrane-associated MMPs. In some
embodiments, such as but not limited to those involving
stem/progenitor cell populations that naturally secrete MMPs,
variables that mute MMP expression--optionally secreted MMP
expression--are controlled in the patch graft. Non-limiting
examples of such variables include variables that result in
maturation of stem/progenitor cells, such as but not limited to
serum supplementation to media or to the graft biomaterials,
hormones or other soluble signals that influence differentiation of
the epithelial and/or mesenchymal cells, oxygen levels (as
anaerobic conditions keep the cells immature, whereas higher oxygen
levels promote differentiation), and the rigidity of graft
materials (as mechanical forces such as shear force and compression
may drive differentiation).
[0201] In some embodiments of the patch graft, the viscosity of the
first layer is about 50 to about 250 Pa. In some embodiments of the
patch graft, the viscosity of the first layer is about 200 Pa. In
some embodiments of the patch graft, the viscosity of the second
layer is about 250 Pa to about 600 Pa. In some embodiments of the
patch graft, the viscosity of the second layer is about 500 Pa. In
some embodiments of the patch graft, the viscosity of the fourth
layer is about 250 to about 500 Pa. In some embodiments of the
patch graft, the viscosity of the fourth layer is about 400 Pa. In
some embodiments of the patch graft, the viscosity of the second
layer is greater than the viscosity of layer 1. In some embodiments
of the patch graft, the viscosity of the second layer is about 1.5
to about 15 fold greater than the viscosity of the first layer. In
some embodiments of the patch graft, the second layer is about 2
fold greater than the viscosity of the first layer.
[0202] In one embodiment, a patch graft comprises, consists of, or
consists essentially of layers starting with that in contact with
the target site and consisting of donor cells embedded into a soft
(<200 Pa) hydrogel prepared in a serum-free, defined medium
(these cells are to engraft and migrate into the tissue); a second
layer of the hydrogel prepared in the same medium and triggered to
have a higher rigidity (e.g. .about.500 Pa or higher) providing a
barrier for the donor cells to migrate in any direction other than
towards the target tissue; a third layer, a biocompatible,
biodegradable, bioresorable backing that is neutral in effects on
the maturational state of the donor cells and can be used
surgically or through other means to tether the graft to the target
site; and a final layer of the hydrogel that is intermediate in
rigidity between the soft hydrogel and the very rigid one and
sufficiently fluid to be painted or coated onto the surface to
minimize adhesions by nearby tissues.
[0203] In some embodiments of the patch graft, the first and second
layers each comprise one or more hyaluronans. In some embodiments
of the patch graft, the fourth layer comprises one or more
hyaluronans.
[0204] In some embodiments of the patch graft, the epithelial cells
and the mesenchymal cells form one or more aggregates. In some
embodiments of the patch graft, the one or more aggregates is an
organoid. In some embodiments of the patch graft, the epithelial
cells comprise epithelial stem cells. In some embodiments of the
patch graft, the epithelial cells comprise biliary epithelial
cells. In some embodiments of the patch graft, the epithelial cells
comprise committed and/or mature epithelial cells. In some
embodiments of the patch graft, the committed and/or mature
epithelial cells comprise mature parenchymal cells. In some
embodiments of the patch graft, the mature parenchymal cells
comprise one or more of hepatocytes, cholangiocytes, and islet
cells.
[0205] In some embodiments of the patch graft, the mesenchymal
cells are supportive mesenchymal cells. In some embodiments of the
patch graft, the mesenchymal cells comprise early lineage stage
mesenchymal cells (ELSMCs). In some embodiments of the patch graft,
the ELSMCs comprise one or more of the group consisting of
angioblast, precursor to endothelia, precursor to stellate cells,
and mesenchymal stem cell (MSC).
[0206] In some embodiments of the patch graft, the epithelial cells
and the mesenchymal cells are not lineage stage partners of one
another. In some embodiments of the patch graft, the epithelial
cells are mature cells. In some embodiments of the patch graft, the
mesenchymal cells are ELSMCs.
[0207] In some embodiments of the patch graft, at least one of the
epithelial cells and the mesenchymal cells are derived from a
donor. In some embodiments, the donor is a subject in need of a
tissue transplant. In some embodiments, the donor is the source of
healthy cells for a tissue transplant.
[0208] In some embodiments of the patch graft, the at least one of
the epithelial cells and the mesenchymal cells are autologous to an
intended recipient of the patch graft. In some embodiments, all of
the cells (i.e. epithelial and mesenchymal) are autologous to the
intended recipient of the graft. In some embodiments, the donor of
cells may be one other than the recipient (allograft) or may also
be the subject (autologous) having the internal organ in a diseased
or dysfunctional condition, optionally, wherein are obtained from a
portion of the internal organ that is not diseased or dysfunctional
and/or that the cells have been genetically modified to restore
function. For establishing a model system to study a disease, the
donor cells can be ones that have the disease and that are
transplanted onto/into normal tissue in an experimental host.
[0209] In some embodiments of the patch graft, at least one of the
epithelial cells or the mesenchymal cells are modified. In some
embodiments, all of the cells are modified. In some embodiments,
the modification is genetic modification. In some embodiments, the
one or more cells is modified to express a therapeutic nucleic acid
or polypeptide. In some embodiments, the one or more cells is
modified to express a wild-type allele of a nucleic acid or
polypeptide.
[0210] In some embodiments of the patch graft, the biocompatible,
biodegradable backing is bioresorbable. In some embodiments of the
patch graft, the biocompatible, biodegradable backing comprises a
porous material. In some embodiments of the patch graft, the
biocompatible, biodegradable backing comprises a scaffold or
membrane. In some embodiments of the patch graft, the scaffold or
membrane comprises silk, amnion, a synthetic textile, or a
combination thereof. In some embodiments, the biocompatible,
biodegradable backing does not comprise any factor that induces or
prevents differentiation in cells. In some embodiments of the patch
graft, the biocompatible, biodegradable backing does not include
one or more components derived from mature extracellular matrix. In
some embodiments of the patch graft, the component derived from
mature extracellular matrix is type I collagen.
[0211] In some embodiments of the patch graft, the patch graft
further comprises one or more matrix metallo-proteinases (MMPs). In
some embodiments of the patch graft, the MMP is a
membrane-associated MMP. In some embodiments of the patch graft,
the membrane-associated MMP is provided by one or more of the
epithelial cells or the mesenchymal cells. In some embodiments of
the patch graft, the MMP is a secreted MMP. The secreted MMPs may
optionally be produced naturally by the one or more of the
epithelial or mesenchymal cells or optionally be produced due to
transformation of the one or more of the epithelial or mesenchymal
cells with a recombinant expression vector for MMP production.
[0212] In some aspects, provided herein is a patch graft
comprising, consisting of, or consisting essentially of multiple
layers including, at least: a soft first layer of hydrogel
comprising biliary tree stem cells; a stiff second layer of
hydrogel; and a third layer comprising a biocompatible,
biodegradable backing.
[0213] In one embodiment, a patch graft consists of layers of
materials and cells that collectively form a "bandaid-like graft"
that can be tethered surgically or otherwise to a target site. The
first layer, that against the target site, comprises a soft
hydrogel (under 200 Pa) into which are seeded a mixture of
epithelial cells and supportive mesenchymal cells suspended in a
defined, serum-free, nutrient-rich medium designed for expansion
and/or survival of the cells; a second layer containing a hydrogel
prepared in the same medium but gelled to a more rigid level (i.e.
higher Pascal levels) and forming a barrier blocking cells from
migrating in a direction other than to the target sites; a third
level comprising a biocompatible, biodegradable backing that does
not affect or minimally affects the differentiation level of the
donor cells but acting as a mechanical support structure for the
patch; a fourth layer comprised of paintable hydrogel (again such
as hyaluronans) that is at a rigidity level intermediate between
that of the soft versus rigid hydrogel and serving to minimize
adhesions to the graft from cells from neighboring tissues. The
hydrogels must consist of a material that is biocompatible,
biodegradable and "tunable", meaning regulatable with respect to
rigidity. One successful material for the hydrogels is
thiol-modified hyaluronan that can be triggered to form hydrogels
when exposed to oxygen and/or to poly (ethylene glycol) diacrylate
(PEGDA) and readily "tunable" by the precise ratios of hyaluronan
and PEGDA concentrations (and/or oxygen levels).
[0214] In another embodiment, a patch graft comprises multiple
layers. The first layer, that against the target site, is of a soft
hydrogel that is minimally sulfated or non-sulfated GAG or other
non-sulfated or neutral biomaterial that can be gelled or
solidified and into which is placed donor cells. A second layer of
a hydrogel or biomaterial that is more rigid and incorporated
into/onto or within a backing, a biocompatible, biodegradable,
bioresorbable backing that allows the patch to be handled for
surgical or other purposes and that serves as a barrier forcing
cells to migrate towards the target tissue. The serosal side of the
backing is coated at the time of surgery with biomaterials such as
hyaluronans (or other minimally or non-sulfated GAGs or other
materials that can be gelled or solidified) and in which the Pascal
levels are at least twice that of the Pascal levels found in the
layer of soft biomaterials; this serves the purpose of minimizing
adhesions from neighboring tissues. The patch graft is tethered to
the target organ or tissue, and the cells are able to migrate into
the tissue or organ and become fully incorporated.
[0215] In a particular embodiment, a patch graft comprises a first
layer of a soft biomaterial (<200 Pa), such as a soft hyaluronan
hydrogel, and into which are placed the donor cells to be
transplanted in a serum-free, defined medium tailored to the
lineage stage of the cells. This layer is placed atop a more rigid
layer (e.g. a more rigid hydrogel) that serves as a barrier forcing
the donor cells to be directed in their migration to the target
tissue. The more rigid layer is prepared ahead of time on a
backing, a biocompatible, biodegradable backing that enables
handling the patch for surgical or other procedures so as to affix
the patch to the target site. The final layer is a biomaterial that
is intermediate in rigidity from that for the donor cells on the
target tissue side and that for the barrier. This layer is added on
the serosal side of the graft and at the time of surgery and serves
to minimize adhesions from neighboring tissues. The biocompatible,
biodegradable backing may be Seri-silk or a derivative thereof.
[0216] Methods of Use and Delivery for Patch Grafts
[0217] Aspects of the disclosure relate to compositions and methods
for engrafting cells into an organ. Efforts to transplant cells
from solid organs into internal organs typically made use either of
direct injection or delivery of cells via a vascular route.
Lanzoni, G. et al. Stem Cells 31, 2047-2060 (2013). These methods
of transplantation result in small numbers of cells being
transplanted to the target site, and in risks of emboli that can be
life threatening. Transplantation is improved if the cells are
delivered by "injection grafting" in which the cells are suspended
in or coated with hyaluronans and then co-injected with a trigger
(PEGDA) that causes the hyaluronan to gel in situ as described in
Turner R. et al. Hepatology 57, 775-784 (2013). Injection grafting
methodologies provide a strategy for localizing cells to a specific
site, albeit in small numbers, typically 10.sup.5-10.sup.7,
10.sup.6-10.sup.7, or 10.sup.5-10.sup.6 cells per injection site.
This strategy eliminates or minimizes ectopic cellular distribution
and optimizes the integration of the cells in the site. However, if
mature functional cells are used, they may be highly immunogenic,
necessitating long-term immunosuppression. Also, the quantity of
cells that are able to be injected may be insufficient to achieve
the requisite clinical results.
[0218] These hurdles and concerns are overcome by "patch grafting"
strategies described herein. In some embodiments, "bandaid-like"
grafts are tethered surgically or otherwise to the surface of an
organ or tissue; the conditions of the graft are such that the
cells engraft fully into the site, migrate throughout the
organ/tissue, and then mature into relevant adult cell types. The
potential for transplantation of large numbers of cells
(>10.sup.8 cells) is shaped or determined by the size of the
patch, the number or mixture of cells within the graft, and the
source of multiple forms of MMPs, ideally cellular sources of the
MMPs. Moreover, in some embodiments the use of organoids
facilitates the ability to stockpile donor cells given the ease by
which the organoids can be cryopreserved under defined, serum-free
conditions.
[0219] The patch graft composition provided herein is directed to
direct grafting of cells onto the tissue or solid organ. The method
is safe, avoids emboli and ectopic cell distribution, and optimizes
cell number engraftment and distribution into and throughout the
tissue.
[0220] Accordingly, provided herein are methods of engrafting cells
into a target tissue comprising, consisting of, or consisting
essentially of contacting the target tissue with a patch graft
disclosed herein above.
[0221] In some embodiments of the methods, the target tissue is
selected from the group consisting of liver, pancreas, biliary
tree, thyroid, thymus, gastrointestine, lung, prostate, breast,
brain, bladder, spinal cord, skin and underlying dermal tissues,
uterus, kidney, muscle, blood vessel, heart, cartilage, tendons,
and bone tissue. In some embodiments of the methods, the target
tissue is liver tissue. In some embodiments of the methods, the
target tissue is pancreatic tissue. In some embodiments of the
methods, the target tissue is biliary tree tissue. In some
embodiments of the methods, the target tissue is gastrointestinal
tissue. In some embodiments, the tissue is diseased, damaged, or
has a disorder. In some embodiments of the methods, the target
tissue is kidney tissue.
[0222] In some embodiments of the methods, the target tissue is an
organ. In some embodiments of the methods, the organ is an organ of
the musculoskeletal system, the digestive system, the respiratory
system, the urinary system, the female reproductive system, the
male reproductive system, the endocrine system, the circulatory
system, the lymphatic system, the nervous system, or the
integumentary system. In some embodiments of the methods, the organ
is selected from the group consisting of liver, pancreas, biliary
tree, thyroid, thymus, gastrointestines, lung, prostate, breast,
brain, bladder, spinal cord, skin and underlying dermal tissues,
uterus, kidney, muscle, blood vessel, heart, cartilage, tendon, and
bone. In some embodiments, the organ is diseased, damaged, or has a
disorder.
[0223] Also provided herein are methods of treating a subject with
a liver disease or disorder, the methods comprising, consisting of,
or consisting essentially contacting the subject's liver a patch
graft disclosed herein above. In some embodiments of the methods,
the liver disease or disorder is liver fibrosis, liver cirrhosis,
hemochromatosis, liver cancer, biliary atresia, nonalcoholic fatty
liver disease, hepatitis, viral hepatitis, autoimmune hepatitis,
fascioliasis, alcoholic liver disease, alpha 1-antitrypsin
deficiency, glycogen storage disease type II, transthyretin-related
hereditary amyloidosis, Gilbert's syndrome, primary biliary
cirrhosis, primary sclerosing cholangitis, Budd-Chiari syndrome,
liver trauma, or Wilson disease.
[0224] In other aspects, provided herein are methods of treating a
subject with a disease or disorder of the pancreas, the methods
comprising, consisting of, or consisting essentially of contacting
the subject's pancreas with a patch graft disclosed herein above.
In some embodiments of the methods, the disease or disorder of the
pancreas is diabetes mellitus, exocrine pancreatic insufficiency,
pancreatitis, pancreatic cancer, sphincter of Oddi dysfunction,
cystic fibrosis, pancreas divisum, annular pancreas, pancreatic
trauma, or hemosuccus pancreaticus.
[0225] In other aspects, provided herein are methods of treating a
subject with a gastrointestinal disease or disorder, the method
comprising, consisting of, or consisting essentially of contacting
one or more of the subject's intestines with a patch graft
disclosed herein above. In some embodiments, the gastrointestinal
disease or disorder is gastroenteritis, gastrointestinal cancer,
ileitis, inflammatory bowel disease, Crohn's disease, ulcerative
colitis, irritable bowel syndrome, peptic ulcer disease, celiac
disease, fibrosis, angiodysplasia, Hirschsprung's disease,
pseudomembranous colitis, or gastrointestinal trauma.
[0226] In some aspects, provided herein are methods of treating a
subject with a kidney disease or disorder, the methods comprising,
consisting of, or consisting essentially of contacting one or more
of the subject's kidneys with a patch graft disclosed herein above.
In some embodiments of the methods, the kidney disease or disorder
is nephritis, nephrosis, nephritic syndrome, nephrotic syndrome,
chronic kidney disease, acute kidney injury, kidney trauma, cystic
kidney disease, polycystic kidney disease, glomerulonephritis, IgA
nephropathy, lupus nephritis, kidney cancer, Alport syndrome,
amyloidosis, Goodpasture syndrome, or Wegener's granulomatosis.
[0227] In some embodiments of the therapeutic methods, at least one
of the epithelial cells and the mesenchymal cells are derived from
a donor. In some embodiments, the donor is a subject in need of a
tissue transplant. In some embodiments, the donor is the source of
healthy cells for a tissue transplant. In some embodiments, at
least one of the epithelial cells and the mesenchymal cells are
autologous to an intended recipient of the patch graft. In some
embodiments, all of the cells (i.e. epithelial and mesenchymal) are
autologous to the intended recipient of the graft. In some
embodiments, the donor of cells may be one other than the recipient
(allograft) or may also be the subject (autologous) having the
internal organ in a diseased or dysfunctional condition,
optionally, wherein are obtained from a portion of the internal
organ that is not diseased or dysfunctional and/or that the cells
have been genetically modified to restore function.
[0228] In some embodiments, the patch graft used in the methods
disclosed herein above is a patch graft comprising multiple layers
including, at least: a first layer of hydrogel comprising
epithelial cells and mesenchymal cells; a second layer of hydrogel;
a third layer comprising a biocompatible, biodegradable backing;
and optionally a fourth layer of hydrogel. In some embodiments, the
methods further comprise allowing the cells contained in the patch
graft to become incorporated into the tissue. In some embodiments
of the methods, the first layer of hydrogel is soft. In some
embodiments of the methods, the second layer of hydrogel is stiff.
In some embodiments of the methods, the mesenchymal cells are
supportive mesenchymal cells.
[0229] In another aspect, this disclosure provides a method for
engrafting cells into an organ comprising use of a patch graft, a
bandaid-like composite with multiple layers of materials and cells
that collectively can be tethered surgically or otherwise to a
target site. The first layer, that against the target site,
comprises a soft hydrogel (under 200 Pa) into which are seeded a
mixture of epithelial cells and supportive mesenchymal cells
suspended in a defined, serum-free, nutrient-rich medium designed
for expansion and/or survival of the cells; a second layer
containing a hydrogel prepared in the same medium but gelled to a
more rigid level (i.e. higher Pascal levels) and forming a barrier
blocking cells from migrating in a direction other than to the
target sites; a third level comprising a biocompatible,
biodegradable backing that does not affect or minimally affects the
differentiation level of the donor cells and hence is "neutral;" a
fourth layer comprised of paintable hydrogel (again such as
hyaluronans) that is at a rigidity level intermediate between that
of the soft versus rigid hydrogel and serving to minimize adhesions
to the graft from cells from neighboring tissues. The hydrogels
must consist of a material that is biocompatible, biodegradable and
"tunable", meaning regulatable with respect to rigidity. One
successful material for the hydrogels is thiol-modified hyaluronan
that can be triggered to form hydrogels when exposed to oxygen
and/or to poly (ethylene glycol) diacrylate (PEGDA) and readily
"tunable" by the precise ratios of hyaluronan and PEGDA
concentrations (and/or oxygen levels). The cells under the
conditions of the biomaterials of the graft produce multiple
matrix-metallo-proteinases (MMPs) that facilitate engraftment,
migration, and integration of the donor cells into the tissue of
the recipient. The microenvironment of the recipient tissue
dictates the adult fate of the transplanted cells.
[0230] In another aspect, this disclosure provides a method for
engrafting cells into an organ comprising contacting a patch graft
comprising multiple layers including, at least, a first layer
comprising a biocompatible, biodegradable backing, a second layer
comprising one or more hyaluronans including a mixture of
epithelial cells and supportive mesenchymal cells and a third layer
comprising one or more hyaluronans, in which the layer in which the
cells are embedded is very soft (under 200 Pa); a layer associated
with the backing is more rigid (.about.500 Pa or more); and a third
layer is intermediate in the level of Pascals and helps to minimize
adhesions from nearby tissues or organs. In yet another aspect, the
cells may be engrafted into an organ selected from the group
consisting of liver, pancreas, biliary tree, thyroid, thymus
intestines, lung, prostate, breast, brain, spinal cord, neural
ganglia, skin and underlying dermal tissues, uterus, bone, thymus,
intestines, uterus, bone, kidney, muscle, blood vessels, or
heart.
[0231] In yet another aspect, the cells may be engrafted into an
organ selected from the group consisting of liver, pancreas,
biliary tree, thyroid, thymus, intestines, lung, prostate, breast,
brain, spinal cord, neural ganglia, skin and underlying dermal
tissues, uterus, bone, tendon, cartilage, kidney, muscle, blood
vessels, or heart.
[0232] A non-limiting example of a patch graft suitable for the
methods disclosed herein is a patch graft comprising: (a) a mixed
population having two or more cell types, at least one of which is
at an early lineage stage that is capable of expressing secreted
and/or membrane-associated and/or secreted matrix
metalloproteinases (MMPs), said mixed population supported in a
medium present in a hydrogel matrix having a viscoelasticity
sufficient to allow for migration of said mixed population,
optionally, within or away from said hydrogel and/or within or away
from the patch graft; (b) a backing comprising a biocompatible,
biodegradable material having a viscoelasticity sufficient to
inhibit or provide a barrier to migration of said mixed population
in a direction of said backing; and, optionally, ((c) a hydrogel
overlaid on a serosal (i.e. outside) surface of said backing, which
is opposite to that in contact with said mixed population and, in
embodiments where the patch graft is tethered to a target site, is
opposite the side in contact with the target site (e.g. organ or
tissue). In some embodiments, this layer prevents or inhibits
adhesions by or from other tissues or organs. In some embodiments,
the patch graft is configured to sustain and maintain said mixed
population while inhibiting said at least one early lineage stage
cell type from differentiating or further maturing to a later
lineage stage that is no longer capable of expressing
membrane-associated and/or secreted MMPs.
[0233] In some embodiments, said backing is porous or non-porous.
In some embodiments, the backing comprises a porous mesh, scaffold,
or membrane. In some embodiments, the backing comprises silk; a
synthetic textile; or a natural material such as aminion, placenta,
or omentum; or a combination thereof. In some embodiments, said
backing comprises a porous mesh infused with a hydrogel. In further
embodiments, such an infusion prevents cell migration away from the
target organ or tissue. In some embodiments, said backing comprises
a solid material.
[0234] In some embodiments, the patch graft further comprises a
hydrogel overlaid on a serosal surface of said backing, which is
opposite to that in contact with said single cell or mixed cell
population.
[0235] In some embodiments, one or more of said hydrogels comprise
hyaluronans.
[0236] In some embodiments, said medium comprises Kubota's medium
or another medium supportive of stem cells and able to maintain
stemness.
[0237] In some embodiments, said mixed population comprises
mesenchymal cells and epithelial cells. In some embodiments, said
epithelial cells may be ectodermal, endodermal, or mesodermal. In
some embodiments, said mesenchymal cells comprise early lineage
stage mesenchymal cells (ELSMCs). In some embodiments, said ELSMCs
comprise one or more of angioblasts, precursors to endothelia,
precursors to stellate cells, and mesenchymal stem cells (MSCs). In
some embodiments, said epithelial cells comprise epithelial
stem/progenitor cells. In some embodiments, said epithelial cells
comprise biliary tree stem cells (BTSCs). In some embodiments, said
epithelial cells comprise committed and/or mature epithelial cells.
In some embodiments, said committed and/or mature epithelial cells
comprise mature parenchymal cells. In some embodiments, said mature
parenchymal cells comprise one or more of hepatocytes,
cholangiocytes, and islet cells. In some embodiments, said
mesenchymal cells and epithelial cells both comprise stem
cells.
[0238] In some embodiment said mixed population comprises
autologous and/or allogeneic cells.
[0239] In some embodiments, one or more cell types are genetically
modified.
EXAMPLES
[0240] The following examples are non-limiting and illustrative of
procedures which can be used in various instances in carrying the
disclosure into effect. Additionally, all reference disclosed
herein below are incorporated by reference in their entirety.
Example 1: Porcine Model for Patch Graft Validation
[0241] Animals
[0242] Animals used as hosts or as donors for cells were maintained
in facilities at the College of Veterinary Medicine at NCSU
(Raleigh, N.C.). Surgeries, necropsies, and the collection of all
biological fluids and tissues were performed at these facilities.
All procedures were approved by the IACUC committee at NCSU. The
pigs being used as recipients were a mixture of six different
breeds: a six-way cross consisting of Yorkshires, Large Whites,
Landraces (from the sows), Durocs, Spots, and Pietrans (from the
boars). This highly heterogeneous genetic background is desirable
in that it parallels the heterogeneous genetic constitutions of
human populations. The host animals were all females, approximately
six weeks of age and .about.15 kg.
[0243] There were two categories. a) male pigs, approximately six
weeks of age and .about.15 kg, were used as donors for cell
transplantation into females; b) transgenic donor animals carrying
a GFP transgene. The GFP+ donor animals were obtained by breeding a
transgenic H2B-GFP boar with a wild type gilt by standard
artificial insemination. The model was developed via CRISPR-Cas9
mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the
endogenous .beta.-actin (ACTB) locus. The transgenic animals show
ubiquitous expression of pH2B-eGFP in all tissues. Fusion of the
GFP to H2B results in localization of the GFP marker to the
nucleosome and allows clear nuclear visualization as well as the
study of chromosome dynamics. The founder line has been analyzed
extensively and ubiquitous and nuclear localized expression has
been confirmed. In addition, breeding has demonstrated transmission
of the H2B-GFP to the next generation. All animals were healthy,
and multiple pregnancies have been established with progeny showing
the expected Mendelian ratio for the transmission of the pH2B-eGFP.
The male offspring were genotyped at birth, and those that were
positive for the transgene were humanely euthanized for tissue
collection, and isolation of donor cells.
[0244] For each donor and recipient animal, the swine leucocyte
antigen class I (SLA-I) and class II (SLA-II) loci have been PCR
amplified using primers designed to amplify known alleles in these
regions based on the PCR-sequence-specific-primer strategy. The
system consists of 47 discriminatory SLA-I primer sets amplifying
the SLA-1, SLA-2, and SLA-3 loci.sup.53, and 47 discriminatory
SLA-II primer sets amplifying the DRB1, DQB1, and DQA loci. These
primer sets have been developed to differentiate alleles by groups
that share similar sequence motifs, and have been shown easily and
unambiguously to detect known SLA-I and SLA-II alleles. When used
together, these primer sets effectively provided a haplotype for
each animal that was tested, thus providing an assay to confirm
easily a matched or mismatched haplotype in donor and recipient
animals.
[0245] Media and Solutions
[0246] All media were sterile-filtered (0.22 .mu.m filter) and kept
in the dark at 4.degree. C. before use. Basal medium and fetal
bovine serum (FBS) were purchased from GIBCO/Invitrogen. All growth
factors were purchased from R&D Systems. All other reagents,
except those noted, were obtained from Sigma.
[0247] A cell wash was formulated with 599 mls of basal medium
(e.g. RPMI 1640; Gibco #11875-093) supplemented with 0.5 grams of
serum albumin (Sigma, #A8896-5G, fatty-acid-free), 10-9 M selenium,
and 5 mls of antibiotics (Gibco #35240-062, AAS). It was used for
washing tissues and cells during processing.
[0248] Collagenase buffer was made and consists of 100 mls of cell
wash supplemented with collagenase (Sigma #C5138) with a final
concentration of 600 U/ml (R1451 25 mg) for biliary tree (ducts)
tissue and 300 U/ml (12.5 mg) for organ-parenchymal tissue (liver,
pancreas).
[0249] Kubota's medium, a defined, serum-free medium designed for
endodermal stem/progenitors was used to prepare cell suspensions,
organoids and HA hydrogels. This medium consists of any basal
medium (here being RPMI 1640) with no copper, low calcium (0.3 mM),
1 nM selenium, 0.1% bovine serum albumin (purified,
fatty-acid-free; fraction V), 4.5 mM nicotinamide, 0.1 nM zinc
sulfate heptahydrate, 5 .mu.g/ml transferrin/Fe, 5 .mu.g/ml
insulin, 10 .mu.g/ml high density lipoprotein, and a mixture of
purified free fatty acids that are presented complexed with fatty
acid free, highly purified albumin. Its preparation is given in
detail in a methods review.sup.57. Also, it is available
commercially from PhoenixSongs Biologicals (Branford, Conn.).
[0250] Soluble, long chain forms of HA (Sigma Catalog #52747) were
used in stabilization of organoid cultures and in cryopreservation
Those used to make the hydrogels, thiol-modified HAs, were obtained
from Glycosan Biosciences, a subsidiary of Biotime. The components
for these thiol-modified HAs were made by a proprietary
bacterial-fermentation process using Bacillus subtilis as the host
in an ISO 9001:2000 process (www.biopolymer.novozymes.com/). The
components were produced by Novozymes under the trade name
HyaCare.RTM. and are 100% free of animal-derived raw materials and
organic solvent remnants. No animal-derived ingredients are used in
the production, and there are very low protein levels and no
endotoxins. The production follows the standards set by the
European Pharmacopoeia) The HA hydrogels were prepared using
Glycosil (HyStem.RTM. HAs, ESI BIO-CG313), the thiol-modified HAs,
that can be trigged to form disulfide bridges using polyethylene
glycol diacrylate (PEGDA). Glycosil.RTM. is reconstituted as a 1%
solution of thiolated HA in 1% phosphate buffered saline (PBS)
using degassed water, or, in our case, in Kubota's Medium. Upon
reconstitution, it remains liquid for several hours but can undergo
some gelation if exposed to oxygen. More precise gelation occurs
with no temperature or pH changes if Glycosil is treated with a
cross-linker such as PEGDA causing gelation to occur within a
couple of minutes.
[0251] The level of cross-linking dictates the level of rigidity,
and can be precisely defined by the ratio of the thiol-modified HAs
to PEGDA. In prior studies, stem cell populations were tested in HA
hydrogels of varying level of rigidity and were found to remain as
stem cells, both antigenically and functionally (e.g. with respect
to ability to migrate), only if the level of rigidity was less than
200 Pa.sup.23. We made use of this finding to design the grafts
with a very soft layer and with more rigid layers of hyaluronan
hydrogels on the serosal side to form a barrier to migration in
directions other than the target tissue as well as to minimize
adhesions from cells from nearby tissues. The 3 versions of the
hydrogels with distinct levels of rigidity are characterized in
FIG. 2, characterizations that included direct measurements of the
rheological properties. The most rigid barrier, that of the
10.times. HA hydrogel (rigidity=760 Pa), was prepared on the
backing ahead of time and could be cryopreserved if desired. At the
time of the surgery, the donor cells were prepared in the soft,
1.times.HA hydrogel (rigidity=60 Pa); placed onto the more rigid
10.times. hydrogel (already on the backing); and the patch tethered
to the target site. After tethering, the serosal side of the graft
was coated or painted with the 2.times.HA hydrogel (rigidity=106
Pa) using a NORM-JECT 4010.200V0 Plastic Syringe with a BD
Micro-Fine.TM. IV permanently attached needle.
[0252] Macro-scale rheological properties of hydrogels were
determined using a stress-controlled cone-and-plate rheometer (TA
Instruments, AR-G2, 40 mm cone diameter, 1.degree. angle). Gels
actively polymerized on the rheometer while oscillating at 1 rad/s
frequency and 0.6 Pa stress amplitude with the modulus monitored
continuously to query for sufficient completion of the
cross-linking reaction. Once equilibrated, the hydrogels were
subjected to an oscillatory frequency sweep (stress amplitude: 0.6
Pa, frequency range: 0.01-100 Hz). The viscoelasticity
(rheological) properties of the 3 versions of hyaluronan hydrogels
that were used are summarized in FIG. 2.
[0253] The most commonly used donor cells were derived from
transgenic H2B-GFP pigs as described above. They offer a
significant advantage for cell transplantation studies in that all
cells are tagged with GFP. The use of fluorescent proteins as
molecular tags enabled the donor cells to be tracked in their
migration and engraftment after transplantation. This fusion
protein is targeted to the nucleosomes resulting in a
nuclear/chromatin GFP signal. In the described grafts, the stem
cells express GFP entirely in the nucleus, but those lineage
restricting to adult cell types can have it in the cytoplasm or
nucleus. Note that the level of cytoplasmic GFP is especially high
in the first week and is reduced with time. This is because the
engraftment/invasion/integration process results in effects on the
cells that can cause the H2B-linked GFP to be found
cytoplasmically. This does not mean that the cells are dying but
rather that they are responding to the high levels of MMPs and
associated signaling that are part of the remodeling zones. Indeed,
the GFP+ cells detected are clearly viable and proliferate, all
expressing various adult functions (e.g. albumin, HNF4a, AFP,
insulin, glucagon, or amylase).
[0254] As described in more detail in the characterizations of the
grafts, autofluorescence both of the backing (spring green color)
and also of lipofuscins (dark forest green color) in mature
hepatocytes presented a challenge given the overlap in wavelengths
with those of GFP. Therefore, Applicants shifted the GFP+ signal to
a pink or rose color using an antibody to GFP and secondarily to an
antibody with a red fluoroprobe. This resulted in the stem cells
being recognized as small cells with pink nuclei (merger of the
nuclear blue DAPI staining with the antibody-tagged-rose colored
GFP+ label). Any donor cells that matured into hepatocytes were
recognized as having a lavender color from the merger of the green
autofluorescence (lipofuscins), the blue (DAPI), and the rose-color
(GFP) (FIG. 4).
[0255] Porcine extrahepatic biliary tree tissue (gall bladder,
common duct, hepatic ducts) were obtained from transgenic pigs.
Tissues were pounded with a sterilized, stainless steel mallet to
eliminate the parenchymal cells, carefully keeping the linkage of
the intra-hepatic and extrahepatic bile ducts. The biliary tree was
then washed with the "cell wash" buffer comprised of a sterile,
serum-free basal medium supplemented with antibiotics, 0.1% serum
albumin, and 1 nM selenium (10.sup.-9 M). It was then mechanically
dissociated with crossed scalpels, and the aggregates enzymatically
dispersed into a cell suspension in RPMI-1640 supplemented with
0.1% bovine serum albumin (BSA), 1 nM selenium, 300 U/ml type IV
collagenase, 0.3 mg/ml deoxyribonuclease (DNAse) and antibiotics.
Digestion was done at 320.degree. C. with frequent agitation for
30-60 minutes. Most tissues required two rounds of digestions
followed by centrifugation at 1100 rpm at 4.degree. C. Cell pellets
were combined and re-suspended in cell wash. The cell suspension
was centrifuged at 30 G for 5 minutes at 4.degree. C. to remove red
blood cells. The cell pellets were again re-suspended in cell wash
and filtered through a 40 .mu.m nylon cell strainer (Becton
Dickenson Falcon #352340) and with fresh cell wash. The cell
numbers were determined and viability was assessed using Trypan
Blue. Cell viability above 90-95% was routinely observed.
[0256] In prior studies, Applicants have defined the antigenic
profile of populations of mesenchymal cells that provide critical
paracrine signals needed for hepatic and biliary tree stem cells
versus others required for mature parenchymal cells. The
mesenchymal cells that partner with BTSCs are subpopulations devoid
of MHC antigens, with low side scatter, and identifiable as
angioblasts (CD117+, CD133+, VEGF-receptor+, and negative for
CD31), precursors to endothelia (CD133+, VEGF-receptor+, and
CD31+), and precursors to stellate cells (CD146+, ICAM1+, VCAM+,
alpha-smooth muscle actin (ASMA)+, and negative for vitamin A).
These 3 subpopulations are referred to collectively as early
lineage stage mesenchymal cells (ELSMCs). By contrast, adult
hepatocytes are associated with mature sinusoidal endothelia
(CD31+++, type IV collagen+, VEGF-receptor+, and negative for
CD117) and those for adult cholangiocytes that are associated with
mature stellate and stromal cells (ICAM-1+, ASMA+, Vitamin A++,
type I collagen+).
[0257] The cell suspensions were added to Multiwell Flat Bottom
Cell Culture Plates (Corning #353043) in serum-free Kubota's Medium
and incubated for .about.an hour at 37.degree. C. to facilitate
attachment of mature mesenchymal cells; Mature mesenchymal cells
attached to the dishes within 10-15 minutes even though the medium
was serum-free. The cells remaining in suspension were transferred
to another dish and again incubated for up to an hour. Repeats of
this resulted in depletion of a significant fraction of the mature
mesenchymal cells. After depletion of mature mesenchymal cells, the
remaining floating cells were seeded at .about.2.times.10.sup.5
cells per wells in serum-free Kubota's Medium in Corning's ultralow
attachment dishes (Corning #3471) and were incubated overnight at
37.degree. C. in a CO2 incubator. Organoids comprised of the
biliary tree stem cells (BTSCs) and of ELMSCs formed overnight
(FIG. 1). These organoid cultures survived for weeks in Kubota's
Medium, especially if the medium was supplemented (0.1%) with
soluble forms of HAs (Sigma); they could also be cryopreserved as
described below. From each gram of neonatal pig biliary tree
tissue, we obtained .about.1.5.times.10.sup.7 cells. We used
.about.3-6.times.10.sup.5 cells per well of a 6-well, ultra-low
attachment plate and incubated in the serum-free Kubota's Medium.
The cells produced, on average, 6000 to 20,000 small organoids
(.about.50-100 cells/organoid/well). For the grafts, we used at
least 100,000 organoids (>10.sup.7 cells). Depending on the size
of the backing, Applicants were able to increase the number of
organoids in the grafts up to 10.sup.8 organoids (i.e.
.about.10.sup.9 cells) or more embedded in .about.1 ml of the soft
hyaluronan hydrogel on a 3 cm.times.4.5 cm backing.
[0258] Isolated stem cell organoids were cryopreserved in CS10, an
isotonic cryopreservation buffer containing antifreeze factors,
dextran and DMSO (Bioliife, Seattle, Wash.;
https://www.stemcell.com/products/cryostor-cs10.html). The
viability of the cells was improved further with supplementation
with 0.1% HAs (Sigma #52747). Cryopreservation was done using
CryoMed.TM. Controlled-Rate Freezers. The viability on thawing was
greater than 90%, and cells after thawing were able to attach, to
expand ex vivo and in vivo and to give rise to the expected mature
cells in vitro and in vivo.
[0259] Isolating the cells and assembling the grafts are
characterized in a schematic in FIG. 1 and with the details
summarized in FIG. 2. The grafts were formed by using a backing
(TABLE 1) onto which were placed the stem cell organoids embedded
in the soft hyaluronan hydrogels. These were readily prepared ahead
of time and maintained in a culture dish in an incubator overnight.
The grafts proved stable at the target site for the duration of the
experiments. Cryopreservation of the organoids was achieved
readily, but that of the organoids when within the soft hydrogel
was not. This meant that embedding the organoids in the soft
hydrogel had to be done just prior to surgery.
[0260] Surgeries
[0261] Anesthesia was induced by administering a combination of
ketamine/xylazine (2-3 mg/kg weight each) injected IV or 20 mg/kg
ketamine plus 2 gm/kg xylazine IM, and was maintained by isoflurane
in oxygen administered via a closed-circuit gas anesthetic
unit.
[0262] The animals were positioned in dorsal recumbency, and the
ventral abdomen was clipped from xyphoid to pubis. The skin was
aseptically prepared with alternating iodinated scrub and alcohol
solutions. After entry into the surgery suite, preparation of the
skin was repeated using sterile technique, and the area was covered
with a topical iodine solution before application of sterile
surgical drapes. The surgeons used appropriate aseptic technique. A
mid-ventral incision was made through the skin, through
subcutaneous tissues and linea alba, starting at the xiphoid
process and extending caudally 8-12 cm. The left hepatic division
was exposed and a 3.times.4.5 cm patch graft was applied to the
ventral surface of the liver and containing 1.times.HA (.about.60
Pa) with embedded organoids placed onto the backing containing
10.times. HA (.about.760 Pa), and the patch was placed in direct
contact onto the surface of the liver capsule. The patch graft was
sutured to the liver using 4-6 simple, interrupted sutures of 4-0
polypropylene. The exposed surface of the graft was then treated
with 2 mls of 2.times.HA hydrogel (.about.106 Pa), a level of
rigidity that was fluid enough to permit it to be painted or coated
onto the serosal side of the graft; it served to further minimize
adhesions from neighboring tissues. Following placement of the
surgical graft, the linea alba was closed with a simple continuous
suture using 0-PDS. The linea was blocked with 2 mg/kg 0.5%
bupivacaine, IM. The subcutaneous tissues and skin were closed with
continuous 2-0 PDS and 3-0 Monocryl sutures, respectively. Tissue
adhesive was placed on the skin surface.
[0263] The graft transplants from the transgenic pigs to the
recipients were allogeneic and so required immunosuppression. The
immune-suppression protocols used were ones established by others.
All pigs received oral dosages of the immunosuppressive drugs
Tacrolimus (0.5 mg/kg) and Mycophenolate (500 mg) twice daily,
beginning 24 hours prior to surgery. The drugs were given
continuously for the entire experimental period. These could be
given to the animals easily if mixed with their favorite foods.
[0264] All animals were humanely euthanized at the designated time
point by sedation with Ketamine/Xylazine, and isofluorane
anesthesia, followed by an intravenous injection of a lethal dose
of sodium pentobarbital. Upon confirmation of death, the carcass
was carefully dissected, and the target organs were removed, and
placed in chilled Kubota's Medium for transportation to the lab. In
addition to the liver, the lungs, heart, kidney, and spleen were
collected and fixed in 10% neutral formalin.
[0265] Characterization of the Grafts
[0266] After 48+ hours of fixation, tissues samples were placed in
labeled cassettes in 70% ethanol and were processed on a long cycle
at 60 degrees in a Leica ASP300S Tissue Processor for approximately
10 hours. After completion of the overnight processing, samples
were embedded using the Leica EG1160 Embedding Station. A mold was
filled with wax and the sample was placed in the correct
orientation so that desired sections could be collected. The
cassette was chilled until the block and tissue sample could be
removed as one unit from the mold. The block was sectioned at 5
microns using a Leica RM2235 Microtome; the sections were floated
in the water bath and placed onto slides. The slides were allowed
to air dry overnight before staining. Sections were stained for
Haematoxylin and Eosin (H&E; Reagents #7211 and #7111) or
Masson's Trichrome (Masson's Trichrome Stain: Blue Collagen Kit
#87019) using Richard Allan Scientific Histology Products and
following the manufacturer's recommended protocol; the protocol is
programed into a Leica Autostainer XL.
[0267] Tissue was embedded and frozen in OCT and flash frozen at
-20.degree. C. for frozen sectioning. Frozen sections were stained
for IHC followed the protocol described above. For
immunofluorescence, frozen sections were thawed for 1 hour at room
temperature and then fixed in 10% buffered formaldehyde, acetone or
methanol according to the antibody specifications. After fixation,
sections were washed 3 times in 1% phosphate buffered saline (PBS),
followed by blocking with 2.5% horse serum in PBS for 1 hour at
room temperature. Primary antibodies diluted in 10% goat serum in
PBS were added and incubated overnight at 4.degree. C. The next
morning, sections were rinsed 3 times with PBS and incubated with
secondary antibodies diluted in 2.5% horse serum in PBS for 2 hours
at room temperature. Images were taken using a Zeiss CLSM 710
Spectral Confocal Laser Scanning microscope (Carl Zeiss
Microscopy). Antibodies are listed in TABLE 3.
[0268] For the images in FIG. 5, sections (3 .mu.m) were stained
with hematoxylin-eosin and Sirius red, according to standard
protocols. For immunohistochemistry, endogenous peroxidase activity
was blocked by a 30 min incubation in methanolic hydrogen peroxide
(2.5%). Antigens were retrieved, as indicated by the vendor, by
applying Proteinase K (code S3020, Dako, Glostrup, Denmark) for 10
min at room temperature. Sections were then incubated overnight at
4.degree. C. with primary antibodies (pan-Cytokeratin, Dako, code:
Z0622, dilution: 1:100; Sox9, Millipore, code: AB5535, dilution:
1:200). Samples were rinsed twice with PBS for 5 min, incubated for
20 min at room temperature with secondary biotinylated antibody
(LSAB+ System-HRP, code K0690; Dako, Glostrup, Denmark) and then
with Streptavidin-HRP (LSAB+ System-HRP, code K0690, Dako,
Glostrup, Denmark). Diaminobenzidine (Dako, Glostrup, Denmark) was
used as substrate, and sections were counterstained with
hematoxylin (PMID: 29248458). For immunofluorescence, non-specific
protein binding was blocked by 5% normal goat serum. Specimens were
incubated overnight at 4.degree. C. with primary antibodies
(chicken anti-GFP, Abcam, code: ab13970, dilution=1:200; rabbit
anti-HNF4a, Abcam, code: 92378, dilution: 1:50, rabbit
anti-albumin, ab2406, dilution=1:500). Specimens were washed and
incubated for 1 h with labeled isotype-specific secondary
antibodies (anti-chicken AlexaFluor-546, anti-mouse Alexafluor-488,
anti-rabbit Alexafluor-488, Invitrogen, Life Technologies Ltd,
Paisley, UK) and counterstained with 4,6-diamidino-2-phenylindole
(DAPI) for visualization of cell nuclei (PMID: 26610370). For all
immunoreactions, negative controls (the primary antibody was
replaced with pre-immune serum) were also included. Sections were
examined in a coded fashion by Leica Microsystems DM 4500 B Light
and Fluorescence Microscopy (Leica Microsystems, Weltzlar,
Germany), equipped with a Jenoptik Prog Res C10 Plus Videocam
(Jena, Germany). Immunofluorescence stains were also analyzed by
Confocal Microscopy (Leica TCS-SP2). Slides were further processed
with an Image Analysis System (IAS--Delta Sistemi, Roma-Italy) and
were independently evaluated by two researchers in a blind fashion.
Immunofluorescence stains were scanned by a digital scanner (Aperio
Scanscope FL System, Aperio Technologies, Inc, Oxford, UK) and
processed by ImageScope.
[0269] Frozen sections were problematic given the high
autofluorescence in hepatocytes (lipofuscin) and the fluorescence
of the Seri-Silk backing. Applicants had greater success by
preparing paraffin sections and staining for the GFP using a rabbit
polyclonal antibody to GFP (Novus Biologicals, NE600-308); the
rabbit anti-GFP antibody was used in combination with a secondary
antibody of donkey anti-rabbit IgG H&L (Alexa Fluor 568;
ab175470, Invitrogen), while Donkey anti-Goat IgG Alexa Fluor 488
antibody was used to exclude non-specific staining of hepatic
autofluorescence. Autofluorescence was reduced by quenching with
the use of dyes and that included Trypan Blue. The Trypan Blue was
used on tissues/cells at 0.4% in PBS. This reduces the background
significantly.
[0270] Total RNA was extracted from the organoids or grafts using
Trizol (Invitrogen). First-strand cDNA synthesized using the
Primescript 1st strand cDNA synthesis kit (Takara) was used as a
template for PCR amplification. Quantitative analyses of mRNA
levels were performed using Faststart Universal Probe Master (Roche
Diagnostics) with ABI PRISM 7900HT Sequence Detection System
(Applied Biosystems). Primers were designed with the Universal
Probe Library Assay Design Center (Roche Applied Science). Primer
sequences are listed in TABLE 4. The primers were annealed at
50.degree. C. for 2 min and 95.degree. C. for 10 min, followed by
40 cycles of 95.degree. C. (15 s) and 60.degree. C. (1 min).
Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
used generally as a control and a standard.
[0271] RNA was purified from cells using the Qiagen RNeasy Kit RNA
integrity (RIN) analysis was performed using an Agilent 2000
Bioanalyzer. The cDNA libraries were generated using the Illumina
TruSeq Stranded mRNA preparation kit and sequenced on the Illumina
HiSeq 2500 platform. Two samples were sequenced per lane, occupying
a total of 8 lanes for all of the samples (one flow cell). Quality
control analysis was completed using FastQ. Mapping of sequence
reads to the human genome (hg19) was performed with MapSplice2
using default parameters. Transcript quantification was carried out
by RSEM analysis, and DESeq was used to normalize gene expression
and identify differentially expressed genes. MapSplice2 was also
used to detect candidate fusion transcripts. Fusion calls were
based on the depth and complexity of reads spanning candidate
fusion junctions. Gene expression profiles were compared using
Pearson's correlation analysis and hierarchical clustering was
performed in R. Hierarchical clustering was performed following
Variance Stabilizing Transformation provided in the DESeq package.
Pathway enrichment analysis was performed with the Ingenuity
Pathway Analysis (IPA) software. Differential gene expression
analysis was conducted only on genes with a minimum average
normalized count >50 in at least one category.
[0272] Statistically significant differences between samples were
calculated by using Student's 2-tailed t test and results are
presented as the mean standard deviation (SD). P values of less
than 0.05 were considered statistically significant.
[0273] Results
[0274] In prior studies on injection grafting, it was found that
engraftment required co-transplantation of epithelial cells with
their lineage-stage-appropriate mesenchymal cell partners. For
hepatic and biliary tree stem cells, these mesenchymal cells are
comprised of angioblasts (CD117+, CD133+, VEGFr+, CD31-negative)
and their immediate descendants, precursors to endothelia (CD133+,
VEGFr+, CD31+, Van Willebrand Factor+) and precursors to stellate
cells (CD146+, ICAM-1+, alpha-smooth muscle actin+(ASMA), vitamin
A-negative). Applicants refer to these collectively as early
lineage stage mesenchymal cells (ELSMCs. Applicants also had
partial success also with isolated porcine mesenchymal stem cells
(MSCs) prepared by the methods of others and isolating cells from
neonatal pig livers.
[0275] In prior studies, Applicants achieved isolating matching
epithelial and mesenchymal cell stages by using multiparametric
flow cytometry to determine the ratios of the lineage stage
partners of epithelial and mesenchymal cells in cell suspensions
and then used those ratios within grafts using immuno-selected
cells. In these studies, Applicants found it more efficient to
deplete cell suspensions of mature mesenchymal cells by repeated
panning procedures followed by culturing remaining cell suspensions
on low attachment dishes and in serum-free Kubota's Medium for 6-8
hours. Organoids self-assembled with each aggregate containing
approximately 50-100 cells. Marker analyses indicated partnering of
BTSCs with ELSMCs (FIG. 1). As summarized in the schematic in FIG.
1A, they were used immediately or were cryopreserved under defined
conditions determined previously and thawed as needed for grafts.
Organoids of BTSCs/ELSMCs were characterized using
immunofluorescence (IF), qRT-PCR and RNA-seq and shown to express
classic traits of BTSCs (FIG. 1) and of ELSMCs (data not shown).
BTSCs in the organoids expressed no mature hepatic or pancreatic
genes but low levels of pluripotency genes (e.g. OCT4, SOX2) and
endodermal stem cell genes (e.g. EpCAM, SOX 9, SOX17, PDX1, LGR5,
CXCR4, MAFA, NGN3 and NIS). Representative qRT-PCR assays confirmed
the findings from IF and from IHC on cells prior to transplantation
(FIG. 1D). IHC assays indicated that more primitive cells (e.g.
ones expressing pluripotency genes) were distributed to the
interiors of the organoids and later maturational lineage stages at
the perimeters (e.g. cells expressing EpCAM or albumin) (FIG.
1C).
[0276] Results from patch grafts were compared with those from
injection grafts with methods established previously and comprised
of injection of cells and with localization to the site by
triggering hyaluronans with polyethylene glycol diacrylate (PEGDA)
to gel within minutes. Injection grafts into the porcine liver
parenchyma resulted in essentially 100% engraftment but with
minimal (if any) migration and with integration into the host
tissue occurring slowly over weeks (data not shown). The findings
were similar to those observed previously with injection grafts of
hepatic stem cells.sup.17. Injection grafts into the mesentery
adjacent to hepatic ducts/portal vein branches immediately caudal
to the liver lobes were feasible with large ducts but caused
smaller ones to occlude from the swelling effects of HA hydrogels
and resulting in cholestasis (FIG. 13). Success with patch grafting
led us to abandon further efforts with injection grafting
strategies.
[0277] The composition of the grafts for stem cells involved use of
conditions with 3 distinct layers of hyaluronans (HA) hydrogels
with precise concentrations of HA to PEGDA to achieve a level of
rigidity assessed by rheological assays (FIG. 2C). Donor cells were
embedded into a soft HA layer (.about.100 Pa) and placed against
the liver/pancreas surface; the soft hydrogels maintained stemness
traits.sup.23 that in these studies proved essential for
engraftment. This layer was placed on top of a rigid (10.times.;
.about.700 Pa) HA layer prepared ahead of time on the backing and
serving as a barrier to migration. The patch was attached to the
target site with sutures or surgical glue. A 2.times. HA hydrogel,
soft enough (rigidity=.about.200 Pa), to permit painting or coating
the serosal surface of the graft at the time of the surgery and
serving to further minimize adhesions from nearby tissues.
[0278] Patch grafts were placed onto the liver surface, i.e.
superficial to the Glisson capsule or pancreatic capsule, and
attached by sutures or by surgical glue at the corners (FIG. 2F).
The stiffness of Seri-Silk resulted in grafts being placed at sites
with minimal curvature and away from sites with significant
mechanical forces (e.g. near the diaphragm). In the grafts onto
pancreas, the graft was wedged between the duodenum and the
pancreas.
[0279] The only variant of patch grafting attempted and then
abandoned was after sharp surgical removal of the capsule.
Hemorrhaging was excessive obviating future use in hosts with
altered hemostasis associated with hepatic failure or even in
normal hosts given the adverse influences of serum on donor cells.
Without such efforts to alter the organ capsules, patch grafts
proved facile for surgical procedures.
[0280] A number of backings were tried with a focus on ones used
clinically in abdominal surgeries (TABLE 1 and TABLE 2). All but
Seri-Silk caused problems that resulted in their elimination for
further consideration. The problems included fragility (e.g.
Seprafilm, Retroglyde); induction of necrosis or fibrosis and
significant levels of adhesions (e.g. Surgisis, Vetrix); and severe
adhesion formations with a filamentous sponge version of Seri-Silk
or any of the backings supplemented with carboxymethylcellulose
("belly jelly") to the abdomen. Of the ones tested, SERI Surgical
Silk.sup.24-26 (Allergan, Inc. Irvine, Calif.) provided the best
combination of mechanical support and minimal adhesions, an effect
further enhanced by application of 2.times.HA to the serosal
surface of SeriSilk after attachment to the target site. The
product is a purified fibroin of Bombyx moth silk and was developed
by David Kaplan (Tuft's University, Boston, Mass.). Applicants
found it to be stiff, a property found useful for surgical
manipulations and placement on flat/rigid organs like the liver.
The stiffness made it difficult to apply to sites with significant
curvature or need for flexibility. Still, its stiffness proved
neutral with respect to maturational effects on the donor cells, a
finding that made this backing acceptable for patch grafting. In
grafts at 3 weeks, Seri-Silk was enveloped by bands of collagen,
suggesting a mild foreign body reaction. Assessment of other
candidate backings, such as synthetic textiles, is ongoing.
[0281] Evidence for remodeling at week one after surgery was
validated with Trichrome staining (FIG. 3, 7) or Safranin O, having
dyes that stain collagens and other extracellular matrix
components. The images of the graft (FIG. 3A-B) that are stained
with Trichrome are compared with ones of the same site and stained
with hematoxylin/eosin (FIG. 3C-D). Reconstitution of the Glisson
capsule and of the lobules occurred by 3 weeks in parallel with HAs
being resorbed. The bands comprising the area of remodeling were
surprisingly large (FIGS. 3-5, 7).
[0282] Donor cells deriving from transgenic GFP+ pigs were
identified readily by GFP expression through IHC assays. In
pancreas, the donor cells were identified by the green
fluorescence. However, in liver, the autofluorescence of the
lipofuscins in hepatocytes peaks at a wavelength overlapping with
that for GFP. Therefore, we identified donor cells in livers with
an antibody to GFP (Rabbit anti-GFP antibody; Novus, NB600-308) and
coupled to a secondary antibody with a red fluoroprobe (Donkey
anti-rabbit 555, Invitrogen) causing donor cells to have pink
nuclei (the red fluoroprobe plus the blue DAPI). Host cells were
recognized given their blue nuclei (DAPI stain) but without GFP
expression (FIG. 4).
[0283] The liver lobules of mature hepatocytes were forest-green
from the autofluorescence (lipofuscins) (FIG. 4B). Donor GFP+ cells
that had matured to aggregates of hepatocytes were a lavender color
and with pink nuclei (FIG. 4C) due to the merger of the red
fluoroprobe from GFP, the blue from DAPI, and the autofluorescent
dark green from lipofuscins. Hepatocytes, whether host or donor
derived, were clustered around by host mesenchymal cells
(endothelia, stellate cells) with bright yellow/green
autofluorescence due, we assume, to vitamin A in the mature
stellate cells (FIG. 4C); the IHC data for the endothelia and
stellate cells are not shown.
[0284] Within a week, patch grafts of BTSCs/ELSMCs organoids
resulted in remodeling of the organ capsule and adjacent lobules
followed by a merger of host and donor cells (FIGS. 3-5, 7).
Finger-like extensions of donor cells extended into the hepatic
lobules of the host tissue; in parallel, host cells extended into
HAs of the grafts (FIG. 4). In the case of the pancreas, the graft
was wedged between the pancreas and the duodenum, and by one week
post surgery, engraftment of donor cells occurred both into the
pancreas and into the Brunner's glands of the submucosa of the
duodenum (FIG. 6). Integration of the cells within large regions of
the liver (or the pancreas) was completed by 2 weeks by which time
the layers of HAs had been mostly resorbed; donor cells had lineage
restricted into adult hepatic parenchymal fates, both
cholangiocytic and hepatocytic (FIG. 5) or into pancreatic fates
(FIG. 6).
[0285] By 3 weeks, the HA layers were resorbed entirely, leaving
only the backing. This correlated with reappearance of the organ
capsule and of the histological structure of the tissue near to the
capsules (FIG. 3, 5, 6) or of the pancreatic capsule and of the
pancreatic histological structures (FIG. 6). In pancreas, mature
cells were identified by functional markers that included insulin
for islet cells (beta cells) and amylase for acinar cells.
[0286] Engraftment efficiency for both the liver and for the
pancreas was close to 100% by a week, since all donor cells
identified were found to be viable and within the liver or
pancreas; not in the remnants of the grafts above the organ
capsules; and with negligible or no evidence of ectopic cell
distribution in other organs (e.g. lung).
[0287] The speed of migration of donor cells in the BTSC/ELSMC
grafts through the liver and through the pancreas proved remarkable
resulting in donor cells in most regions of the organ (liver or
pancreas) by the end of a week and with uniformly dispersed cells
throughout the tissue (liver/pancreas) by 2-3 weeks (FIGS.
3-6).
[0288] Correlated with the dissolution and remodeling of the
Glisson capsule (or pancreatic capsule) and neighboring liver
lobules (or pancreatic tissue) and correlating with significant
engraftment was elevated expression of multiple MMPs, enzymes known
to dissolve extracellular matrix components and to be associated
with cell migration. In FIG. 7 are summarized data from RNA-seq
studies and IHC assays on MMPs expressed by stem/progenitors versus
adult cells. BTSCs expressed high levels of multiple MMPs,
comprised of both secreted forms (e.g. MMP2, MMP7) as well as
membrane-associated forms (e.g. MMP14 and MMP15). The ELSMCs,
precursors of endothelia and of stellate cells, also contributed to
multiple MMPs.
[0289] The findings from RNA-seq data were confirmed by IHC assays
for the proteins encoded by MMP genes (FIG. 7). IHC assays
confirmed the presence of the secreted forms of MMPs (e.g. MMP1,
MMP2, MMP7, MMP9) especially in the regions of remodeling. Protein
expression of MMP1 was found in BTSCs/ELSMCs organoids and also in
remodeling regions of grafts; however, existing data banks of
RNA-seq findings do not include MMP1 because of a lack of an
annotated species of porcine MMP1 to be used for the analyses.
Therefore, recognition of its expression is based on IHC
assays.
[0290] Variables causing differentiation of donor cells resulted in
a muting of expression of MMPs, especially the secreted forms, and,
in parallel, a loss in potential for engraftment and migration
(data not shown). These factors included serum, various soluble
regulatory signals (growth factors, cytokines, hormones) known to
influence differentiation of the donor cells, extracellular matrix
components whether in the hydrogels or in the backings (especially
type I collagen-containing backings), and the stiffness of the HA
hydrogels (i.e. the Pa levels). If differentiation of the ELSMCs
progressed preferentially to stroma, the grafts became fibrotic; if
to endothelia, the grafts retained viable cells and tissue but
remained superficial to the organ capsule (data not shown).
[0291] Organoids of BTSCs/ELSMCs proved the most successful
arrangement for the cells for grafting. In the past, we had
co-transplanted epithelial-mesenchymal partners by immuno-selecting
them from cell suspensions by flow cytometry using their
distinctive surface antigens, and then mixing them according to the
ratios found in cells suspensions from freshly isolated
tissues.sup.17. Here we found that letting them self-select into
organoids, after removal by panning of mature mesenchymal cells,
proved more efficient and effective in establishing lineage-stage
appropriate epithelial-mesenchymal partners with relevant paracrine
signaling for the grafts and yielding organoids under defined
(serum-free) conditions, that made them easily and safely
cryopreserved.
[0292] The primary design of the grafts consisted of mixing of
cells with appropriate biomaterials that can become insoluble and
keep cells localized to the target site. For grafts, the ideal
biomaterials proved to be non-sulfated or minimally sulfated
glycosaminoglycans (GAGs), such as hyaluronans (HAs), found in all
stem cell niches, with receptors to HAs being classic stem cell
traits. Maintenance of cells as stem/progenitors optimized
expression of secreted and membrane-associated MMPs effective for
engraftment.
[0293] Evidence of engraftment processes was particularly dramatic
within regions of remodeling that occurred at the interface of the
graft and the host tissue. To validate the findings of remodeling,
Trichrome staining and Safranin O were used having dyes that stain
extracellular matrix components and analyzed in parallel with
adjacent sections stained with hematoxylin/eosin (FIG. 3, 7). It
confirmed remodeling of the organ capsule and of adjacent tissue
within a week after surgery. By 3 weeks post-surgery, these assays
demonstrated reconstitution of the organ capsules and of the normal
tissue histology following clearance of HAs. The remodeling zone
was surprisingly large (FIG. 3, 7), especially at one week after
surgery and was shown to involve multiple forms of MMPs (FIG.
7)
[0294] Although there are many sources and types of HAs, among the
most useful are thiol-modified ones established by Glenn Prestwich
(University of Utah, Salt Lake City, Utah) and that can be
triggered with PEGDA to form a hydrogel with precise biochemical
and mechanical properties. These properties of HAs confer perfect
elasticity, allow access into the graft of all soluble signals in
blood, lymph or interstitial fluid, and minimize the maturation of
donor cells until engraftment and migration have occurred. The
ability to vary the rheological factors with simple changes in HA
and PEGDA concentrations provided additional advantages in guiding
the direction of migration of the cells and in minimizing
adhesions. Soft HA hydrogels, ones mimicking properties in stem
cell niches, were permissive for expression of the stem/progenitor
cell-associated repertoire of MMPs. Thus, the mechanical properties
of HAs, studied for years in the functions of skeletal tissues, are
important also in managing grafting strategies.sup.23.
[0295] Patch grafts containing stem/progenitors resulted in a
striking phenomena of grafts "melting" into tissues within a few
days, followed by a merger of donor and host cells, and a
distribution of cells throughout most regions of the organ by one
to two weeks. Thereafter, maturation of donor cells and restoration
of the organ capsules occurred in parallel with the tissue
clearance of HAs.
[0296] The engraftment and integration process correlated with
expression of multiple MMPs, a family of calcium-dependent,
zinc-containing endopeptidases that degrade extracellular matrix
components. Using RNA-seq studies, we found a pattern of
stem/progenitor-associated MMPs, comprised of high levels of
secreted forms (e.g. MMP2, MMP7) as well as membrane-associated
forms (e.g. MMP14, MMP15). IHC assays indicated that protein levels
of secreted MMPs (e.g. MMP1, MMP2, MMP7) were found richly
expressed in areas of remodeling (FIG. 7). Conditions (soluble
growth factors, cytokines, serum, matrix components, mechanical
forces) that caused donor cells to differentiate resulted in
reduction in MMPs, especially the secreted forms, and, in parallel,
abrogation of the engraftment process.
[0297] The biomaterials of the grafts, especially the HAs, have
been shown ex vivo and in vivo to maintain stemness traits in
cells. Since the grafts are devoid of known signals that can
trigger fate determination, the findings of donor cells that had
matured into distinct adult fates, depending whether the graft was
placed onto the liver or the pancreas, implicate the local
microenvironment of the host tissue as the logical source of
relevant factors for the maturational processes.
[0298] The numbers of cells that can be engrafted are considerable
(>10.sup.8) and dictated by the dimensions of the graft, the
numbers of cells, and the repertoire of secreted and
plasma-membrane-associated MMPs. These findings are in contrast to
the limited numbers of cells (e.g. 10.sup.5-10.sup.6) feasible with
vascular delivery or by injection grafting.
[0299] Patch grafting is a safe strategy by which to transplant
large numbers of cells into a solid organ, including internal
organs, and may prove useful for treatment of patients especially
if engraftment can occur sufficiently under disease conditions.
Although, there is concern that aberrant engraftment may occur
where tissue is fibrotic or affected by cirrhosis. Accordingly,
examples are provided herein to determine efficacy of patch grafts
for the method aspects.
Example 2: Treatment of Liver Disease
[0300] This example describes an exemplary method of treating a
subject having a liver disease or disorder using a patch graft.
Donor cells are prepared as organoids of biliary tree stem cells
(BTSCs), precursors to liver and to pancreas, aggregated with early
lineage stage mesenchymal cells (ELSMCs) consisting of angioblasts
and their early lineage stage descendants, precursors to endothelia
and precursors to stellate cells as described herein. The
BTSC/ELSMCs organoids are embedded into soft hyaluronan hydrogels
(<200 Pa) placed onto a backing that is tethered to a target
site of the subject's liver.
[0301] Following administration of the patch graft, the subject is
monitored for improvement in liver function. Commonly used tests to
check liver function include but are not limited to the alanine
transaminase (ALT), aspartate aminotransferase (AST), alkaline
phosphatase (ALP), albumin, and bilirubin tests. The ALT and AST
tests measure enzymes that are released by the liver in response to
damage or disease. The albumin and bilirubin tests measure how well
the liver creates albumin, a protein, and how well it disposes of
bilirubin, a waste product of the blood. It is expected that after
about 2 weeks to about 36 weeks, an improvement in liver function
will be detected. Improvement is determined by detecting an
improved value of one or more of the liver function tests relative
to the value prior to administration of the graft and/or an
improvement or amelioration of one or more symptoms of the liver
disease or disorder.
Example 3: Treatment of Pancreatic Disease
[0302] This example describes an exemplary method of treating a
subject having a disease or disorder of the pancreas using a patch
graft. Donor cells are prepared as organoids of biliary tree stem
cells (BTSCs), aggregated with early lineage stage mesenchymal
cells (ELSMCs) consisting of angioblasts and their early lineage
stage descendants, precursors to endothelia and precursors to
stellate cells as described herein. The BTSC/ELSMCs organoids are
embedded into soft hyaluronan hydrogels (<200 Pa) placed onto a
backing that is tethered to a target site of the subject's
pancreas.
[0303] Following administration of the patch graft, the subject is
monitored for improvement in pancreatic function. Commonly used
tests to check pancreatic function include but are not limited to
blood tests for levels of the pancreatic enzymes amylase and
lipase, the direct pancreatic function test following
administration of secretin or cholecystokinin, fecal elastase test,
CT scan with contrast dye, abdominal ultrasound, endoscopic
retrograde cholangiopancreatography (ERCP), endoscopic ultrasound,
and magnetic resonance cholangiopancreatography. It is expected
that after about 2 weeks to about 36 weeks, an improvement in
pancreatic function will be detected. Improvement is determined by
detecting an improved value of one or more of the pancreatic
function tests relative to the value prior to administration of the
graft and/or an improvement or amelioration of one or more symptoms
of the disease or disorder of the pancreas.
Example 4: Treatment of Kidney Disease
[0304] This example describes an exemplary method of treating a
subject having a disease or disorder of the kidney using a patch
graft. Donor cells are prepared as organoids of biliary tree stem
cells (BTSCs), aggregated with early lineage stage mesenchymal
cells (ELSMCs) consisting of angioblasts and their early lineage
stage descendants, precursors to endothelia and precursors to
stellate cells as described herein. The BTSC/ELSMCs organoids are
embedded into soft hyaluronan hydrogels (<200 Pa) placed onto a
backing that is tethered to a target site of the subject's
kidney.
[0305] Following administration of the patch graft, the subject is
monitored for improvement in kidney function. Commonly used tests
to check pancreatic function include but are not limited to
clinically relevant endpoints of kidney function known in the art.
It is expected that after about 2 weeks to about 36 weeks, an
improvement in kidney function will be detected. Improvement is
determined by detecting an improved value of one or more of the
kidney function tests relative to the value prior to administration
of the graft and/or an improvement or amelioration of one or more
symptoms of the disease or disorder of the kidney.
Example 5: Treatment of GI Disease
[0306] This example describes an exemplary method of treating a
subject having a gastrointestinal disease or disorder using a patch
graft. Donor cells are prepared as organoids of biliary tree stem
cells (BTSCs), aggregated with early lineage stage mesenchymal
cells (ELSMCs) consisting of angioblasts and their early lineage
stage descendants, precursors to endothelia and precursors to
stellate cells as described herein. The BTSC/ELSMCs organoids are
embedded into soft hyaluronan hydrogels (<200 Pa) placed onto a
backing that is tethered to a target site of the subject's
intestines.
[0307] Following administration of the patch graft, the subject is
monitored for improvement in intestinal function. Commonly used
tests to check intestinal function include but are not limited
clinically relevant endpoints of intestinal function known in the
art. It is expected that after about 2 weeks to about 36 weeks, an
improvement in intestinal function will be detected. Improvement is
determined by detecting an improved value of one or more of the
intestinal function tests relative to the value prior to
administration of the graft and/or an improvement or amelioration
of one or more symptoms of the gastrointestinal disease or
disorder.
Sequence CWU 1
1
28121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1atcctgggct acactgagga c 21221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2aagtggtcgt tgagggcaat g 21320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3ttccttcctc catggatctg
20420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4atctgctgga ggctgaggta 20520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5gccctgcagt acaactccat 20620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6gctgatcatg tcccgtaggt
20720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7cgaagctgga caaggagaag 20820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8gctgaacacc ttcccaaaga 20922DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 9accagagaat gctatccaga ac
221019DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10ctcactcgct ccaaacagg 191120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11ccttggccct gaacaaaata 201220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 12atttctttcc cagggagtgg
201320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13cggttcgagc aagaataagc 201420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14gtaatccggg tggtccttct 201520DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 15tcattgatgc cacaaccatt
201620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16tgaaaagccc cggaactaat 201720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17ggctgctcat tgagaggagt 201819DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 18atgttcccga actccaagg
191922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19agaaccccca ggtctctgtc tt 222022DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20cagtccgaaa cactccctca ca 222121DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 21cgcgtttctg gttgcttaca c
212221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22acttcttgct cttggccttg g 212320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23agtctgccaa gctgctgata 202420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 24agccttggga aatctctggc
202521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25agtgatactg gattggcgtt g 212620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26tagggagcct tccaatgtgt 202720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 27gcttcagcaa ggaggaggtc
202820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28tctcgctctc cagaatgtgc 20
* * * * *
References