U.S. patent application number 14/774045 was filed with the patent office on 2016-01-28 for tissue engineered intestine.
The applicant listed for this patent is NANOFIBER SOLUTIONS LLC, RESEARCH INSTITUTE AT NATIONWIDE CHILDREN'S HOSPITAL, INC.. Invention is credited to Gail E. BESNER, Jed JOHNSON, Yanchun LIU.
Application Number | 20160022873 14/774045 |
Document ID | / |
Family ID | 50680159 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160022873 |
Kind Code |
A1 |
BESNER; Gail E. ; et
al. |
January 28, 2016 |
TISSUE ENGINEERED INTESTINE
Abstract
The invention provides for engineered intestinal construct and
methods of making these constructs. The invention also provides for
methods of treating short bowel syndrome or methods of repairing an
intestine after resection comprising inserting an engineered
intestinal construct into the intestine of a subject in need.
Inventors: |
BESNER; Gail E.; (Dublin,
OH) ; LIU; Yanchun; (Columbus, OH) ; JOHNSON;
Jed; (Columbus, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RESEARCH INSTITUTE AT NATIONWIDE CHILDREN'S HOSPITAL, INC.
NANOFIBER SOLUTIONS LLC |
Columbus
Columbus |
OH
OH |
US
US |
|
|
Family ID: |
50680159 |
Appl. No.: |
14/774045 |
Filed: |
March 14, 2014 |
PCT Filed: |
March 14, 2014 |
PCT NO: |
PCT/US2014/028186 |
371 Date: |
September 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61783655 |
Mar 14, 2013 |
|
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Current U.S.
Class: |
424/423 ;
435/180; 435/325 |
Current CPC
Class: |
A61L 27/38 20130101;
A61L 27/3826 20130101; A61L 27/3891 20130101; A61L 2430/22
20130101; A61L 27/56 20130101; A61L 27/3882 20130101; A61L 2400/12
20130101; A61L 27/3886 20130101; A61L 27/227 20130101; A61L 27/3895
20130101; A61L 27/18 20130101; A61L 27/3873 20130101; A61L 27/383
20130101 |
International
Class: |
A61L 27/38 20060101
A61L027/38; A61L 27/18 20060101 A61L027/18; A61L 27/22 20060101
A61L027/22 |
Claims
1. An engineered intestine construct comprising a nanofiber
scaffold seeded with neural stem cells, smooth muscle cells and
intestinal stem cells, and wherein the nanofiber scaffold comprises
HB-EGF polypeptide or a fragment thereof.
2. An engineered intestine construct comprising a nanofiber
scaffold seeded with neural stem cells, smooth muscle cells and
intestinal stem cells, wherein at least one of the neural stem
cells, smooth muscle cells or intestinal stem cells overexpress
HB-EGF polypeptide or a fragment thereof.
3. An engineered intestine construct comprising a multilayer
nanofiber scaffold, wherein the multilayer nanofiber scaffold
comprises at least an inner layer and an outer layer, wherein the
outer layer comprises neural stem cells and smooth muscle cells,
and wherein the inner layer comprises intestinal stem cells.
4. The engineered intestine construct of claim 3 wherein at least
of the layers comprises HB-EGF polypeptide or a fragment.
5. The engineered intestine construct of claim 3 or 4 wherein the
construct further comprises at least one middle layer.
6. The engineered intestine construct of any one of claims 1-5
wherein the nanofiber scaffold comprises Poly(glycolic acid)(PGA)
nanofibers, Poly(.epsilon.-caprolactone) (PCL) nanofibers,
Poly(-caprolactone-co-lactic acid) (PLC) nanofibers, Poly(L-lactic
acid) (PLLA) nanofibers, Poly(D-lactic acid-co-glycolic acid)
(PDLGA) nanofibers, Poly(D-lactic acid-co-glycolic acid) (PLGA)
nanofibers, Polyurethane (PU) nanofibers, Polydioxanone (PDO)
nanofibers or a combination thereof.
7. The engineered intestine construct of any one of claims 3-6
wherein the construct comprises a layer of macrofibers between two
layers.
8. The engineered intestine construct of claim 7 where the layer of
macrofibers comprises PGA.
9. A method of generating an engineered intestine construct
comprising a) preparing a nanofiber scaffold by electrospinning a
polymer to a target fiber diameter and porosity, b) embedding an
HB-EGF polypeptide or fragment thereof on the scaffold, c) seeding
the scaffold with intestinal stem cells, neural stem cells and
smooth muscles cells, and d) culturing the cells in the scaffold to
form a construct that will form a mature intestine upon insertion
into a subject.
10. The method of claim 10 wherein the culturing step is carried
out in a bioreactor.
11. The method of claim 10 or 11 wherein the nanofiber scaffold
comprises at least an outer and an inner layer.
12. The method of claim 11 wherein the nanofiber scaffold further
comprises a middle layer.
13. The method of claims 9-12 wherein the intestinal stem cells are
seeded on the inner layer.
14. The method of any one of claims 9-13 wherein the neural stem
cells and smooth muscle cells are seeded on the outer layer.
15. The method of any one of claims 9 to 14 wherein the polymer is
Poly(glycolic acid)(PGA) nanofibers, Poly(.epsilon.-caprolactone)
(PCL) nanofibers, Poly(-caprolactone-co-lactic acid) (PLC)
nanofibers, Poly(L-lactic acid) (PLLA) nanofibers, Poly(D-lactic
acid-co-glycolic acid) (PDLGA) nanofibers, Poly(D-lactic
acid-co-glycolic acid) (PLGA) nanofibers or Polyurethane (PU)
nanofibers, Polydioxanone (PDO) nanofibers or a combination
thereof.
16. The method of any one of claims 9-15 wherein the polymer is
PDLGA or PGA.
17. A method of treating short bowel syndrome in a subject
comprising attaching the engineered intestine construct of any one
of claims 1-9 under conditions wherein the construct will implant
within the intestine of the subject.
18. A method of repairing the intestine of a subject undergoing
intestinal resection comprising attaching the engineered intestine
construct of any one of claims 1-8 under conditions wherein the
construct will implant within the intestine of the subject.
19. The method of claim 17 or 18, wherein the subject is suffering
from inflammatory bowel disease, trauma, mesenteric vascular
disease, vovlulus, congenital atresias, neonatal necrotizing
enterocolitis, Crohn's disease, ischemia, intestinal blockage,
bowel obstruction, regional ileitis, regional enteritis, colorectal
cancer, carcinoid tumor, Merkel's diverticulum, precancerous
polyps, diverticulitis, intestinal bleeding, intussusceptions, or
ulcerative colitis.
20. A method of enriching a cell sample for a particular cell type
comprising contacting a cell sample with multiple sieve membranes
wherein the membranes are aligned in descending order according to
pore size, wherein the cell sample contacts the membrane with the
largest pore size first and wherein the cell sample comprises
multiple cell types, filtering the cell sample through the
membranes and recovering the enriched cell sample.
21. The method of claim 20 wherein the enriched cell sample is
enriched for intestinal stem cells in crypts.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/783,655 filed Mar. 14, 2013, which is
incorporated by reference herein in its entirety.
FIELD OF INVENTION
[0002] The invention provides for engineered intestinal construct
and methods of making these constructs. The invention also provides
for methods of treating short bowel syndrome or methods of
repairing an intestine after resection comprising inserting an
engineered intestinal construct into the intestine of a subject in
need.
BACKGROUND
[0003] Heparin-binding epidermal growth factor (HB-EGF) was first
identified in the conditioned medium of cultured human macrophages
(Besner et al., Growth Factors, 7: 289-296 (1992), and later found
to be a member of the epidermal growth factor (EGF) family of
growth factors (Higashiyama et al., Science. 251:936-9, 1991). It
is synthesized as a transmembrane, biologically active precursor
protein (proHB-EGF) composed of 208 amino acids, which is
enzymatically cleaved by matrix metalloproteinases (MMPs) to yield
a 14-20 kDa soluble growth factor (sHB-EGF). Pro-HB-EGF can form
complexes with other membrane proteins including CD9 and integrin
.alpha.3.beta.1; these binding interactions function to enhance the
biological activity of pro-HB-EGF. ProHB-EGF is a juxtacrine factor
that can regulate the function of adjacent cells through its
engagement of cell surface receptor molecules.
[0004] sHB-EGF is a potent mitogenic and chemoattractant protein
for many types of cells. Similar to all members of the EGF family,
HB-EGF binds to the "classic" or prototypic epidermal growth factor
receptor (EGFR; ErbB-1). However, while the mitogenic function of
sHB-EGF is mediated through activation of ErbB-1, its
migration-inducing function involves the activation of ErbB-4 and
the more recently described N-arginine dibasic convertase (NRDc,
Nardilysin). This is in distinction to other EGF family members
such as EGF itself, transforming growth factor (TGF)-.alpha. and
amphiregulin (AR), which exert their signal-transducing effects via
interaction with ErbB-1 only. In fact, the NRDc receptor is totally
HB-EGF-specific. In addition, unlike most members of the EGF
family, which are non-heparin binding, sHB-EGF is able to bind to
cell-surface heparin-like molecules (heparan sulfate proteoglycans;
HSPG), which act as low affinity, high capacity receptors for
HB-EGF. The differing affinities of EGF family members for the
different EGFR subtypes and for HSPG may confer different
functional capabilities to these molecules in vivo. The combined
interactions of HB-EGF with HSPG and ErbB-1/ErbB-4/NRDc may confer
a functional advantage to this growth factor. Importantly,
endogenous HB-EGF is protective in various pathologic conditions
and plays a pivotal role in mediating the earliest cellular
responses to proliferative stimuli and cellular injury.
[0005] Short bowel syndrome (SBS) is a consequence of massive small
bowel resection performed in patients presenting with various
diseases including inflammatory bowel disease, trauma, mesenteric
vascular disease, volvulus, congenital atresias and neonatal
necrotizing enterocolitis (NEC). Many thousands of patients with
SBS each year depend on total parenteral nutrition (TPN) for
survival, with the cost exceeding $50,000 per year per patient.
Mortality remains high at 30% in the pediatric population.
Approximately 50% of these deaths are attributable to hepatic
failure from TPN-induced liver disease. In adults with SBS, 5% of
deaths are related to complications of TPN. Surgical strategies in
patients presenting with SBS include lengthening of the remnant
small bowel, construction of intestinal valves to delay passage of
intestinal material, and tapering procedures to improve
peristalsis. However, such approaches are rarely feasible in adults
owing to clinical complications including fistula formation and
sepsis typically associated with Crohn's disease, the most
prevalent underlying pathology within this patient subpopulation.
In pediatric SBS patients treated with intestinal lengthening
procedures, long-term survival is only 45%. Allogeneic
transplantation of small bowel offers the potential for definitive
functional rescue but may be associated with technical
complications including high rates of graft rejection and
complications associated with long-term immunosuppression. Clearly,
there is a crucial clinical need for novel approaches to the
treatment and management of SBS.
[0006] Tissue engineering and regenerative medicine technologies
represent a next logical step towards the development of small
intestinal substitutes. Such approaches require a cell source and
biodegradable scaffold combined to produce a construct implanted
into the body. The cell source and biodegradable scaffold must be
capable of catalyzing the body's innate regenerative potential. The
feasibility of a tissue engineered intestine begins with the
remarkable regenerative ability of the intestinal epithelium. When
a synthetic material is used to patch a full-thickness defect
created in the small intestine of a rodent, enteric cells at the
interface between the patch and the native mucosa migrate into the
bare area and form organized epithelium. This observation led to
efforts to implant enteric cells attached to polymer materials into
the omentum of a rodent. The implantation of neonatal rodent
intestinal OUs (partially digested pieces of the intestine)
attached to biodegradable polymer scaffolds in the rodent omentum
produces cystic structures lined by epithelial cells (Choi et al.
Journal of pediatric surgery. 1998; 33:991-6). Patches of such
tissue-engineered structures were successfully anastomosed to the
native small intestine of rodents (Grikscheit et al. Annals of
surgery. 2004; 240:748-54). After anastomosis, the rudimentary
epithelium in the cystic structures developed into mature crypts
and villi. When these tissue-engineered cysts were anastomosed to
the side of the proximal small intestine in a rodent model of SBS,
animals lost less weight and recovered sooner than the controls
(Choi et al. Journal of pediatric surgery. 1998; 33:991-6).
[0007] Such tissue-engineered treatments would avoid problems
associated with intestinal transplantation, including donor
availability and complications of immunosuppressive therapy. Before
such a strategy can be brought into clinical practice, however,
considerable obstacles need to be overcome. The first obstacle is
the source of cells to be used. In the last decade, studies have
focused on using organoid units (OU) as the cell source for
engineered intestine. OU are cell clusters that are isolated from
full-thickness intestine, and represent a mixed population of
differentiated and undifferentiated cells. This cell source is not
efficient for tissue regeneration because differentiated epithelial
cells no longer have the capacity to proliferate, and will likely
undergo apoptosis. The second obstacle is the need to recreate
peristaltic motion of the small intestine. Although tissues
generated from intestinal organoids histologically resemble the
mucosa, functional smooth muscle layers and neural plexuses are
absent. The motility of enteric smooth muscle is primarily
controlled by the myenteric plexuses, which comprise the enteric
nervous system (ENS), and the ENS needs to be generated in tissue
engineered intestine (TEI) to produce peristalsis.
SUMMARY OF INVENTION
[0008] The invention provides for an engineered intestine construct
comprising a nanofiber scaffold seeded with neural stem cells
(NSC), smooth muscle cells (SMC) and intestinal stem cells (ISC),
wherein the nanofiber scaffold comprises HB-EGF polypeptide or a
fragment thereof.
[0009] In addition, the invention provides for an engineered
intestine construct comprising a nanofiber scaffold seeded with
NSCs, SMCs and ISCs, wherein at least one of the neural stem cells,
smooth muscle cells or intestinal stem cells overexpress HB-EGF
polypeptide or a fragment thereof. The engineered intestine
constructs of the invention may comprise a single layer nanofiber
scaffold or a multilayer nanofiber scaffold.
[0010] In addition, the invention provides for the use of a custom
designed cell filtration system to enrich intestinal stem
cell-containing crypts to enhance mucosa engraftment for TEI; 2)
incorporation of growth factors into scaffolds to improve the
morphological and functional properties of TEI; 3) use of
state-of-the-art technology to fabricate tissue engineering
scaffolds that mimic the architecture and properties of native
intestine and enhance the bio-environment for cell adhesion,
proliferation, and differentiation.
[0011] The invention provides for engineered intestine construct
comprising a multilayer nanofiber scaffold, wherein the multilayer
nanofiber scaffold comprises at least an inner layer and an outer
layer, wherein the outer layer comprises NSCs and SMCs, and wherein
the inner layer comprises ISCs. The inclusion of multiple types of
stem cells in the generation of the engineered intestine construct,
e.g. NSCs and ISCs, allows for the generation of full thickness
intestine with peristaltic and absorptive function. The ISC may be
provided by seeding the scaffold with crypts and the inner layer of
the scaffold may comprise crypts and ISCs.
[0012] The engineered intestine constructs of the invention may
comprise a nanofiber scaffold wherein the scaffold comprises any
biodegradable polymers. For example, may comprise nanofibers and/or
macrofibers of one or more biodegradable polymer such as
poly(glycolic acid)(PGA) nanofibers, Poly(.epsilon.-caprolactone)
(PCL) nanofibers, Poly(-caprolactone-co-lactic acid) (PLC)
nanofibers, Poly(L-lactic acid) (PLLA) nanofibers, Poly(D-lactic
acid-co-glycolic acid) (PDLGA) nanofibers, Polydioxanone (PDO)
nanofibers, Polyurethane (PU) nanofibers and PGA macrofibers or
combinations thereof.
[0013] The multiple layers of the nanofiber scaffold allow for the
use of polymers with varying pore size and strength through a
single scaffold. For example, the presence of layer of different
scaffold materials allow for multiple layers with a pore size
gradient applied through the sidewall starting from the innermost
layer with the biggest pores to the outermost layer with the
smallest pores. This allows for the delivery of cells of different
sizes to the scaffold and delivery of intact crypts to regenerate
the mucosa layer.
[0014] The engineered intestine constructs of the invention may
comprise a multilayer nanofiber scaffold comprising at least three
layers, at least four layers, at least five layer, at least six
layers, at least seven layers or at least eight layers. For example
in one embodiment, the construct comprises an outer layer, at least
one middle layer and an inner layer. In another embodiment, the
engineered intestine construct comprises a multilayer nanofiber
scaffold comprising an outer layer, a layer adjacent to the outer
layer, a middle layer, a layer adjacent to the inner layer, such a
layer of macrofibers, and an inner layer.
[0015] The multiple layers within the nanofiber scaffold of the
invention may comprise the same or different biodegradable polymers
and the layers include those comprising nanofibers and those
comprising macrofibers.
[0016] In one embodiment, the engineered intestine construct
comprises a nanofiber scaffold having an inner layer, at least one
middle and the outer layer comprises of PDLGA. In a further
embodiment, the engineered intestine construct comprises a scaffold
having a layer of macrofibers, such as PGA macrofibers, between the
inner layer and the middle layer, or adjacent to the inner layer.
In another embodiment, the engineered intestine construct comprises
a scaffold having a layer of PCL is in between the middle layer and
the outer layer, or adjacent to the outer layer.
[0017] Any of the engineered intestine constructs of the invention
may comprise a nanofiber scaffold wherein at least one of the
layers comprises a HB-EGF polypeptide or an active fragment
thereof. In some embodiments, the engineered intestine construct
comprises a nanofiber scaffold wherein all the layers comprise
HB-EGF polypeptide or an active fragment thereof. Alternatively,
one or more the cell types seeded on the nanofiber scaffold may be
transfected to express HB-EGF at a level above endogenous
expression, including overexpression.
[0018] The HB-EGF polypeptide comprises the amino acid sequence of
SEQ ID NO: 2 or is a fragment thereof that competes with HB-EGF for
binding to the ErbB-1 receptor and has ErbB-1 agonist activity. A
preferred HB-EGF fragments comprises amino acids of 74-148 of SEQ
ID NO: 2 (human HB-EGF(74-148). Other fragments of HB-EGF which may
be used to construct the engineered intestine constructs are
fragments of SEQ ID NO: 2 which induce epithelial cell or somatic
stem cell, such as NSC, MSC or ISC, proliferation, fragments of SEQ
ID NO: 2 that induce epithelial cell or somatic stem cell, such as
MSC or ISC, migration, fragments of SEQ ID NO: 2 that promote
epithelial cell or somatic stem cell, such as MSC or ISC,
viability, and a fragment of HB-EGF that protects epithelial cells
or somatic stem cells, such as MSC or ISC form apoptosis or other
types of cellular injury.
[0019] The HB-EGF polypeptide or fragments thereof include
recombinant HB-EGF produced in E. coli and HB-EGF produced in
yeast. The development of expression systems for the production of
recombinant proteins is important for providing a source of protein
for research and/or therapeutic use. Expression systems have been
developed for both prokaryotic cells such as E. coli, and for
eukaryotic cells such as yeast (Saccharomyces, Pichia and
Kluyveromyces spp) and mammalian cells.
[0020] In one embodiment, the engineered intestine construct
comprise an inner layer of nanofibers, e.g. of PLLA or PDLGA, that
has a smooth lumen surface that prevent crypts from leaking out and
comprises ISC, a layer of macrofibers, such as PGA, adjacent to the
inner layer which serves to deliver cypts to regenerate the mucosa
layer, a middle layer of nanofibers, such as PLLA or PDLGA, which
serves to separate the crypts and SMCs, a layer of nanofibers, such
as PCL, adjacent to the outer layer which serves to deliver SMCs
and NSCs to regenerate the functional smooth muscle layer and an
outer layer of nanofibers, such as PLLA or PDLGA, which serves to
prevent SMCs from leaking out.
[0021] Exemplary multilayer scaffolds comprising either an inner
and an outer layer or an inner, middle and outer layer are provided
in Table 1. These exemplary scaffolds may comprise PGA as a
nanofiber or macrofiber. In addition, these exemplary scaffolds may
comprise one or more additional layers that are adjacent to the
inner or outer layer. The invention is not limited to these
exemplary scaffolds and the invention contemplates any combination
of biodegradable polymers may be used to construct the
scaffold.
TABLE-US-00001 TABLE 1 Exemplary Two Layer Scaffolds Outer Layer
Inner Layer PGA PGA PCL PCL PLC PLC PLLA PLLA PDLGA PDLGA PU PU POD
PDO PGA PCL PGA PLC PGA PLLA PGA PDLGA PGA PU PGA PDO PLC PGA PLC
PCL PLC PLLA PLC PDLGA PLC PU PLC PDO PCL PGA PCL PLC PCL PLLA PCL
PDLGA PCL PU PCL POD PLLA PGA PLLA PLC PLLA PCL PLLA PDLGA PLLA PU
PLLA PDO PDLGA PGA PDLGA PLC PDLGA PCL PDLGA PLLA PDLGA PU PDGLA
PDO PU PGA PU PLC PU PCL PU PLLA PU PDLGA PU PDO PDO PGA PDO PCL
PDO PLC PDO PLLA PDO PDLGA PDO PU Exemplary Three Layer Scaffold
Outer Layer Middle Layer Inner Layer PGA PGA PGA PGA PGA PCL PGA
PGA PLC PGA PGA PLLA PGA PGA PDLGA PGA PGA PU PGA PGA PDO PGA PGA
PGA PGA PCL PGA PGA PLC PGA PGA PLLA PGA PGA PDLGA PGA PGA PU PGA
PGA PDO PGA PCL PCL PCL PCL PCL PGA PCL PCL PCL PCL PCL PLLA PCL
PCL PDLGA PCL PCL PU PCL PCL PDO PCL PGA PCL PCL PCL PCL PCL PLLA
PCL PCL PDLGA PCL PCL PU PCL PCL PDO PCL PLC PLC PLC PLC PLC PGA
PLC PLC PCL PLC PLC PLLA PLC PLC PDLGA PLC PLC PU PLC PLC PDO PLC
PGA PLC PLC PCL PLC PLC PLLA PLC PLC PDLGA PLC PLC PU PLC PLC PDO
PLC PLLA PLLA PLLA PLLA PLLA PGA PLLA PLLA PCL PLLA PLLA PLC PLLA
PLLA PDLGA PLLA PLLA PU PLLA PLLA PDO PLLA PGA PLLA PLLA PCL PLLA
PLLA PLC PLLA PLLA PDLGA PLLA PLLA PU PLLA PLLA PDO PLLA PDLGA
PDLGA PDLGA PDLGA PDLGA PGA PDLGA PDLGA PCL PDLGA PDLGA PLC PDLGA
PDLGA PLLA PDLGA PDLGA PU PDLGA PDLGA PDO PDLGA PGA PDLGA PDLGA PCL
PDLGA PDLGA PLC PDLGA PDLGA PLLA PDLGA PDLGA PU PDLGA PDLGA PDO
PDLGA PU PU PU PU PU PGA PU PU PCL PU PU PLC PU PU PLLA PU PU PDLGA
PU PU PDO PU PGA PU PU PCL PU PU PLC PU PU PLLA PU PU PDLGA PU PU
PDO PU PDO PDO PDO PDO PDO PGA PDO PDO PCL PDO PDO PLC PDO PDO PLLA
PDO PDO PDLGA PDO PDO PU PDO PGA PDO PDO PCL PDO PDO PLC PDO PDO
PLLA PDO PDO PDLGA PDO PDO PU PDO
[0022] In another aspect, the invention provides for methods of
generating an engineered intestine construct comprising a)
preparing a nanofiber scaffold by electrospinning a polymer to a
target fiber diameter and porosity, b) embedding an HB-EGF
polypeptide or fragment thereof on at least one layer of the
scaffold, c) seeding the scaffold with intestinal stem cells,
neural stem cells and smooth muscles cells, and d) culturing the
cells in the scaffold to form a construct that will form a mature
intestine upon insertion into a subject. A number of endpoints
could be used to determine when an engineered intestine construct
of the invention is in condition for insertion into a subject in
need, such as the number of cells engrafted on the scaffold, a
decrease in cell apoptosis within the construct, formation of
mature intestine within the construct. Mature intestine is refers
to intestine that contains mucosa, smooth muscle layers and an
enteric nervous system.
[0023] "Target fiber diameter and porosity" refers to fibers that
exhibit one or more of the following qualities: matches mechanical
requirements of the engineered organ, allow for uniform cell
seeding, promotes cell attachment, communication and signaling,
prevents cells from penetrating the scaffold and mimics the native
organ.
[0024] The invention provides for methods of generating an
engineered intestine construct wherein the culturing step is
carried out in a bioreactor such as a perfusion system bioreactor.
In addition, the invention provides for method of generating an
engineered intestine construct wherein the nanofiber scaffold
comprises at least an outer and an inner layer, and these
constructs may further comprise a middle layer.
[0025] In any of the method of the invention, the intestinal stem
cells are seeded on the inner layer and may be provided by seeding
crypts on the inner layer. Furthermore, in any of the methods of
the invention, the neural stem cells and smooth muscle cells are
seeded on the outer layer.
[0026] The methods of the invention may generate engineered
intestine constructs that comprise multiple layers within the
nanofiber scaffold, and these layers may comprise the same or
different biodegradable polymers and the layers include those
comprising nanofibers and those comprising macrofibers. The layers
of the scaffold may comprise nanofibers and/or macrofibers of a
biodegradable polymer such as poly(glycolic acid)(PGA) nanofibers,
Poly(.epsilon.-caprolactone) (PCL) nanofibers,
Poly(-caprolactone-co-lactic acid) (PLC) nanofibers, Poly(L-lactic
acid) (PLLA), Poly(D-lactic acid-co-glycolic acid) (PDLGA),
Polyurethane (PU) nanofibers, Polydioxanone (PDO) nanofibers and
PGA macrofibers or combinations thereof. In particular, the
nanofibers may comprise PDLGA or PLLA.
[0027] In another aspect, the invention provides for methods of
method of treating short bowel syndrome in a subject comprising
attaching an engineered intestine construct of the invention under
conditions wherein the construct will implant within the intestine
of the subject.
[0028] The invention also provides for use of the engineered
intestine construct of the invention for the preparation of a
medicament for treating short bowel syndrome wherein the medicament
is administered to subject under conditions wherein the intestine
implants within the subject. The invention also provides for
engineered intestine constructs of the invention for use in
treating short bowl syndrome wherein the engineered intestine
construct implants within the intestine of the subject.
[0029] The invention also provides for methods of repairing the
intestine of a subject undergoing intestinal resection comprising
attaching the engineered intestine construct of the invention under
conditions wherein the construct will implant within the intestine
of the subject. The term "intestinal resection" includes small
bowel resection, large bowel resection and colectomy.
[0030] The invention also provides for use of the engineered
intestine construct of the invention for the preparation of a
medicament for repairing the intestine of subject undergoing
intestinal resection wherein the medicament is administered under
conditions wherein the engineered intestine construct of the
invention attaches to and implants within the intestine of the
subject. The invention also provides for an engineered intestine
construct of the invention for use in repairing the intestine of a
subject undergoing intestinal resection wherein the engineered
intestine construct of the invention attaches to and implants
within the intestine of the subject.
[0031] Furthermore, the invention provides for methods of treating
short bowel syndrome in a subject or repairing the intestine of a
subject suffering from inflammatory bowel disease, trauma,
mesenteric vascular disease, vovlulus, congenital atresias,
neonatal necrotizing enterocolitis, Crohn's disease, ischemia,
intestinal blockage, bowel obstruction, regional ileitis, regional
enteritis, colorectal cancer such as colorectal cancer and other
tumors that invade the intestine, carcinoid tumor, Merkel's
diverticulum, precancerous polyps, diverticulitis, intestinal
bleeding, intussusceptions, or ulcerative colitis.
[0032] The invention also provides for use of the engineered
intestine construct of the invention for the preparation of a
medicament for treating short bowl syndrome in a subject or for
repairing the intestine of a subject wherein the subject is
suffering from inflammatory bowel disease, trauma, mesenteric
vascular disease, vovlulus, congenital atresias, neonatal
necrotizing enterocolitis, Crohn's disease, ischemia, intestinal
blockage, bowel obstruction, regional ileitis, regional enteritis,
colorectal cancer such as colorectal cancer and other tumors that
invade the intestine, carcinoid tumor, Merkel's diverticulum,
precancerous polyps, diverticulitis, intestinal bleeding,
intussusceptions, or ulcerative colitis.
[0033] The invention also provides for engineered intestine
constructs for use in treating short bowl syndrome in a subject or
repairing the intestine of a subject, wherein the subject is
suffering from inflammatory bowel disease, trauma, mesenteric
vascular disease, vovlulus, congenital atresias, neonatal
necrotizing enterocolitis, Crohn's disease, ischemia, intestinal
blockage, bowel obstruction, regional ileitis, regional enteritis,
colorectal cancer such as colorectal cancer and other tumors that
invade the intestine, carcinoid tumor, Merkel's diverticulum,
precancerous polyps, diverticulitis, intestinal bleeding,
intussusceptions, or ulcerative colitis.
[0034] In another aspect, the invention provides for methods of
enriching a cell sample for a particular cell type comprising
contacting a cell sample with multiple sieve membranes wherein the
membranes are aligned in descending order according to pore size,
wherein the cell sample contacts the membrane with the largest pore
size first and wherein the cell sample comprises multiple cell
types, filtering the cell sample through the membranes and
recovering the enriched cell sample. In particular, the methods of
the invention are used to enrich intestinal cells in crypts for use
in generating the TEI of the invention. The cell sample comprising
multiple cell types includes samples comprising at least two
different cell types or a sample comprising differentiated and
undifferentiated cells or cells at different states of
differentiation.
[0035] An enriched cell sample may comprise one cell type or two
cells types or three cells type or more. The term "enriched" refers
to improving the quality of the cell sample by removing unneeded
cell types or reducing the number of cell types within a sample. An
exemplary enriched sample comprises intestinal stem cells and their
neighboring cells including paneth cells within crypts. Another
example of an enriched cell sample consists of differentiated
cell-containing villi, which are separated from the stem
cell-containing crypts by their larger size.
[0036] The invention provides for engineered tissue construct
comprising a nanofiber construct seeded with NSCs, SMCs and ISCs.
An innovation of the invention is the use of multiple stem cell
types in order to generate full thickness intestine with
peristaltic and absorptive function. Another innovation of the
invention is the use of state-of-the-art technology to fabricate
tissue engineering scaffolds that mimic the architecture and
properties of native intestine and enhance the bio-environment for
cell adhesion, proliferation, and differentiation. The present
invention combines cell therapy with novel nanofiber technology to
regenerate full-thickness, functional intestine.
[0037] The engineered intestine constructs of the invention avoid
problems associated with intestinal transplantation, including
donor availability and complications of immunosuppressive therapy.
In addition, the engineered tissue constructs avoid the use of
organoid units (OU), or cell clusters that comprise a mixed
population of differentiated and undifferentiated cells, and the
presence of differentiated epithelial cells do not have the
capacity to proliferate which does not promote tissue
regeneration.
[0038] The present invention utilizes intestinal crypts enriched in
ISC delivers concentrated ISC in order to enhance mucosa formation.
Intestinal crypts are a gland located in the epithelial ling of the
small intestine and the colon. The crypts comprise an epithelium
that comprises goblet cell and enterocytes. New epithelium is
formed within the crypts and therefore the basal portion of the
crypt comprises multipotent stem cells, e.g. ISCs.
[0039] The second obstacle is the need to recreate peristaltic
motion of the small intestine. Although tissues generated from
intestinal organoids histologically resemble the mucosa, functional
smooth muscle layers and neural plexuses are absent. The motility
of enteric smooth muscle is primarily controlled by the myenteric
plexuses, which comprise the enteric nervous system (ENS). The ENS
needs to be generated in tissue engineered intestine (TEI) to
produce peristalsis. The present invention overcomes this obstacle
by using specially designed electrospun nanofiber scaffolds modeled
after native intestinal architecture with circumferentially and
longitudinally aligned nanofibers in the middle and outside layer
of the scaffold, respectively, for delivering a mixture of smooth
muscle cells (SMCs) and NSCs. The seeded SMC migrate and
proliferate along the nanofibers and will eventually be
encapsulated by extracellular matrix (ECM) to form muscularis
interna and externa. The implanted NSC in the newly regenerated
intestine will differentiate to form myenteric plexuses which will
communicate with the native ENS to perform peristalsis.
Nanofiber Scaffolds
[0040] The engineered intestine constructs of the invention
comprise a nanofiber scaffold. The nanofiber scaffolds used in the
invention are specially designed electrospun nanofiber scaffolds
seeded with a mixture of smooth muscle cells (SMCs) and neural stem
cells (NSCs), which is one of the innovations and goals of this
proposal. The nanofiber scaffolds may comprise one layer or
multiple layers of nanofibers or macrofibers of biodegradable
polymers.
[0041] Electrospinning has been used to fabricate tissue engineered
scaffolds comprising non-woven, three-dimensional, porous,
nanoscale fiber-based matrices. The characteristics of fibrous
scaffolds, such as high surface area to volume ratio with similar
structural morphology to the fibrillar extracellular matrix (ECM)
found in vivo, suggest that they may serve as effective tissue
engineering scaffolds. Moreover, the alignment of nanofibers and
the porosity of the scaffold can be tailored to match the
mechanical requirements of the target organ.
[0042] Electrospinning of nanofibers for tissue engineering has a
number of benefits (Lannutti et al. Mat. Sci. Engin: C 27: 504-509,
2007). These include cost-effectiveness and that the scaffolds can
be created in a nanoscaled form resembling the extracellular matrix
allowing for more natural cellular proliferation. Electrospinning
also allows for the adjustment of fiber diameter and alignment to
guide cellular infiltration. Pore sizes can also be adjusted and
the scaffolds tend to have large surface areas with open, connected
porous arrangements of 70-90% relative porosity. This allows for
both enhanced drug delivery and room for cell adhesion and
proliferation. Finally, multiple different polymers and blends of
polymers can be used to create the ideal mechanical and degradative
features for tissue engineering. These alterations in scaffold
structure allow for improved cell-scaffold interactions and may
promote cell migration and proliferation to optimize the tissue
engineered structure or organ. Electrospinning of nanofiber
scaffolds also allows for polymers to be blended or layered to
produce a more biomimetic scaffold than is possible using a single
material. Ultimately, a blend or layering of these polymers may
produce the most ideal scaffold composition in this application.
The nanofiber scaffold may comprise a biodegradable polymers
including poly(glycolic acid) (PGA), polyesters such as
poly(.epsilon.-caprolactone) (PCL) and
poly(.epsilon.-caprolactone-co-lactic polylactic acid (PCL), and
polylactic acid copolymers such as poly(L-lactic acid (PLLA) and
poly(D-lactic acid-co-glycolic acid (PDLGA), polyurethane (PU),
polyanhydrides; Polydioxanone (PDO) nanofibers; poly alkyl
cyanoacrylates such as n-butyl cyanoacrylate and isopropyl
cyanoacrylate; polyacrylamides; poly(orthoesters);
polyphosphazenes; polypeptides; polyurethanes; and combinations of
such polymers.
[0043] While the multilayer nanofiber scaffolds of the invention
are primarily composed of nanofibers, the invention provides for
scaffolds that include layers of macrofibers such as PGA.
[0044] In one aspect, the nanofiber scaffold comprises the
copolymer of glycolic acid and lactic acid (PLGA), such as
Poly(D-lactic acid-co-glycolic acid) (PDLGA) having a proportion
between the lactic acid/glycolic acid units ranging from about
100/0 to about 25/75. The average molecular weight ("MW") of the
polymer will typically range from about 6,000 to 700,000 and
preferably from about 30,000 to 120,000, as determined by
gel-permeation chromatography using commercially available
polystyrene of standard molecular weight, and have an intrinsic
viscosity ranging from 0.5 to 10.5.
[0045] Poly(.epsilon.-caprolactone) (PCL) is a semicrystalline
material with good mechanical properties. PCL is one of the most
widely used biodegradable polyesters for medical applications
because of its biocompatibility, biodegradability, and flexibility.
Scaffolds fabricated using PCL are more resistant to hydrolysis,
and consequently they are capable of supporting the viability,
proliferation, and differentiation status of implanted cells.
Poly(c-caprolactone-co-lactic acid) (PLC), a poly(lactic acid)(PLA)
and PCL copolymer, has a similar Young's modulus (the ratio of the
uniaxial stress over the uniaxial strain in the range of stress in
which Hooke's Law holds), to PCL and native intestine as
demonstrated in our preliminary data, but possesses better
mechanical strength in vitro, allowing the tubular structure to be
maintained in a hydrated state. This is especially beneficial for
maintaining continuity of the lumen with native intestine. In this
proposal, PCL and PLC will be used to fabricate tri-layer scaffolds
with a pore size gradient applied through the sidewall starting
from the innermost layer with the biggest pores to the outermost
layer with the smallest pores. A non-woven innermost layer with the
biggest pore size will allow for the accommodation of crypts and
the regeneration of mucosa. Circumferentially and longitudinally
aligned nanofibers in the middle and outermost layers,
respectively, are intended for the residence of SMCs and NSCs and
eventually the formation of muscularis interna and externa. Others
have demonstrated increased SMC attachment and proliferation on
aligned nanofibers compared to randomly oriented nanofiber matrices
(Levin et al. Expert review of medical devices. 2011; 8:673-5).
This can be explained by the "contact guidance" theory, which
illustrates that a cell has the maximum probability of migrating in
directions that are associated with chemical, structural, and/or
mechanical properties of the substratum. Aligned nanofibers
represent an effective approach to control cell orientation and
migration in tissue engineering.
[0046] In the present invention, different scaffold materials were
used to fabricate multi-layered scaffolds with a pore size gradient
applied through the sidewall starting from the innermost layer with
the biggest pores to the outermost layer with the smallest pores. A
non-woven innermost layer with the biggest pore size will allow for
the accommodation of SC-containing crypts and the regeneration of
mucosa. Circumferentially and longitudinally aligned nanofibers in
the middle and outermost layers, respectively, are intended for the
residence of SMC and NSC and eventually the formation of muscularis
interna and externa. Others have demonstrated increased SMC
attachment and proliferation on aligned nanofibers compared to
randomly oriented nanofiber matrices. This can be explained by the
"contact guidance" theory, which illustrates that a cell has the
maximum probability of migrating in directions that are associated
with chemical, structural, and/or mechanical properties of the
substratum. Aligned nanofibers represent an effective approach to
control cell orientation and migration in tissue engineering. The
use of multi-layered nanofiber scaffolds, with seeding of the inner
layer with SC-containing crypts, and seeding of the outer layer
with SMC and NSC, represents a completely novel methodology that
has never been previously described.
[0047] In order to accelerate and mature tissue formation in vitro
and in vivo, heparin binding EGF-like growth factor (HB-EGF) will
be embedded into the tri-layer scaffold. Multiple experiments have
shown that administration of HB-EGF protects the intestines from
experimental NEC, intestinal ischemia/reperfusion injury, and
hemorrhagic shock and resuscitation. HB-EGF protects the intestines
from experimental NEC and other forms of intestinal injury by
protecting ISC from injury, by promoting the proliferation and
migration of enterocytes, by increasing microvascular villous blood
flow, by increasing gut barrier function, and by reducing
intestinal apoptosis. It also known that HB-EGF promotes the
migration and proliferation of mesenchymal stem cells (MSC), and
protects MSC from anoxia/reoxygenation-induced apoptosis in vitro.
To generate the engineered intestine constructs, HB-EGF was
embedded into the nanofibers using CO2-assisted infusion of HB-EGF
into electrospun tri-layer nanofiber scaffolds will mimic the
native structural and chemical environment of the intestine,
improve tissue regeneration, and provide a significant advancement
in the production of tissue engineered intestine. Lastly, to
facilitate cell adhesion and inclusion, the temperature-sensitive
hydrogel pluronic F-127 will be mixed with cells prior to seeding
of nanofiber scaffolds. Others have shown that -30-40% of seeded
cells pass through or are released from open spaces on the surface
of polymer scaffolds upon cell seeding (Yu et al. J Surg Res. 2012;
172:165-76). Pluronic F-127 is a triblock copolymer composed of a
central hydrophobic chain of poly(propylene oxide) flanked by two
hydrophilic chains of poly(ethylene oxide). It exhibits low
viscosity below room temperature and changes to a viscous soft gel
at body temperature (-37.degree. C.). The combination of nanofiber
scaffolds and hydrogel will prevent cells from detaching. It is
expected that the tissue engineered intestine constructs of the
invention have similar intestinal morphology and function when
compared to with native intestine
Fabrication of Nanofiber Scaffolds
[0048] The nanofiber scaffolds of the invention can be fabricated
using any method known in the art. In one embodiment, the
multilayer nanofiber scaffolds are fabricated using
electrospinning. This fabrication method allows the generation of
nanofibers with a pore size gradient that allows for seeding of
different sized cells. In particular, nanofibers with greater pore
size assists seeding the scaffold with cypts.
Electrospinning Multi-Layer Fiber Scaffolds
[0049] Electrospinning is driven by the application of a high
voltage, typically between 0 and 30 kV, to a droplet of a polymer
solution or melt. The liquid polymer is typically ejected from a
capillary at a flow rate between 0 and 50 ml/h to create a
condition of charge separation between two electrodes and within
the polymer solution to produce a jet of polymer. A typical polymer
solution would consist of a polymer such as polycaprolactone,
polystyrene, or polyethersulfone and a solvent such as
1,1,1,3,3,3-Hexafluoro-2-propanol, N,N-Dimethylformamide, Acetone,
or Tetrahydrofuran in a concentration range of 5-50 wt %. As the
jet of polymer solution travels toward the electrode it is
elongated into sub-micron diameter fibers typically in the range of
0.1-50 .mu.m.
Dual Layer Electrospinning
[0050] During electrospinning polymer fibers are driven toward a
collector by charge separation caused by applied voltage. The
collector is typically a conductive surface such as aluminum or
copper, but can also be covered by a thin layer of plastic between
0.001-0.1 inches thick. The charge that drives electrospinning
toward the collector comes from mobile ions within the polymer
solution or melt [ ]. The jet of polymer that is produced will have
a net positive or negative charge depending upon the polarity of
the DC voltage applied to the electrode(s). When the jet solidifies
on the collector surface the charge will build up as subsequent
fiber layers are collected. It is believed that as the charge
builds up on the surface fiber with similar charge will be repelled
leading to a lower density and lower uniformity of fiber collected.
A means of collecting a large uniform layer of fiber onto a
collector is to electrospin for a period of time using one voltage
polarity i.e. negative followed by electrospinning with the
opposite polarity i.e. positive for a period of time. Since
opposite charges attract an increased thickness of fiber may be
deposited or two different materials/fiber diameters can be used in
one scaffold.
Stem Cell Environment
[0051] In cell culture it is desirable to have a three dimensional
surface on which the cells will differentiate and expand. In stem
cell culture a three dimensional surface may help the cells
maintain their "stemness" as opposed to differentiating down an
undesired pathway. When cultured on a two dimensional surface i.e.
flat polystyrene the cells often differentiate into adipocytes or
other undesired cell phenotypes. Ideally, the three dimensional
scaffold would have a porosity greater than 10 .mu.m to allow cell
penetration, but could have layers with pore sizes smaller than 10
.mu.m to prevent cells from penetrating through the scaffold. Using
multi-layer electrospinning a dense layer of small diameter fiber
with average pore diameter less than 10 .mu.m is deposited followed
by the deposition of large porosity fibers with an average pore
diameter greater than 10 .mu.m. This configuration will allow the
cells to penetrate the large porosity fiber to the small pore size
fiber.
Material Selection
[0052] Choosing a material that can accurately mimic the mechanical
properties of the native organ/tissue is critical to promote proper
stem cell differentiation and facilitating normal tissue function.
Materials may be non-resorbable for permanent implantation or may
be designed to slowly degrade while the host body rebuilds the
native tissue until the implanted prosthesis is completely
resorbed. Permanent polymers may include polyurethane,
polycarbonate, polyester terephthalate and degradable materials may
include polycaprolactone, polylactic acid, polyglycolic acid,
gelatin, collagen, or fibronectin. The fibers may be electrospun
onto a form with the desired prosthesis shape.
Fiber Orientation and Composite Structure
[0053] Closely mimicking the structure of the native organ is
necessary to replicate the organ function. By controlling the
orientation of the fibers and assembling a composite structure of
different materials and/or different fiber orientations it is
possible to control and direct cell orientation and
differentiation.
Scaffold Porosity
[0054] The scaffold needs to allow complete cellular penetration
and uniform seeding for proper function and prevention of necrotic
areas developing. If the fiber packing is too dense, then cells
will not be able to penetrate or migrate from the exposed surfaces
into the inner portions of the scaffold. However, if the fiber
packing is not close enough, then the attached cells will not be
able to properly fill the voids, communicate and signal each other
and a complete tissue or organ will not be developed. Controlling
the fiber diameter is one way to change the scaffold porosity as
the porosity scales with fiber diameter. Alternatively, blends of
different polymers may be electrospun together and one polymer
preferentially dissolved to increase scaffold porosity.
[0055] Fiber orientation can be altered in each layer of a
composite or sandwich scaffold in addition to the material and
porosity to most closely mimic the native tissue.
Tissue Culture Vessels
[0056] Those of ordinary skill in the art will readily appreciate
that the cell culture and bioengineering methodologies described
herein may be carried out in on a variety of environments or
substrates (i.e., vessels or containers). SMCs, NSCs and ISCs are
anchorage dependent, and therefore to grow in culture these cells
require a nontoxic, biologically inert, and optically transparent
surface that will allow cells to attach and allow movement for
growth. Tissue culture vessels or plates include specially-treated
polystyrene plastic that are supplied sterile and are disposable.
These include Petri dishes, multi-well plates, microtiter plates,
roller bottles, screwcap flasks (T-25, T-75, T-150 cm2 of surface
area), culture bags or any container capable of holding cells,
preferably in a sterile environment.
[0057] In one embodiment of the present invention, a bioreactor is
also useful for bioengineering the engineered intestine constructs.
In particular, the invention contemplates generation of the
engineered intestine constructs using a perfusion provides
bioreactor. For example, several manufacturers currently make
devices that can be used to grow cells and be used in combination
with the methods of the present invention. See for example, Celdyne
Corp., Houston, Tex.; Unisyn Technologies, Hopkinton, Mass.;
Synthecon, Inc. Houston, Tex.; Aastrom Biosciences, Inc. Ann Arbor,
Mich.; Wave Biotech LLC, Bedminster, N.J. Further, patents covering
such bioreactors include U.S. Pat. Nos. 6,096,532; 6,001,642,
5,985,653; 5,888,807; 5,688,687, 5,605,835, 5,190,878, which are
incorporated herein by reference.
[0058] There are a number of different kinds of bioreactors,
devices designed to provide a low-shear, high nutrient perfusion
environment, available on the market. For example, the invention
may be carried out in a rotating wall bioreactor, which consists of
a small inner cylinder, the substrate for the electrospinning
process, positioned inside a larger outer cylinder. Although the
electrospun matrix can be fabricated on the inner cylinder, other
locations within the bioreactor also may be used for placement of
the matrix for seeding. The gap between the inner and outer
cylinders serves as the culture vessel space for cells. Culture
medium is oxygenated via an external hydrophobic membrane. The low
shear environment of the rotating bioreactor promotes cell-cell and
cell-extracellular matrix (ECM) interactions without the damage or
"washing away" of nutrients that occurs with active stirring.
[0059] The cells that are seeded on the nanofiber scaffold may be
grown in a hydrogel or another extracellular matrix. Hyrdogels may
are formed from synthetic (e.g., poly(ethylene glycol),
poly(hydroxyethyl methacrylate)) and naturally occurring polymers
(e.g., collagen, hyaluronan, heparin), and are useful 3D models of
tissue culture due to their high water content and ability to form
in the presence of cells, proteins and DNA.
[0060] Fibrin gel is a suitable material that may be used for organ
replacement. Fibrin gel is a network made up of monomeric fibrin
molecules generated by activation of fibrinogen by thrombin. This
biopolymer is known to be involved in hemostatis and wound healing.
Fibrin is a biodegradable material that has been used for temporary
tissue replacement and as an absorbable implant material.
[0061] Another particular example of a suitable material is fibrous
collagen, which may be lyophilized following extraction and partial
purification from tissue and then sterilized. Matrices may also be
prepared from tendon or dermal collagen as may be obtained from a
variety of commercial sources, such as, e.g., Sigma and Collagen
Corporation.
[0062] In addition, lattices made of collagen and glycosaminoglycan
(GAG) such as that described in Yannas & Burke, U.S. Pat. No.
4,505,266, may be used in the practice of the invention. The
collagen/GAG matrix may effectively serve as a support or
"scaffolding" structure into which repair cells may migrate.
[0063] The various collagenous materials may also be in the form of
mineralized collagen. For example, the fibrous collagen implant
material termed UltraFiber.TM., as may be obtained from Norian
Corp., (1025 Terra Bella Ave., Mountain View, Calif., 94043) may be
used for formation of matrices. U.S. Pat. No. 5,231,169,
incorporated herein by reference, describes the preparation of
mineralized collagen through the formation of calcium phosphate
mineral under mild agitation in situ in the presence of dispersed
collagen fibrils.
[0064] At least 20 different forms of collagen have been identified
and each of these collagens may be used in the practice of the
invention. For example, collagen may be purified from hyaline
cartilage, as isolated from diarthrodial joints or growth plates.
Type II collagen purified from hyaline cartilage is commercially
available and may be purchased from, e.g., Sigma Chemical Company,
St. Louis. Type I collagen from rat tail tendon may be purchased
from, e.g., Collagen Corporation. Any form of recombinant collagen
may also be employed, as may be obtained from a collagen-expressing
recombinant host cell, including bacterial yeast, mammalian, and
insect cells. When using collagen as a matrix material it may be
advantageous to remove what is referred to as the "telopeptide"
which is located at the end of the collagen molecule and known to
induce an inflammatory response.
Somatic Stem Cells
[0065] Stem cells are cells with the ability to divide for
indefinite periods in culture to give rise to specialized cells.
The term "somatic stem cell" or "adult stem cell" refers to
undifferentiated cells, found among differentiated cells within a
tissue or organ, which has the capacity for self-renewal and
differentiation. The somatic stem cells can differentiate to yield
some or all of the major specialized cell types of the renewable
tissue or organ. The primary role of somatic stem cells is to
maintain and repair the tissue in which they are found.
[0066] Somatic stem cells may be used for transplantation. For
example, the invention provides for methods of transplanting
somatic stem cells to treat intestinal injury or to reduce the
damage to the intestine in a patient suffering from an intestinal
injury. Exemplary somatic stem cells include hematopoietic stem
cells, mesenchymal stem cells, intestinal stem cells, skeletal stem
cells, hepatocyte stem cells, neural stem cells, skin stem cells,
endothelial stem cells, mammary stem cells, intestinal stem cells
and neural crest stem cells.
[0067] The stem cells seeded on the scaffolds of the invention may
be isolated from a subject to generate the engineered intestine
construct. In addition, established cell lines may be used to seed
to the scaffolds of the invention to generate the engineered
intestine construct. Alternatively, intestine tissue is minced and
organoids are grown and these cells are used to seed the scaffolds
for generation of the intestine scaffold construct.
Mesenchymal Stem Cells
[0068] "Mesenchymal stem cells" (MSC) are non-hematopoietic,
pluripotent, self-renewing progenitor cells with a characteristic
spindle-shaped morphology. These cells are derived from immature
embryonic connective tissue (mesoderm layer).
[0069] Mesenchymal stem cells (MSC) have the ability to
differentiate into different cell lineages and can stimulate wound
healing via paracrine signaling pathways. Preclinical studies have
shown that MSC can regulate the host immune response, thus avoiding
recognition and subsequent rejection by recipients. The robust,
self-renewing, multilineage differentiation potential of MSC makes
these cells very desirable candidates for possible clinical
cellular therapy. Baksh et al., J Cell Mol Med 2004; 8(3):301-16.
Ongoing investigations are exploring ways to optimize MSC efficacy
prior to therapeutic delivery, including preconditioning by
exposure to hypoxia, Hu et al., J Thorac Cardiovasc Surg 2008;
135(4):799-808, growth factors, Hahn et al., J Am Coll Cardiol
2008; 51(9):933-43, and various cytokines. Gui et al., Mol Cell
Biochem 2007; 305(1-2):171-8, Pasha et al., Cardiovasc Res 2008;
77(1):134-42, Liu et al., Acta Pharmacol Sin 2008;
29(7):815-22.
[0070] MSC have been shown to contribute to the maintenance and
regeneration of various connective tissues. (Pittenger et al.,
Science 1999; 284(5411):143-7) MSC differentiate into a number of
cell types, including chondrocytes, bone, fat, cells that support
the formation of blood, and fibrous connective tissue.
[0071] MSC are mobilized from bone marrow in response to tissue
injury to aid in repair after a variety of end organ injury-models
including models of myocardial infarction (Kawada et al., Blood
2004; 104:3581-7), spinal cord injury (Koda et al., Neuroreport
2005; 16:1763-7), renal ischemia/reperfusion injury (Togel et al.,
Am J Physiol Renal Physiol 2005; 289:F31-42) and intestinal
radiation injury (Zhang et al., J Biomed Sci 2008; 15:585-94).
[0072] Mesenchymal stem cells may be isolated from various tissues
including but not limited to bone marrow, peripheral blood, blood,
placenta, and adipose tissue and amniotic fluid (denoted as AF-MCS
herein) Exemplary methods of isolating mesenchymal stem cells from
bone marrow are described in (Phinney et al., J Cell Biochem 1999;
72(4):570-85), from amniotic fluid (Baghaban et al., Arch Iran Med
2011; 14(2):96-103), from peripheral blood are described by Kassis
et al. (Bone Marrow Transplant. 2006 May; 37(10):967-76), from
placental tissue are described by Zhang et al. (Chinese Medical
Journal, 2004, 117 (6):882-887), from adipose tissue, placental and
cord blood mesenchymal stem cells are described by Kern et al.
(Stem Cells, 2006; 24:1294-1301).
[0073] The mesenchymal stem cells may be characterized using
structural phenotypes. For example, the cells of the present
invention may show morphology similar to that of mesenchymal stem
cells (a spindle-like morphology). Alternatively or additionally,
the MSC may be characterized by the expression of one or more
surface markers. Exemplary MSC surface markers include but are not
limited to CD105+, CD29+, CD44+, CD90+, CD73+, CD105+, CD166+,
CD49+, SH(1), SH(2), SH(3), SH(4), CD14-, CD34-, CD45-, CD19-,
CD5-, CD20-, CD11B-, FMC7- and HLA class 1 negative. Other
mesenchymal stem cell markers include but are not limited to
tyrosine hydroxylase, nestin and H-NF.
[0074] Examples of cells derived from mesenchymal cells include (1)
cells of the cardiovascular system such as endothelial cells or
cardiac muscle cells or the precursor cells of the cells of the
cardiovascular system, and cells having the properties of these
cells; (2) cells of any one of bone, cartilage, tendon and skeletal
muscle, the precursor cells of the cells of any one of bone,
cartilage, tendon, skeletal muscle and adipose tissue, and the
cells having the properties of these cells; (3) neural cells or the
precursor cells of neural cells, and the cells having the
properties of these cells; (4) endocrine cells or the precursor
cells of endocrine cells, and the cells having the properties of
these cells; (5) hematopoietic cells or the precursor cells of
hematopoietic cells, and the cells having the properties of these
cells; and (6) hepatocytes or the precursor cells of hepatocytes,
and the cells having the properties of these cells.
[0075] Methods of mesenchymal cell culture are well known in the
art of cell culturing (see, for example, Friedenstein et al., Exp
Hematol 1976 4, 267-74; Dexter et al. J Cell Physiol 1977,
91:335-44; and Greenberger, Nature 1978 275, 7524). For example,
mesenchymal cells are derived from a source selected from the group
consisting of endothelial cells, cardiac muscle cells, bone cells,
cartilage cells, tendon cells, skeletal muscle cells, bone cells,
cartilage cells, tendon cells, adipose tissue cells, neural cells,
endocrine cells, hematopoietic cells, hematopoietic precursor
cells, bone marrow cells, and the precursor cells thereof,
hepatocytes, and hepatocyte precursor cells.
[0076] The marrow or isolated mesenchymal stem cells can be
autologous, allogeneic or from xenogeneic sources, and can be
embryonic or from post-natal sources. Bone marrow cells may be
obtained from iliac crest, femora, tibiae, spine, rib or other
medullary spaces. Other sources of human mesenchymal stem cells
include embryonic yolk sac, placenta, umbilical cord, periosteum,
fetal and adolescent skin, and peripheral, circulating blood.
Intestinal Stem Cells
[0077] The lining of the intestines is composed of millions of
villi and crypts, which form a barrier against bacterial invasion.
The intestinal epithelium is the most rapidly proliferating tissue
in adult mammals. Intestinal stem cells (ISCs) are responsible for
self-renewal of the epithelium, and also represent a reserve pool
of cells that can be activated after injury. The estimated number
of stem cells is 4-6 per crypt. (Barker et al., Gastroenterology
2007; 133:1755-1760) Stem cells have been proven to be crucial for
the recovery and regeneration of several tissues including the
intestinal epithelium. (Vaananen et al., Ann Med 2005; 37:469-479).
In the past, ISCs were identified at position +4 from the crypt
bottom, directly above the Paneth cells. It is now thought that
there may be two populations of ISCs, a slowly cycling quiescent
reserve population above the Paneth cells (upper stem cell zone,
USZ) (the +4 cells), and a more rapidly cycling (every 24 hours)
active pool of crypt base columnar (CBC) cells located between the
Paneth cells (lower stem cell zone, LSZ). The more active ISCs may
maintain homeostatic regenerative capacity of the intestine with
the more quiescent ISCs held in reserve. (Scoville et al.,
Gastroenterology 2008 136: 849-864) Several signaling pathways
including the Wnt/b-catenin, BMP, RTK/PI3K and Notch cascades are
critical to ISC self-renewal and proliferation. Among them,
Wnt/b-catenin is the signature/signaling pathway, and its
downstream regulated genes represent potential ISC markers. The
Wnt/b-catenin target gene LGR5 has been recently identified as a
marker for CBC ISCs. (Sato et al., Nature 2009; 459:262-265)
Prominin-1 is also expressed in ISC. (Snippert et al.,
Gastroenterology 2009; 136:2187-2194, Zhu et al., Nature 2009; 457:
603-607).
[0078] The integrity of the intestinal epithelium is ensured by
pluripotent, self-renewing and proliferative stem cells. Barker et
al., Gastroenterology 2007; 133:1755-1760, Potten et al., Cell
Prolif 2009; 42:731-750. These cells have only recently been
identified using special markers such as Leucine-rich
repeat-containing G-protein coupled receptor 5 (LGR5) and
prominin-1/CD133, in addition to classic +4 long retention cell
characteristics. Barker et al., Nature 2007; 449:1003-1007,
Snippert et al., Gastroenterology 2009; 136:2187-2194. Between 4
and 6 stem cells at each crypt base generate epithelial progenitor
cells in the transit-amplifying (TA) zone, which subsequently
differentiate and maintain intestinal homeostasis. Barker et al.,
Gastroenterology 2007; 133:1755-1760, Potten et al., Cell Prolif
2009; 42:731-750. They provide a fast-paced renewal of the four
differentiated epithelial cell lineages, each of which has distinct
important physiologic functions: enterocytes that absorb nutrients,
goblet cells that produce protective mucus, Paneth cells that
secrete antibacterial proteins and neuroendocrine cells that
produce hormones. Scoville et al., Gastroenterology 2008;
134:849-864. Stresses such as intestinal ischemia can harm the
intestinal epithelial cell (IEC) lineages, particularly the stem
cells, thereby disrupting normal homeostasis and gut barrier
function. Stem cells in some organs, including the intestines, have
been shown to respond to stress and to promote recovery from
injury. Markel et al., J Pediatr Surg 2008; 43:1953-1963. A
previous study showed that bone marrow-derived progenitor cells
have the ability to regenerate the intestine after injury. Gupta et
al., Biomacromolecules 2006; 7:2701-2709. However, the role of
intestinal stem cells (ISCs) in recovery from NEC is unknown. The
ability to protect ISCs in the face of stress may represent a
critical step in the prevention and treatment of NEC.
[0079] Identification of ISCs and their markers is an active area
of research. In 1998, a G-protein-coupled receptor with 16
leucine-rich repeats was discovered. Additional leucine-rich
G-protein receptors were identified and, ultimately, Hans Clevers
of the Hubrecht Institute demonstrated that cells expressing Lgr5,
located in the crypt base adjacent to the Paneth cells, are
multipotent ISCs [14]. Cell surface markers for ISC include but are
not limited to LGR5 and prominin-1 (Barker et al., Nature 2007;
449:1003-1007, Snippert et al., Gastroenterology 2009;
136:2187-2194, Lee et al., Nat Neurosci 2005; 8:723-729, Zhu et
al., Nature 2009; 457: 603-607, Chen et al., Growth Factors 2010;
28:82-97).
[0080] In the present invention, crypts will be isolated from
transgenic Lgr5EGFP mice, and used as cell source of ISC for the
regeneration of intestinal epithelium. This protocol will deliver a
high percentage of ISC while still maintaining their relationship
with mesenchymal cells.
Neural Stem Cells
[0081] Neural stem cells (NSCs) are stem cells in the nervous
system that can self-renew and give rise to differentiated
progenitor cells to generate lineages of neurons as well as glia,
such as astrocytes and oligodendrocytes.
[0082] NSCs are generated throughout an adult's life via the
process of neurogenesis. Since neurons do not divide within the
central nervous system (CNS), NSCs can be differentiated to replace
lost or injured neurons or in many cases even glial cells. In vivo,
NSCs are differentiated into new neurons within the SVZ of lateral
ventricles, a remnant of the embryonic germinal neuroepithelium, as
well as the dentate gyrus of the hippocampus.
[0083] Adult NSCs were first isolated from mouse striatum in the
early 1990s. They are capable of forming multipotent neurospheres
when cultured in vitro. Neurospheres can produce self-renewing and
proliferating specialized cells. These neurospheres can
differentiate to form the specified neurons, glial cells, and
oligodendrocytes.
[0084] Intracellualar markers for NSC include but are not limited
to galectin-1, nestin, SOX2. Nestin is expressed predominantly in
stem cells of the central nervous system and it is absent from
nearly all mature CNS cells. Cell surface markers for NSC include
but are not limited to ABCG2, MXR, BCRP, ABCP, FGF R4, Frizzled-9,
CD 133 and Musashi 1.
Embryonic Stem Cells
[0085] Embryonic stem cells (ESC) are derived from embryos that
were developed from eggs that have been fertilized using in vitro
fertilization. Procedures for isolating and growing human
primordial stem cells are described in U.S. Pat. No. 6,090,622.
Human embryonic stem cells (hESCs) can be isolated from human
blastocysts obtained from human in vivo preimplantation embryos, in
vitro fertilized embryos, or one-cell human embryos expanded to the
blastocyst stage (Bongso et al., Hum. Reprod. 4:706, 1989). Human
embryos can be cultured to the blastocyst stage in G1.2 and G2.2
medium (Gardner et al., Fertil. Steril. 69:84, 1998). The zona
pellucida is removed from blastocysts by brief exposure to pronase.
The inner cell masses can be isolated by immunosurgery or by
mechanical separation, and are plated on mouse embryonic feeder
layers, or in an appropriate culture system. Inner cell
mass-derived outgrowths are then dissociated into clumps using
calcium and magnesium-free phosphate-buffered saline (PBS) with 1
mM EDTA, using dispase, collagenase, or trypsin, or by mechanical
dissociation with a micropipette. The dissociated cells are then
replated for colony formation. Colonies demonstrating
undifferentiated morphology are individually selected by
micropipette, mechanically dissociated into clumps, and replated.
Embryonic stem cell-like morphology is characterized as compact
colonies with apparently high nucleus to cytoplasm ratio and
prominent nucleoli.
[0086] The ESC may be cultured under conditions that support the
substantially undifferentiation growth of the primordial stem cells
using any suitable cell culture technique known in the art. For
example, the ESCs may be grown on synthetic or purified
extracellular matrix using methods standard in the art.
Alternatively, the ESC may be grown on extracellular matrix that
contains laminin or a growth-arrested murine or human feeder cell
layer (e.g., a human foreskin fibroblast cell layer) and maintained
in a serum-free growth environment.
[0087] Cell surface markers for ESC include, but are not limited
to, alkine phosphatase, CD30, Cripto (TDGF-1), GCTM-2, Genesis,
Germ cell nuclear factor, OCT-4/POU5F1, SSEA-3, SSEA-4, stem cell
factor (SCF or c-kit ligand), TRA-1-60 and TRA-1-81.
Smooth Muscle Cells
[0088] Smooth muscle surrounds the supports of many of the hollow
organs. For example, in the gut smooth muscle surrounds the stomach
and intestinal track. Contraction of this muscle mixes food and
propels it along the digestive track. Smooth muscle lacks the
nearly uniform cell shape and lattice-like distribution of skeletal
and cardiac muscle cells. However, smooth muscle cells do exhibit
an elongated, bipolar cell shape. As a population, smooth muscle
cells are organized along a similar axis in a series of overlapping
cellular layers. This pattern of organization allows smooth muscle
to exert contractile forces in a complex pattern.
[0089] The present invention can be employed using isolated primary
smooth muscle cells or cell lines derived from such primary cells,
tumors and the like. For example, cell lines derived from muscle
may be obtained from a cell line depository such as the American
Type Culture Collection (ATCC, Bethesda, Md.). Such cell smooth
muscle cell lines include human iliac vein smooth muscle cells
(HIVS-125; ATCC accession no. CRL-2482), Syrian Golden Hamster
ductus deferens smooth muscle cells (DDT1; CRL-1701), human umbical
vein smooth muscle cells (HUVS-112D: CRL-2481), rat aorta smooth
muscle cells (Hep-Sa; CRL-2018), and human aortic smooth muscle
cells (T/G HA-VSMC; CRL-2498). The conditions for growth of the
specific cell line purchased will depend on the biological source
and generally instructions for the growth of the cells are made
available along with the cell lines from ATCC. Cell surface markers
for smooth muscle cells include but are not limited to
.alpha.-smooth muscle actin, calponin, SM22 and heavy chain
myosin,
HB-EGF
[0090] The cloning of a cDNA encoding human HB-EGF (or HB-EHM) is
described in Higashiyama et al., Science, 251: 936-939 (1991) and
in a corresponding international patent application published under
the Patent Cooperation Treaty as International Publication No. WO
92/06705 on Apr. 30, 1992. Both publications are hereby
incorporated by reference herein in their entirety. In addition,
uses of human HB-EGF are taught in U.S. Pat. No. 6,191,109 and
International Publication No. WO 2008/134635 (Intl. Appl. No.
PCT/US08/61772), also incorporated by reference in its
entirety.
[0091] The sequence of the protein coding portion of the HB-EGF
cDNA is set out in SEQ ID NO: 1 herein, while the deduced amino
acid sequence is set out in SEQ ID NO: 2. Mature HB-EGF is a
secreted protein that is processed from a transmembrane precursor
molecule (pro-HB-EGF) via extracellular cleavage. The predicted
amino acid sequence of the full length HB-EGF precursor represents
a 208 amino acid protein. A span of hydrophobic residues following
the translation-initiating methionine is consistent with a
secretion signal sequence. Two threonine residues (Thr75 and Thr85
in the precursor protein) are sites for O-glycosylation. Mature
HB-EGF consists of at least 86 amino acids (which span residues
63-148 of the precursor molecule), and several microheterogeneous
forms of HB-EGF, differing by truncations of 10, 11, 14 and 19
amino acids at the N-terminus have been identified. HB-EGF contains
a C-terminal EGF-like domain (amino acid residues 30 to 86 of the
mature protein) in which the six cysteine residues characteristic
of the EGF family members are conserved and which is probably
involved in receptor binding. HB-EGF has an N-terminal extension
(amino acid residues 1 to 29 of the mature protein) containing a
highly hydrophilic stretch of amino acids to which much of its
ability to bind heparin is attributed. Besner et al., Growth
Factors, 7: 289-296 (1992), which is hereby incorporated by
reference herein, identifies residues 20 to 25 and 36 to 41 of the
mature HB-EGF protein as involved in binding cell surface heparin
sulfate and indicates that such binding mediates interaction of
HB-EGF with the EGF receptor.
[0092] As used herein, "HB-EGF product" includes HB-EGF proteins
comprising about amino acid 63 to about amino acid 148 of SEQ ID
NO: 2 (HB-EGF(63-148)); HB-EGF proteins comprising about amino acid
73 to about amino acid 148 of SEQ ID NO: 2 (HB-EGF(73-148)); HB-EGF
proteins comprising about amino acid 74 to about amino acid 148 of
SEQ ID NO: 2 (HB-EGF(74-148)); HB-EGF proteins comprising about
amino acid 77 to about amino acid 148 of SEQ ID NO: 2
(HB-EGF(77-148)); HB-EGF proteins comprising about amino acid 82 to
about amino acid 148 of SEQ ID NO: 2 (HB-EGF(82-148)); HB-EGF
proteins comprising a continuous series of amino acids of SEQ ID
NO: 2 which exhibit less than 50% homology to EGF and exhibit
HB-EGF biological activity, such as those described herein; fusion
proteins comprising the foregoing HB-EGF proteins; and the
foregoing HB-EGF proteins including conservative amino acid
substitutions. In a specific embodiment, the HB-EGF product is
human HB-EGF(74-148). Conservative amino acid substitutions are
understood by those skilled in the art. The HB-EGF products may be
isolated from natural sources known in the art (e.g., the U-937
cell line (ATCC CRL 1593)), chemically synthesized, or produced by
recombinant techniques such as disclosed in WO92/06705, supra, the
disclosure of which is hereby incorporated by reference. In order
to obtain HB-EGF products of the invention, HB-EGF precursor
proteins may be proteolytically processed in situ. The HB-EGF
products may be post-translationally modified depending on the cell
chosen as a source for the products.
[0093] The HB-EGF products of the invention are contemplated to
exhibit one or more biological activities of HB-EGF, such as those
described in the experimental data provided herein or any other
HB-EGF biological activity known in the art. One such biological
activity is that HB-EGF products compete with HB-EGF for binding to
the ErbB-1 receptor and has ErbB-1 agonist activity. In addition,
the HB-EGF products of the invention may exhibit one or more of the
following biological activities: cellular mitogenicity, cellular
chemoattractant, endothelial cell migration, acts as a pro-survival
factor (protects against apoptosis), decrease inducible nitric
oxide synthase (iNOS) and nitric oxide (NO) production in
epithelial cells, decrease nuclear factor-.kappa.B (NF-.kappa.B)
activation, increase eNOS (endothelial nitric oxide synthase) and
NO production in endothelial cells, stimulate angiogenesis and
promote vasodilatation.
[0094] The present invention provides for the HB-EGF products
encoded by the nucleic acid sequence of SEQ ID NO: 1 or fragments
thereof including nucleic acid sequences that hybridize under
stringent conditions to the complement of the nucleotides sequence
of SEQ ID NO: 1, a polynucleotide which is an allelic variant of
any SEQ ID NO: 1; or a polynucleotide which encodes a species
homolog of SEQ ID NO: 2.
Expression of HB-EGF by Cells within the Construct
[0095] The invention provides for transforming or transfecting
somatic stem cells, such as MSC, NSC and ISC, and/or SMC with a
nucleic acid encoding the amino acid sequence of a HB-EGF product.
The transformed somatic stem cells are then administered to a
patient suffering from an intestinal injury in any of the methods
of the invention which results in administration of the HB-EGF
product and the somatic stem cell concurrently.
[0096] A nucleic acid molecule encoding the amino acid sequence of
an HB-EGF product may be inserted into an appropriate expression
vector that is functional in stem cells using standard ligation
techniques. Exemplary vectors that function in somatic stem cells
include bacterial vectors, eukaryotic vectors, plasmids, cosmids,
viral vectors, adenovirus vectors and adenovirus associated
vectors.
[0097] The expression vectors preferably may contain sequences for
cloning and expression of exogenous nucleotide sequences. Such
sequences may include one or more of the following nucleotide
sequences: a promoter, one or more enhancer sequences, an origin of
replication, a transcriptional termination sequence, a complete
intron sequence containing a donor and acceptor splice site, a
sequence encoding a leader sequence for polypeptide secretion, a
ribosome binding site, a polyadenylation sequence, a polylinker
region for inserting the nucleic acid encoding the polypeptide to
be expressed, and a selectable marker element.
[0098] The vector may contain a sequence encoding a "tag", such as
an oligonucleotide molecule located at the 5' or 3' end of the
HB-EGF product coding sequence; an oligonucleotide sequence
encoding polyHis (such as hexaHis), FLAG, hemaglutinin influenza
virus (HA) or myc or other tags for which commercially available
antibodies exist. This tag may be fused to the HB-EGF product upon
expression. A selectable marker gene element encoding a protein
necessary for the survival and growth of a host cell grown in a
selective culture medium may also be a component of the expression
vector. Exemplary selection marker genes include those that encode
proteins that complement auxotrophic deficiencies of the cell; or
supply critical nutrients not available from complex media.
[0099] A leader, or signal, sequence may be used to direct the
HB-EGF product out of the stem cell after administration. For
example, a nucleotide sequence encoding the signal sequence is
positioned in the coding region of the HB-EGF product nucleic acid,
or directly at the 5' end of the HB-EGF coding region. The signal
sequence may be homologous or heterologous to the HB-EGF product
gene or cDNA, or chemically synthesized. The secretion of the
HB-EGF product from the stem cell via the presence of a signal
peptide may result in the removal of the signal peptide from the
secreted HB-EGF product. The signal sequence may be a component of
the vector, or it may be a part of the nucleic acid molecule
encoding the HB-EGF product that is inserted into the vector.
[0100] The expression vectors used in the methods of the invention
may contain a promoter that is recognized by the host organism and
operably linked to the nucleic acid sequence encoding the HB-EGF
product. Promoters are untranscribed sequences located upstream to
the start codon of a structural gene that control the transcription
of the structural gene. Inducible promoters initiate increased
levels of transcription from DNA under their control in response to
some change in culture conditions, such as the presence or absence
of a nutrient or a change in temperature. Alternatively,
constitutive promoters initiate continual gene product production
with little or no control over gene expression. A large number of
promoters, recognized by a variety of potential host cells, are
well known. A suitable promoter is operably linked to the nucleic
acid molecule encoding the HB-EGF product. The native HB-EGF gene
promoter sequence may be used to direct amplification and/or
expression of a HB-EGF product nucleic acid molecule. A
heterologous promoter also may be used to induce greater
transcription and higher yields of the HB-EGF product expression as
compared to HB-EGF expression induced by the native promoter.
[0101] In addition, an enhancer sequence may be inserted into the
vector to increase the transcription of a DNA encoding the HB-EGF
product. Enhancers are cis-acting elements of DNA, usually about
10-300 bp in length, that act on the promoter to increase
transcription. Enhancer sequences available from mammalian genes
include globin, elastase, albumin, alpha-feto-protein and insulin.
Exemplary viral enhancers that activate eukaryotic promoters
include the SV40 enhancer, the cytomegalovirus early promoter
enhancer, the polyoma enhancer, and adenovirus enhancers. While an
enhancer may be spliced into the vector at a position 5' or 3' to a
nucleic acid molecule encoding the HB-EGF product, it is typically
located at a site 5' from the promoter.
[0102] The transformation of an expression vector encoding a HB-EGF
product into a stem cell may be accomplished by well-known methods
such as transfection, infection, calcium chloride, electroporation,
microinjection, lipofection or the DEAE-dextran method or any other
technique known in the art. These methods and other suitable
methods are well known in the art, for example, in Sambrook,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press; 3rd ed., 2001.
BRIEF DESCRIPTION OF DRAWING
[0103] FIG. 1 depicts in vitro degradation rate. The percent of
weight loss for each scaffold over 12 weeks of incubation in
stimulated intestinal fluid. Only PGA-nanofiber displays
significant weight loss over this period.
[0104] FIG. 2 depicts the scaffold implantation in vivo. Panel A is
a schematic of scaffold orientation in vivo. Panel B) depicts the
abdominal wall after harvesting at 4 weeks showing scaffolds
secured in their location against the underside of the abdominal
wall. Poly(glycolic acid)-macrofiber (PGA-M), Poly(glycolic
acid)-nanofiber (PGA-N), Poly(-caprolactone-co-lactic
acid)-nanofiber (PLC-N), Poly(-caprolactone)-nanofiber (PCL-N),
Poly(D-lactic acid-co-glycolic acid) (PDLGA-N) Poly(L-lactic
acid)-nanofiber (PLLA-N), and Polyurethane-nanofiber (PU-N).
[0105] FIG. 3 depicts histologic examination of scaffolds.
Representative photomicrographs of H&E stained sections of each
of the 7 scaffolds A-G) 1 week and H-N) 4 weeks after implantation.
A,H) PGA-nanofiber: significant tissue infiltration begins at 1
week and no fibers are visible at 4 weeks; B,I) PGA-macrofiber:
significant tissue infiltration is visible starting at 1 week; some
fibers remain visible at 4 weeks; C,J. PCL-nanofiber: significant
tissue infiltration and retained fiber structure are visible at 4
weeks; D,K) PLC-nanofiber: minimal tissue infiltration and minimal
degradation; E,L) PLLA-nanofiber: almost no tissue infiltration and
minimal degradation; F,M) PDLGA-nanofiber: less tissue infiltration
at 1 week but rapid degradation by 4 weeks; G,N. PU-nanofiber: some
tissue infiltration and no fiber degradation at 4 weeks.
[0106] FIG. 4 depicts in vivo degradation rate, Weight loss over a
12 week incubation period after intra-peritoneal implantation. Both
the PGA and PDLGA nanofiber as well as the PGA macrofiber display
significant weight loss over the period.
[0107] FIG. 5 provides scanning electron microscopic examination of
scaffolds. SEM images (500.times.) of scaffolds A-G) prior to
implantation and H-N) 4 weeks days after implantation. A,H)
PGA-nanofiber; B,I) PGA-macrofiber; C,J) PCL-nanofiber; D,K)
PLC-nanofiber; E,L) PLLA-nanofiber; F,M) PDLGA-nanofiber; and G,N)
PU-nanofiber. Insets show gross scaffold appearance.
[0108] FIG. 6 depicts tensile strength measurements for native
small bowel, and for scaffolds both prior to and after 3 weeks of
implantation. A) ultimate tensile strength; B) percent elongation;
C) Young's modulus. SB, small bowel; M, macrofiber; N,
nanofiber.
[0109] FIG. 7 depicts suture retention strength of the implanted
scaffolds. Suture strength measurements (in Newtons) for native
small bowel and for scaffolds prior to and after 3 weeks of
implantation. SB, small bowel; M, macrofiber; N, nanofiber.
[0110] FIG. 8 depicts the filtration system used to enrich
intestinal stem cells in crypts. This system consists of a bottom
filter, top cups, sieve membranes, and cell scraper (a). The
smallest size (8 .mu.m) has been loaded first and placed on the top
of base filter cup (b). The second top cup has been placed on the
base filter cup to secure the sieve membrane (c). Similarly, all
other sizes of sieve membranes have been loaded in order from
smaller to larger pore sizes, and a vacuum tube is connected
[0111] FIG. 9 depicts localization of ISC in filtered
fractions.
[0112] FIG. 10 depicts the appearance of TEI produced from
ISC-enriched compared to non-enriched seeding.
[0113] FIG. 11 depicts the effect of ISC-enriched cell seeding on
mucosa production.
[0114] FIG. 12 depicts the scheme used to seed cells in the PGA
scaffolds.
[0115] FIG. 13 depicts the HB-EGF release kinetics from the PGA
scaffolds.
[0116] FIG. 14 depicts the biopotency of the HB-EGF released from
the PGA scaffold.
[0117] FIG. 15 depicts the histology of the native intestine
compared to the TEI of the invention. TEI produced from in vivo
incubation of crypt-seeded scaffolds (panels b-f) was
histologically very similar to that of native intestine (panel a).
PAS staining, V=villi, C=crypts, SM=smooth muscle, scale bar=200
.mu.m
[0118] FIG. 16 depicts the villous height of native intestine
compared to the TEI of the invention. HB-EGF infusion of scaffolds
led to TEI with increased villous height. The villous height of
native intestine was 212.9.+-.16.8 .mu.m. no HB-EGF 88.7.+-.32.3
.mu.m; HB-EGF (1 .mu.g) 101.8.+-.32.5 .mu.m; HB-EGF (1 .mu.g)+CO2
infusion 186.0.+-.23.4 .mu.m; HB-EGF (10 .mu.g) 222.6.+-.34.6
.mu.m; HB-EGF (10 .mu.g)+CO2 infusion 406.34.+-.37 .mu.m.
DETAILED DESCRIPTION
[0119] Example 1 describes the preliminary studies that analyzed
different scaffold materials. Example 2 describes scaffold
fabrication. Example 3 describes in vitro characterization of the
nanofiber scaffolds. Example 4 describes cell seeding of the
nanofiber scaffolds and Example 5 describes biological
characterization of nanofiber scaffolds. Example 6 describes a cell
filtration system to enrich intestinal stem cells in crypts.
Example 7 describes HB-EGF incorporation into PGA enhances the
formation of tissue engineered intestine.
EXAMPLES
Example 1
Analysis of Scaffold Materials
[0120] This detailed evaluation of the numerous potential scaffold
materials was carried out to determine potential scaffold materials
for the construction of a multilayer nanofiber scaffold for use in
generating engineered intestine constructs. The purpose of this
study was to characterize seven different scaffold materials
according to degradation rates, histologic changes, and tensile
strength to determine which would be best suited for the production
of the engineered intestine constructs.
[0121] Initially, the seven different single tube scaffolds were
fabricated using electrospinning as described in Example 2 and
above. These scaffolds were comprised of poly(glycolic acid)(PGA)
nanofibers, Poly(-caprolactone) (PCL) nanofibers,
Poly(-caprolactone-co-lactic acid) (PLC) nanofibers, Poly(L-lactic
acid) (PLLA), Poly(D-lactic acid-co-glycolic acid) (PDLGA),
Polyurethane (PU) nanofibers and PGA macrofibers. The physical and
chemical characteristics of the nanofiber scaffolds are provided in
Table 2.
TABLE-US-00002 TABLE 2 Characteristics of Polymers Tensile modulus
Tensile Elongation Degradation Density Melting of elasticity
strength at break time Polymer Molecular Formula (g/cm.sup.3) point
(GPa) (MPa) (%) (months) PGA (C.sub.2H.sub.2O.sub.2).sub.n 1.5
225-230.degree. C. 6.5-7.0 90-110 1-2 6-12 PCL
(C.sub.6H.sub.10O.sub.2).sub.n 1.2 60.degree. C. 0.2-0.3 25-35
>300 >24 PLC
((C.sub.3H.sub.4O.sub.2)--(C.sub.6H.sub.10O.sub.2)).sub.n 1.25
g/cm.sup.3 110-120.degree. C. 0.02-0.04 18-22 >100 12-24 PLLA
(C.sub.3H.sub.4O.sub.2).sub.n 1.3 150-160.degree. C. 3.1-3.7 60-70
2-6 >24 PDLGA
((C.sub.3H.sub.4O.sub.2)--(C.sub.2H.sub.2O.sub.2)).sub.n 1.4
amorphous 3.4-3.8 40-50 1-4 1-2 PU
((C.sub.16H.sub.14O.sub.3).sub.x--(C.sub.15H.sub.14O.sub.2)).sub.n
1.2 180.degree. C. 0.03 45-50 >500 biostable
[0122] Prior to implantation, the in vitro degradation of the
scaffolds was analyzed in simulated intestinal fluid (SIF). The
degradation rate of each scaffold type was assessed by weekly
measurements of change in scaffold weight over the 12-week
incubation period. The PGA-nanofiber was the only composition that
underwent significant, measurable change and showed complete
degradation by week 8. All other materials displayed little weight
change. Several of the weekly measurements suggested a slight
weight gain (see FIG. 1). This was attributable to solute from SIF
solution that remained trapped in the nanoscaled scaffold even
after multiple rinses.
In Vivo Studies
[0123] The scaffolds were sterilized and maintained at -20.degree.
C. until implantation. PGA-nanofiber, PGA-macrofiber,
PLLA-nanofiber and PU-nanofiber scaffolds were sterilized via
exposure to hydrogen peroxide gas (Sterrad). PC-nanofiber and
PLC-nanofiber scaffolds were sterilized via immersion in 70%
ethanol solution for 30 minutes. The sterilized scaffolds were
implanted onto the interior surface of the abdominal wall of adult
Lewis rats.
[0124] Under general anesthesia with inhalation of isofluorane, a
midline laparotomy was performed and a 1 cm length of each of the
seven scaffolds was secured to the anterior abdominal wall of the
peritoneal cavity using 5-0 polypropylene suture. Three scaffolds
were placed on either side of the midline and one in the pelvis
(FIG. 2A). Each scaffold was secured with two 5-0 polypropylene
sutures passed through the lumen of the scaffold and then secured
to the fascia. Animals were euthanized by CO.sub.2 asphyxiation and
scaffolds harvested at each of 6 time points (1, 2, 3, 4, 8 and 12
weeks) (Figure) were used for histological evaluation (n=3), weight
changes and SEM examination (n=3).
[0125] Upon harvesting, these scaffolds were also tested for
ultimate tensile strength, elongation, and modulus. Modulus (Young
modulus) was measured as the ratio of the uniaxial stress over the
uniaxial strain in the range of stress in which Hooke's Law holds.
Elongation was measured as the percentage of original dimensions.
Furthermore, ultimate tensile strength (UTS) was measured within
the limitations inherent to standard tensile evaluation of
component properties at these scales.
Histology
[0126] The scaffolds were subjected to H&E staining and
examined histologically at 1 week and 4 weeks after implantation.
Scaffolds were harvested en bloc, cut in a cross-sectional fashion
across the center of the scaffold, fixed in 10% neutral buffered
formalin and embedded in paraffin. Three sections were obtained
from each of three levels at 200 .mu.m intervals, deparaffinized in
Americlear (Cardinal Health, Dublin, Ohio), and stained with
hematoxylin and eosin (H&E) dye. Slides were examined and
assessed microscopically. The histologic examination is depicted in
FIG. 3.
[0127] Histologically, PGA-nanofiber scaffolds had both significant
tissue infiltration as well as fiber degradation at early time
points (1 and 2 weeks) with no fibers left at 4 weeks. There was
marked tissue reaction with granulomatous inflammation at 2 weeks
post-implantation, with numerous macrophages and a few foreign body
giant cells. A reduction in the inflammatory reaction was observed,
as the fibers were absorbed at 4 weeks post-implantation.
[0128] PGA-macrofiber scaffolds also had significant tissue
infiltration at early time points, but maintained structural
integrity longer (some fibers still visible at 4 weeks). Fiber
degradation was observed beginning at 21 days post-implantation.
There was marked foreign body reaction at 2 weeks post-implantation
again characterized by numerous foreign body giant cells and
macrophages. Fibrosis located within the midpoint of the scaffold
wall was observed at 2 weeks.
[0129] PCL-nanofibers had slower tissue infiltration that became
more prominent at 2 to 3 weeks, and maintained structural integrity
after 4 weeks. PLC-nanofibers had minimal degradation and poor
tissue infiltration. There was foreign body reaction by 2 weeks
post-implantation and fibrosis was observed starting at 3 weeks,
and remained visible up to 12 weeks post-implantation.
[0130] PLC-nanofiber scaffolds showed poor tissue infiltration. The
tissue reaction was characterized generally by chronic inflammation
and fibrosis. Mild chronic inflammation was also present at 4, 8
and 12 weeks.
[0131] PLLA also had slower tissue infiltration that did not occur
until at least 3 weeks post-implantation. The tissue reaction was
characterized by mild chronic inflammation present throughout all
time points; marked fibrosis was observed beginning at 3 weeks
post-implantation. Fibers remained visibly intact up to 12 weeks
post-implantation.
[0132] PDLGA-nanofibers underwent slightly slower tissue
infiltration (present at 2 weeks) but rapid structural loss at 3 to
4 weeks. The tissue reaction was characterized by inflammation and
fibrosis, both of which were mild at 1-2 weeks post implantation
and more chronic at 3-4 weeks. This was followed by a reduction in
the inflammatory reaction as fibers were absorbed beginning at 4
weeks post-implantation. Degradation of fibers was visible at 1
week; fibers were essentially completely absorbed by 4 weeks
post-implantation.
[0133] PU-nanofibers had tissue infiltration at 3 to 4 weeks but
maintained structural integrity at all time points. The tissue
reaction was characterized generally by chronic inflammation and
fibrosis. The chronic tissue reaction transitioned to a foreign
body reaction and fibrosis at 4 weeks post-implantation. Visibly
undamaged fibers were present up to 12 weeks post-implantation.
In Vivo Degradation after Peritoneal Implantation
[0134] In addition, PGA-macrofiber and PDLGA caused the least
amount of tissue reaction at and around the implant sites compared
to the other materials. Scaffolds were cut into 4-6 pieces and
placed into 4 ml of 5% sodium hypochlorite (Sigma-Aldrich, St
Louis, Mo.) diluted with phosphate buffered saline (PBS), to remove
in-growth tissues. After digestion of adherent biological tissue in
sodium hypochlorite, the scaffolds were rinsed five times in
distilled water and freeze dried overnight. Each sample was then
weighed to determine the amount of scaffold degradation as assessed
by the change in weight pre-implantation. Each of the seven
scaffold materials were examined in triplicate at each time point
and the percentage of weight loss calculated.
[0135] Significant weight loss was identified for PGA-nanofiber
(92.2%.+-.9.3%), PGA-macrofiber (67.6%.+-.28.8%), and PDLGA
76.9%.+-.31.0%) scaffolds as opposed to PU-nanofiber
(1.5%.+-.3.4%%), PCL-nanofiber (10.7%.+-.20.6%%), PLC-nanofiber
(9.4%.+-.11.8%) and PLLA-nanofiaber (7.6%.+-.5.7%) (all with
p<0.05) when combining the percent weight loss for scaffolds at
all time pointes. See FIG. 4. In addition to having significantly
higher weight loss, PGA-nanofiber, PDLGA-nanofiber, and
PGA-macrofiber had the bulk of the weight loss during the earlier
time points, when compared to the other scaffold materials.
Scanning Electron Microscopy (SEM)
[0136] Scanning electron microscopy was also performed for each of
the scaffolds at each time point. See FIG. 5. Individual fibers
were indistinguishable for all PLC scaffolds, for PGA-nanofiber
scaffolds after 1 week, for PGA-macrofiber scaffolds at 2 weeks,
and for PDLGA-nanofiber scaffolds at 4 weeks. Little change in
fiber size, structure, or pore size was seen in PCL-nanofiber,
PLLA-nanofiber and PU-nanofiber scaffolds. PDLGA-nanofiber
scaffolds underwent significant microstructural changes including
increased pore size and individual fiber breakage at 2 weeks.
[0137] The percent weight loss compared to the baseline weight of
each of the seven scaffolds at 1 week, 2 weeks, 3 weeks, 4 weeks, 8
weeks and 12 weeks is determined from e SEM images. A portion of
each of the samples used for scaffold degradation studies was gold
sputtuer-coated (Emitech K550X, Quorum Technologies Ltd, Ashford,
Kent, England) and examined by SEM (Hitachi S-4800, Hitachi High
Technologies America, Inc., Dallas, Tex.) at a voltage of 7 kV at
100, 500, and 1000.times. magnification. Measurements of fiber
diameter were taken from the SEM micrographs at random locations at
500.times. magnification using Image J software (National
Institutes of Health, Bethesda, Md.) from three different scaffold
samples representing each time point. PGA-nanofiber had the most
rapid degradation, followed by PGA-macrofiber and PDLGA-nanofiber.
PCL-nanofiber, PLC-nanofiber, PLLA-nanofiber had much slower
degradation. PU-nanofiber had no significant degradation even at 12
weeks.
[0138] SEM was performed for each of the scaffolds at each time
point. Fiber width was measured from SEM images. Individual fibers
were not distinguished for PLC-nanofiber scaffolds at any time,
PGA-nanofiber at 1 week, PGA-macrofiber at 2 weeks, and
PDLGA-nanofiber at 4 weeks. Little change in fiber size,
structures, or pore size was seen in PCL-nanofiber, PLLA-nanofiber
and PU-nanofiber scaffolds. PGA-macrofiber had an increase in fiber
size at 3 weeks followed by subsequent decline. PDLGA-nanofiber
scaffolds underwent significant changes including increased pore
size and individual fiber breakage at 2 weeks.
Tensile Strength
[0139] Tensile strength measurements were taken for native small
intestine, as well as for each of the six scaffold materials
(PGA-macrofiber, PCL-nanofiber, PLLA-nanofiber, and PU-nanofiber)
at baseline and after 3 weeks of intra-abdominal implantation (FIG.
6) and after 3 weeks of intra-abdominal implantation (FIG. 6).
PGA-nanofiber scaffolds were not used for testing of tensile
strength due to near complete degradation at the 3 week time point.
There is a statistically significant reduction in ultimate tensile
strength (UTS) after implantation compared to baseline for
PGA-macrofiber (p<0.001), PCLnanofiber (p=0.001),
PDLGA-nanofiber (p<0.001), and PU-nanofiber (p=0.01). There was
also a statistically significant reduction in percent elongation
after implantation compared to baseline for PGA-macrofiber
(p=0.002), PCL-nanofiber (p=0.003), PDLGA-nanofiber (p=0.018), and
PU-nanofiber (p<0.001). There was a statistically significant
reduction in Young's modulus after implantation compared to
baseline for PGA-macrofiber (p<0.001), PCL-nanofiber
(p<0.001), and PDLGA-nanofiber (p=0.003).
[0140] Ultimate tensile strength is the highest point on the
stress-strain curve, and represents the maximum amount of stress
that a material can withstand before breaking or failing. Young's
modulus, on the other hand, is the linear portion of the
stress-strain curve, and corresponds to the ability of the scaffold
to withstand alterations in length when exposed to tension. These
factors are critical to the evaluation of our scaffolds as they
determine how the scaffold would respond to a bolus of food or
peristalsis compared to the surrounding native small bowel. It has
been shown that deposited ECM and tissue infiltration can have
significant effects on the tensile properties of these nanofiber
scaffolds (Johnson et al., J. Appl. Polymer Sci. 104(5):2919-2927,
2007; Johnson et al., J. Biomat. Sci.--Polymer Ed. 20(4):467-481,
2009). The mechanical response of the scaffolds depends upon the
rearrangement and alignment of the nanofibers in the direction of
strain and the biological milieu can prohibit that fiber
rearrangement.
[0141] In terms of tensile strength and suture retention testing,
all scaffolds initially displayed equal or better strength and
suture retention strength (see below) than the native small bowel.
PLLA-nanofiber and PDLGA-nanofiber were much stiffer than the other
scaffolds. The percent elongation was not statistically different
as compared to the small bowel due to the relatively low strength
of the small bowel compared to the PLLA-nanofiber and
PDLGA-nanofiber scaffolds. This lack of stiffness has some benefit,
however, in that it can help to maintain structural architecture
during the formation of new tissues (Lee et al., Biomed. Mater.
8(1): 0101201, 2012). After 3 weeks of implantation, PGA-macrofiber
and PDLGA-nanofiber most closely resembled the mechanical
characteristics of small intestine, with PCL-nanofiber being the
next closest in regards to mechanical characteristics of the small
intestine.
[0142] In addition, the mechanical properties of native small
intestine were compared to each of the scaffolds at 3 weeks; no
statistically significant differences in UTS were apparent. There
was a statistically significant difference between percent
elongation and PU-nanofiber compared to native small bowel, as well
as each of the other 5 scaffolds (p<0.03), but no significant
differences between the other scaffolds and the native small bowel.
PLLA-nanofiber and PLC-nanofiber had significantly higher Young's
modulus when compared to native small bowel (p=0.003), but no other
significant differences could be identified.
Suture Retention
[0143] Suture retention strength (SRS) was evaluated for native
small intestine and each of 6 scaffolds at baseline and after 3
weeks of intra-abdominal implantation (FIG. 7). PGA-nanofiber
scaffolds were not used for SRS testing due to nearly complete
degradation at the 3-week time point. Maximum force was calculated
in Newtons for the small intestine samples as well as for each of
the samples. There was a statistically significant reduction in SRS
after 3 weeks of implantation for PGAmacrofiber (p<0.001),
PDLGA-nanofiber (p<0.001), and PU-nanofiber (p=0.02).
[0144] The maximum force for each scaffold after 3 weeks of
implantation was also compared to that of the small intestine
samples. PLC-nanofiber, PLLA-nanofiber, and PU-nanofiber all had
significantly higher SRS than native small bowel following 3 weeks
of intra-abdominal implantation (p<0.05 for each). There was no
statistically significant difference between native small intestine
and the other 3 scaffolds after 3 weeks of implantation
(PGA-macrofiber, PCL-nanofiber, and PDLGA-nanofiber) (FIG. 7).
[0145] This analysis of the seven scaffolds indicated that
PGA-macrofiber and PDLGA appear to be the most appropriate
scaffolds for the production of tissue engineered intestine due to
their degradation in approximately 3 weeks and their
biocompatibility.
[0146] In this specific environment, the PDLGA-nanofiber and
PLLA-nanofiber scaffolds appear to strike the appropriate balance
of properties needed to maintain structural integrity while
allowing for the appropriate rate of tissue replacement of the
synthetic scaffold. However, PDLGA-nanofiber appeared to be more
biocompatible displaying a minimal foreign body response and a more
ideal degradation rate. PU-nanofiber and PLC-nanofiber are much
less ideal in this context due to their longer degradation rates,
decreased porosity, and ongoing foreign body response.
PCL-nanofiber causes significant tissue reaction but slower
degradation making it appear less ideal. PGA-nanofiber scaffolds
degraded too rapidly and did not allow sufficient ECM production,
making it difficult to completely assess the state of the scaffold,
as we were unable to test the characteristics of this scaffold.
Finally, PGA-macrofiber causes significant tissue reaction but does
have a more ideal degradation rate and tensile strength compatible
with the production of TEI.
Example 2
Scaffold Fabrication
[0147] A multilayer nanofiber scaffold was fabricated for
generation of an engineered intestine constructs. Multilayer
scaffolds were constructed in order to facilitate the delivery of
cells of different sizes, e.g. neural stem cells, smooth muscle
cells and crypt cells. In addition, the multilayer scaffold allows
for different mechanical properties within the scaffold, allows for
a smooth lumen and allows for separation of different cell types
which allowed for the generation of different types of tissue.
[0148] To seed the scaffolds with cells, the scaffolds are coated
onto a cell culture plate for three dimensional cell culture. Human
smooth muscle cells are plated and within these cultures the cells
migrated along the nanofibers after 5 days in culture. The
migration of the smooth muscle cells (SMC) demonstrates that upon
seeding of smooth muscle cells into a circumferentially or
longitudinally aligned nanofiber tubular layer, the SMCs will
align, orientate, migrate, and proliferate along the nanofibers to
form muscularis interna and externa. Tubular nanofiber scaffolds
are fabricated with PCL and have similar physical properties to
native. By controlling the size of the fibers and pores, cell
clusters (crypts) as well as individual cells can be seeded on
separate layers.
[0149] Based on the modulus test described above and in vitro and
in vivo degradation studies PGA-macrofiber and PDLGA are good
polymers for fabrication of electrospun multilayer nanofiber
scaffold. An exemplary multilayer nanofiber scaffold structure is
as follows: outer layer of PDLGA, a layer of PCL, a middle layer of
PDLGA, a layer of macrofiber PGA and an internal layer of
PDLGA.
[0150] Polymer solutions were prepared by dissolving polymers in an
optimal amount of organic solvent mixture and mixing by a magnetic
stir bar overnight (12 hours). The solvent type used and the
concentration of each polymer solution prepared for electrospinning
were optimized to achieve the desired fiber diameter and scaffold
porosity in order to promote cell attachment and infiltration
through the scaffold. A pore size gradient was applied through the
sidewall starting from the innermost layer with the biggest pores
(100-300 pm) and ending at the outermost layer with the smallest
pores (50-100 pm). This pore size gradient is designed for loading
crypts (cell clusters) and a mixture of MSCs and NSCs separately in
order to generate mucosa internally surrounded by smooth muscle/ENS
externally.
[0151] Briefly, a 5 wt % solution of biodegradable polymer, such as
PDLGA, PLC or PCL in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), was
prepared by continuous stirring at room temperature overnight to
dissolve the polymer. This solution was then placed in a 60 ml
syringe with a 20 gauge blunt tip needle and electrospun using a
high voltage DC power supply (Glassman High Voltage, Inc., High
Bridge, N.J.) set to +16 kV, a 20 cm tip-to-substrate distance and
a 5 ml/hr flow rate. The fiber was deposited onto a rotating 4.76
mm diameter stainless steel rod until the desired wall thickness
was achieved. The arrangement of fibers was controlled as nonwoven
in the innermost layer, circumferential alignment in the middle
layer, and longitudinal alignment in the outermost layer, in order
to recapitulate the natural alignment of native intestinal smooth
muscle layers. The thickness ratio of the three layers was 100 pm
(outer layer), 200 pm (middle layer), and 200 pm (inner layer) for
a total sidewall thickness of 0.5 mm, based on the anatomic
structure of native intestine. The scaffold tubes were then removed
from the rod, heated, and placed under vacuum to ensure removal of
residual organic solvent. Finally, the scaffold tubes were plasma
treated, which increases the surface energy and promotes cellular
attachment to the fibers.
[0152] HB-EGF is a potent intestinal protective agent that promotes
intestinal epithelial cell (IEC) and SMC proliferation and
migration. Therefore, to accelerate and mature tissue formation in
vitro and in vivo, HB-EGF was embedded on the scaffold nanotubes
via subcritical CO.sub.2 infusion. The dose of HB-EGF to be used
for each 5 cm long scaffold was calculated based on the dosages per
surface area efficacious in our multiple animal models of
intestinal injury. For example, 40 pg of HB-EGF is typically
administered enterally to rat pups over five days, with an average
length of intestine of 18 cm and diameter of 2 mm. This equates to
a dose of HB-EGF per surface area of 1.77 pg/cm.sup.2, which is
equivalent to 15.7 pg of HB-EGF in a 5 cm long scaffold tube. These
amounts were used as a reference range for the coating of scaffold
tubes. Different scaffold tubes are uniformly hydrated with 1, 10,
or 100 pg of HB-EGF in DPBS buffer and then placed into a chamber
in which the pressure is maintained at 900 psi via 002 for 1 hour
followed by gradual pressure release at 90 psi per minute.
HB-EGF-embedded compared to non-HB-EGF-embedded scaffolds then
underwent in vitro and in vivo characterization.
Example 3
In Vitro Characterization of the Nanofiber Scaffolds
[0153] A total of seven nanofiber scaffolds (PCL, PLC, PCL-FHB-EGF,
PLC-FHB-EGF) are fabricated as described in Example 1 and each of
these scaffolds are analyzed using the following in vitro
characterizations. Any nanofiber scaffold of the invention will be
characterized using one or more of the following analyses.
Scanning Electron Microscopy (SEM)
[0154] Scaffolds are sputter coated with gold and then observed
under a scanning electron microscope at an accelerated voltage of
15 kV. Fiber and pore size of the inner and outer layers are
measured using Image J software and continuity between pores are
assessed. Fiber and pore size between HB-EGF-coated and noncoated
scaffolds are compared using Student t-test, with p<0.05
considered statistically significant. The scanning electron
microscopy studies provide guidance on how modify the fiber size
and pore size to best accommodate the cells to be seeded into the
scaffold.
Modulus Determination
[0155] Tensile properties are determined utilizing a 1-kg load cell
(Model 31, Sensotec) and a strain rate of 50 mm/min on an Instron
load frame using a lightweight carbon fiber. The nanofiber tubes
are cut into 5 cm lengths, and the same lengths of rat intestine
are prepared. Grips will be modified to incorporate 80-grit
sandpaper affixed with heavy-duty double-sided tape to securely fix
scaffold tubes during tensile testing. All scaffolds are weighed
and their width and thickness in flat state measured with a digital
micrometer prior to testing. After mounting, the gauge length of
samples are measured and a small tare load applied (-0.05 lbs) to
ensure proper seating. Ten sinusoidal pre-conditioning cycles are
then carried out to 1% of the gauge length at a strain rate of
0.1%/s. After pre-conditioning, a constant strain of 0.1%/s will be
applied until sample failure or 50% strain are achieved. In cases
where samples do not fail, the non-recoverable deformation will be
assessed by releasing the applied deformation until the measured
load becomes negligible at equilibrium. The Young's modulus of
samples in tension are calculated from the slope of the
stress-strain curve in the linear region (i.e., below the yield
stress) and the initial sample geometry. Yield stress and yield
strain for each sample are determined from the intersection of the
experimental data with a line parallel to the linear region of the
stress strain curve and offset by +0.2% strain. Comparisons between
the nanofiber scaffolds and native intestine are conducted using
one-way ANOVA, with p<0.05 considered statistically
significant.
Suture Retention Strength
[0156] Suture retention strength (SRS) are used to measure the
force necessary to pull sutures through the wall of the material
being tested. Nanofiber scaffolds and rat intestine (n=5 each) are
cut into 5 cm lengths, and three silk sutures (6.0 Ethicon with a
cutting edge needle) are inserted 2 mm from the end at 90.degree.
angles, looped, and tied with seven knots. The suture loop and the
other end of the tube are secured to the grips of the tensile
machine using a 22.7-kg (50 lbs) load cell and pulled at 50 mm/min
until the suture pulls through the sample wall. The maximum force
required is the SRS. Comparisons between nanofiber scaffolds and
native intestine are conducted using one-way ANOVA, with p<0.05
considered statistically significant.
Degradation in Simulated Intestinal Fluid (SW)
[0157] In vitro degradation studies in simulated intestinal fluid
are conducted in 2 ml filter tubes with 0.22 .mu.m pore size
filters at the bottom. Nanofiber scaffolds are cut into 0.5 cm
segments and the outside diameter and sidewall thickness are
measured and recorded. Segments of each material area added into
individual tubes followed by the addition of 1.8 ml of SIF, which
is prepared according to US Pharmacopeia. Tubes are mounted on a
rotating system and were kept rotating for 1 hour. The SIF is
completely removed, and the tube together with the scaffold were
weighed, which is used as the Day 0 baseline weight. After all
samples are weighed, 1.8 ml of fresh SIF are added to each tube
followed by continuous rotating until the next time point of sample
collection. Samples are collected every 24 hours followed by
complete SIF exchange. The percentage of weight loss are calculated
and compared using two-way ANOVA (Fisher's LSD method), with
p<0.05 considered statistically significant.
[0158] HB-EGF release kinetics HB-EGF coated nanofiber scaffolds
are cut into 0.5 cm length segments, completely submerged in 2 mL
of DPBS solution in filter tubes (molecular weight cutoff 3,000),
and incubated at 37.degree. C. on a shaker. On days 1, 4, 7, 10,
14, 17 and 21, samples are transferred to new filter tubes and the
supernatant in the original tube is spun down to concentrate
HB-EGF, which were quantified by ELISA. Cumulative HB-EGF release
are calculated and compared between different scaffolds using
two-way ANOVA with Fisher's LSD method, with p<0.05 considered
statistically significant.
HB-EGF Biopotency
[0159] To examine the biological activity of released HB-EGF, a
cell proliferation assay are performed using NIH 3T3 cells.
Briefly, 10,000 cells are added into each well of a 96-well plate
in 150 pl of Assay Medium [(DMEM/F-12 containing 1% fetal bovine
serum (FBS) and 0.5% bovine serum albumin (BSA)]. Released HB-EGF
is diluted to a detectable range based on the concentration
obtained by ELISA. Serial dilutions of the HB-EGF samples are
prepared, with a total of 12 dilutions needed to establish standard
curves. After 4 hours of culture, each cell-seeded well receive 100
pl of either released HB-EGF sample or standard. All samples are
assayed in duplicate. After 48 hours, 5-bromo-2'-deoxyuridine
(BrdU) incorporation is assessed to determine after compared with
HB-EGF standard. Comparison of HB-EGF biopotency are conducted
using Student's West, with p<0.05 considered statistically
significant.
Example 4
Cell Seeding of the Nanofiber Scaffolds Biological Characterization
of Nanofiber Scaffolds
Isolation of Crypts, Smooth Muscle Cells, and Neural Stem Cells
[0160] Crypts containing stem cells are isolated from 6-7 day old
Lgr5-EGFP transgenic (TG) mice on a C57BL/6 background described in
Chen et al. (Lab. Invest. 2012; 92:331-44), and are quantified
using hemocytometry with Trypan blue. Lgr5-EGFP TG mice were used
since these mice have been genetically engineered so that their
native intestinal stem cells are fluorescently labeled (Barker et
al., Nature 2007; 449:1003-7), which allows for tracking of the
intestinal stem cells (ISC)s by fluorescent microscopy. Crypts are
used immediately after isolation for in vitro characterization and
in vivo intestine formation. Neural stem cells (NSCs) are isolated
from fetal intestine at E11.5. Embryos are removed via C-section
followed by harvesting of the intestines. Small and large bowel are
dissected, minced and digested with collagenase (0.5 mg/ml) and
dispase (0.5 mg/ml) for 60 minutes at 37.degree. C. Cells will be
triturated through a siliconized Pasteur pipette with the tip
barely fore-polished. After filtration through 40 pm cell
strainers, neural precursor cells are harvested by magnetic bead
immunoselection using anti-P75 antibodies. NSCs are expanded in
culture medium prior to in vitro characterization and in vivo
intestine formation. SMCs are also harvested by magnetic bead
immunoselection, but using anti-SMMHC antibodies. MSCs are expanded
in culture medium prior to in vitro characterization and in vivo
intestine formation.
Cell Seeding and Characterization
[0161] Crypts are encapsulated in pluronic F-127 hydrogel and then
painted on the inner layer of nanofiber scaffolds. SMCs and NSCs
are mixed in pluronic F-127 hydrogel followed by pressure
infiltration into the scaffold from the outer surface. Both HB-EGF
coated and non-coated scaffolds are seeded with cells and cultured
in the customized dynamic bioreactor system that enables the
dynamic culture of cell-scaffolds, which are used for ex vivo organ
formation. At various time points, samples are observed
microscopically and then fixed for SEM and histology. ISCs are
detected by fluorescence microscopy. To ensure formation of all
intestinal epithelial cell lineages, goblet cells are detected by
alcian blue staining. Paneth cells are detected by lysozyme
immunostaining, and enteroendocrine cells are detected by
Chromogranin A immunostaining.
[0162] In the NSC cultures, colonies grow in size and formed
neurospheres between days 1 to 21. Nestin immunostaining confirmed
the presence of significant numbers of neuronal precursor cells in
the neurospheres. The crypts are isolated and grown in ex vivo
culture. In addition, amniotic fluid derived mesenchymal cells
attached to the surface of a nanofiber scaffold.
Example 5
In Vivo Characterization of Engineered Intestine
[0163] In vivo characterization of HB-EGF-embedded nanofiber
scaffolds of are conducted via anastomosis of the scaffold with
native rat intestine in a defunctionalized Roux-en-Y intestinal
limb, as described by Jwo et al. (Br J Surg. 2008; 95:657-63) with
modifications. In this model, the mesentery are detached from a
resected intestinal segment and used to wrap the cell-seeded
scaffold for provision of blood supply after the scaffold has been
anastomosed to the native intestine. Compared to published animal
models, this model will provide a reliable blood supply for the
engineered intestine.
[0164] Under general anesthesia, a midline laparotomy is performed
on athymic nude rats (rnu/rnu) (n=12/group/time point) with a 3-5
cm incision, and the ligament of Treitz and ileocecal junction are
identified. After dividing the proximal jejunum 10 cm from the
ligament of Treitz, the Roux-en-Y bypass technique is used to make
an end-to-side jejunoileostomy anastomosis with 8/0 nylon sutures
between the proximal cut end and the side wall of the ileum, 20 cm
away from the ileocecal junction. The distal cut end of the
dysfunctional limb (Roux limb) is closed. A 1 mm silicon tube is
inserted into the intestinal lumen and the other end of the tube is
folded, tied, and buried in the right abdominal wall. A 10 cm
central segment of the Roux limb is removed and substituted with a
2-cm scaffold tube anastomosed to native intestine with 8/0 nylon
sutures The mesentery from the resected segment of intestine is
wrapped around the scaffold tube as a source of blood supply to the
implanted scaffold, and is immobilized with 8/0 silk sutures.
Animals are radiographed at weeks 4 and 8 post-operatively, with
contrast reagent given orally and via the tube buried in the right
abdominal wall, and sacrificed immediately afterwards. Any signs of
inflammation or adhesion formation are recorded during necropsy.
The implant site with adjacent tissues are excised and fixed in 10%
neutral buffered formalin, and processed for paraffin embedding.
Tissue sections are evaluated for inflammation and tissue
regeneration based on established grading scales, and scaffold
degradation are assessed as well using Mann-Whitney U test, with
p<0.05 considered statistically significant intestine.
Example 6
Cell Filtration System to Enrich Intestinal Stem Cells in
Crypts
[0165] In the last decade, studies have focused on using organoid
units (OU) as the cell source for TEL OU are cell clusters that are
isolated from full-thickness intestine, and represent a mixed
population of differentiated and undifferentiated cells. This cell
source is not efficient for tissue regeneration because
differentiated epithelial cells no longer have the capacity to
proliferate, and will likely undergo apoptosis.
[0166] The present invention provides for a novel cell filtration
system using multiple sieve membranes with different pore sizes.
With one filtration step, different cell populations are obtained
in a convenient and efficient cell recovery method from the
removable sieve membrane. Use of donor intestine from an Lgr5
transgenic mice (which has fluorescent labeling of all intestinal
stem cells (ISC)), allowed for the development of a custom-designed
filtration system (FIG. 8) to determine which filtration fraction
is enriched in ISC. This system consists of a bottom filter, top
cups, sieve membranes, and cell scraper (FIG. 8a). The smallest
size (8 .mu.m) has been loaded first and placed on the top of base
filter cup (FIG. 8b). The second top cup has been placed on the
base filter cup to secure the sieve membrane (FIG. 8c). Similarly,
all other sizes of sieve membranes have been loaded in order from
smaller to larger pore sizes, and a vacuum tube is connected.
[0167] Using the method set out in FIG. 8, an optimal cell
population between 8-70 .mu.m was identified as highly enriched in
fluorescently-labeled ISC (FIG. 9). Fluorescently-labeled
intestinal stem cells (ISC) appear first in the 50-70 .mu.m
population, increase in the 25-50 .mu.m population, and disappear
in the 8-25 .mu.m and smaller populations. Based on these
observations, an optimal cell population of 8-70 .mu.m was selected
for in vivo implantation studies as described in Example 7.
[0168] Briefly, the cells from Lewis rat pups were filtered with
the filtration system, and two cell populations (8-70 .mu.m and
70-200 .mu.m) were seeded into PGA scaffolds and implanted into the
peritoneal cavity of the dam rat by suturing to the undersurface of
the abdominal wall. After 4 weeks of in vivo incubation, the
samples were harvested and embedded in paraffin for Periodic
acid-Schiff (PAS) staining (see FIG. 9). TEI formed from scaffolds
seeded with the 8-70 .mu.m cell population (enriched in ISC) showed
a higher percentage of mucosal engraftment compared to TEI from
scaffolds seeded with the 70-200 .mu.m cell population (see FIG.
10) The percent of mucosal engraftment from scaffolds seeded with
the 8-70 .mu.m cell population is significantly higher than that
from the 70-200 .mu.m cell population (p<0.01). Five sections
were measured at 200 .mu.m intervals from each sample and 12
samples were measured from each cell population (see FIG. 11)
[0169] To confirm the sizes of the enriched intestinal stem cells,
frozen tissue sections from Lgr5EGFP mice was mounted onto glass
slide with VectaShield medium containing DAPI. This staining
verified that intestinal stem cells reside in crypts underneath
villi. The length of villi and crypts was measured using Zeiss LSM
image browser (Version 4.2.0.121). Scale bar. Exemplary
measurements are provided in Table 3.
TABLE-US-00003 TABLE 3 Representative measure of villi and crypts
length.sup.1 Villi Crypts Length Length (.mu.m) (.mu.m) 91.69 33.75
177.00 28.79 176.70 28.73 96.99 44.20 Mean 135.60 33.87 STDEV 47.69
7.28
Example 7
HB-EGF Incorporation into PGA Enhances the Formation of Tissue
Engineered Intestine
[0170] Preparation of PGA scaffolds were prepared as described in
Example 2. Briefly, tubular PGA scaffolds were prepared with PGA
BioFelt (Biomedical Structures, thickness=1 mm, density=60 mg/ml)
and hydrated with or without HB-EGF (0, 1 or 10 .mu.g in 100 .mu.l
PBS). Select scaffolds were subjected to subsequent CO.sub.2
infusion (900 psi) for 1 h to increase HB-EGF incorporation.
[0171] Small intestines were harvested from Lewis rat pups, minced,
and digested in dispase and collagenase for 30 minutes. Intestinal
stem cell (ISC) enriched crypts were obtained by filtration with
100 .mu.m and 40 .mu.m cell strainers. Scaffolds (1 cm length) were
then seeded with 1.5-2 million crypts and implanted into the
peritoneal cavity of the dam of the donor rat pups, on the interior
surface of the abdominal wall. After 4 weeks of in vivo incubation,
explants were assessed histologically and villous length measured
as described in Example 1 and depicted in FIG. 12.
[0172] As shown in FIG. 13, HB-EGF release kinetic studies were
assessed using an HB-EGF ELISA. Increased amounts of HB-EGF were
released from PGA scaffolds with CO.sub.2 infusion compared to
those without CO.sub.2 infusion. The biopotency of HB-EGF released
from scaffolds was assessed using a cell proliferation assay, and
was very similar to that of control HB-EGF, as confirmed by an EC50
fitting curve (Graphpad Prism 6 software) (p=0.209; see FIG.
14)
[0173] Histology was carried out on native intestine and compared
to TEI and depicted in FIG. 15. TEI produced from in vivo
incubation of crypt-seeded scaffolds was histologically very
similar to that of native intestine. HB-EGF infusion of scaffolds
led to TEI with increased villous height, increased crypt numbers,
and well-developed smooth muscle layers. In addition, villious
height of the native intestine was compared with the TEL HB-EGF
infusion of scaffolds led to TEI with increased villous height. The
villous height of native intestine was 212.9.+-.16.8 .mu.m. The
villous height of TEI increased with increasing concentrations of
HB-EGF and with the use of CO.sub.2 infusion as shown in FIG.
16.
[0174] These experiments demonstrate that HB-EGF incorporation into
scaffolds improves the quality of the TEI produced. In addition,
CO.sub.2 infusion improves the efficacy of HB-EGF incorporation
into TEI scaffolds. The use of HB-EGF in the production of TEI may
be beneficial for the future treatment of patients with short bowel
syndrome.
Sequence CWU 1
1
21624DNAHomo sapiensCDS(1)..(624) 1atg aag ctg ctg ccg tcg gtg gtg
ctg aag ctc ttt ctg gct gca gtt 48Met Lys Leu Leu Pro Ser Val Val
Leu Lys Leu Phe Leu Ala Ala Val 1 5 10 15 ctc tcg gca ctg gtg act
ggc gag agc ctg gag cgg ctt cgg aga ggg 96Leu Ser Ala Leu Val Thr
Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly 20 25 30 cta gct gct gga
acc agc aac ccg gac cct ccc act gta tcc acg gac 144Leu Ala Ala Gly
Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr Asp 35 40 45 cag ctg
cta ccc cta gga ggc ggc cgg gac cgg aaa gtc cgt gac ttg 192Gln Leu
Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu 50 55 60
caa gag gca gat ctg gac ctt ttg aga gtc act tta tcc tcc aag cca
240Gln Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro
65 70 75 80 caa gca ctg gcc aca cca aac aag gag gag cac ggg aaa aga
aag aag 288Gln Ala Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg
Lys Lys 85 90 95 aaa ggc aag ggg cta ggg aag aag agg gac cca tgt
ctt cgg aaa tac 336Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys
Leu Arg Lys Tyr 100 105 110 aag gac ttc tgc atc cat gga gaa tgc aaa
tat gtg aag gag ctc cgg 384Lys Asp Phe Cys Ile His Gly Glu Cys Lys
Tyr Val Lys Glu Leu Arg 115 120 125 gct ccc tcc tgc atc tgc cac ccg
ggt tac cat gga gag agg tgt cat 432Ala Pro Ser Cys Ile Cys His Pro
Gly Tyr His Gly Glu Arg Cys His 130 135 140 ggg ctg agc ctc cca gtg
gaa aat cgc tta tat acc tat gac cac aca 480Gly Leu Ser Leu Pro Val
Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr 145 150 155 160 acc atc ctg
gcc gtg gtg gct gtg gtg ctg tca tct gtc tgt ctg ctg 528Thr Ile Leu
Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175 gtc
atc gtg ggg ctt ctc atg ttt agg tac cat agg aga gga ggt tat 576Val
Ile Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185
190 gat gtg gaa aat gaa gag aaa gtg aag ttg ggc atg act aat tcc cac
624Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His
195 200 205 2208PRTHomo sapiens 2Met Lys Leu Leu Pro Ser Val Val
Leu Lys Leu Phe Leu Ala Ala Val 1 5 10 15 Leu Ser Ala Leu Val Thr
Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly 20 25 30 Leu Ala Ala Gly
Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr Asp 35 40 45 Gln Leu
Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu 50 55 60
Gln Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro 65
70 75 80 Gln Ala Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg
Lys Lys 85 90 95 Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys
Leu Arg Lys Tyr 100 105 110 Lys Asp Phe Cys Ile His Gly Glu Cys Lys
Tyr Val Lys Glu Leu Arg 115 120 125 Ala Pro Ser Cys Ile Cys His Pro
Gly Tyr His Gly Glu Arg Cys His 130 135 140 Gly Leu Ser Leu Pro Val
Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr 145 150 155 160 Thr Ile Leu
Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175 Val
Ile Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185
190 Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His
195 200 205
* * * * *