U.S. patent application number 14/840653 was filed with the patent office on 2016-01-28 for methods and combination comprising eukaryotic cells and recombinant spider silk protein.
This patent application is currently assigned to SPIBER TECHNOLOGIES AB. The applicant listed for this patent is SPIBER TECHNOLOGIES AB. Invention is credited to My HEDHAMMAR, Jan JOHANSSON, Ulrika JOHANSSON, Anna RISING, Mona WIDHE.
Application Number | 20160024464 14/840653 |
Document ID | / |
Family ID | 44798897 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160024464 |
Kind Code |
A1 |
JOHANSSON; Jan ; et
al. |
January 28, 2016 |
METHODS AND COMBINATION COMPRISING EUKARYOTIC CELLS AND RECOMBINANT
SPIDER SILK PROTEIN
Abstract
A method and a combination for the cultivation of eukaryotic
cells are provided, as well as a method for preparation of
eukaryotic cells. The methods comprise providing a sample of
eukaryotic cells to be cultured, applying said sample to a cell
scaffold material; and maintaining said cell scaffold material
having cells applied thereto under conditions suitable for cell
culture. The combination comprises eukaryotic cells and a cell
scaffold material. The cell scaffold material comprises a polymer
of a spider silk protein.
Inventors: |
JOHANSSON; Jan; (Stockholm,
SE) ; RISING; Anna; (Uppsala, SE) ; HEDHAMMAR;
My; (Stockholm, SE) ; JOHANSSON; Ulrika;
(Uppsala, SE) ; WIDHE; Mona; (Uppsala,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SPIBER TECHNOLOGIES AB |
Uppsala |
|
SE |
|
|
Assignee: |
SPIBER TECHNOLOGIES AB
Uppsala
SE
|
Family ID: |
44798897 |
Appl. No.: |
14/840653 |
Filed: |
August 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13639763 |
Jan 17, 2013 |
9157070 |
|
|
PCT/SE2011/050448 |
Apr 12, 2011 |
|
|
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14840653 |
|
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61323226 |
Apr 12, 2010 |
|
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Current U.S.
Class: |
435/398 |
Current CPC
Class: |
C12N 5/0676 20130101;
A61L 27/56 20130101; A61L 27/3834 20130101; C12N 5/0629 20130101;
C12N 5/0691 20130101; C12N 2539/00 20130101; C12N 2500/82 20130101;
C12N 5/067 20130101; A61L 27/3804 20130101; C07K 14/005 20130101;
C12N 5/0068 20130101; C12N 5/0647 20130101; C12N 5/0606 20130101;
A61L 27/227 20130101; C07K 14/43518 20130101; C12N 5/0623 20130101;
D01F 4/02 20130101; C12N 2533/50 20130101; C12N 5/0656
20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 12, 2010 |
EP |
10159694.8 |
Feb 7, 2011 |
EP |
11153543.1 |
Claims
1. A method for the cultivation of eukaryotic cells, comprising
applying a sample of eukaryotic cells to be cultured to a cell
scaffold material; and maintaining said cell scaffold material
having cells applied thereto under conditions suitable for cell
culture; wherein said cell scaffold material comprises a polymer of
a spider silk protein consisting of from 140 to 600 amino acid
residues and comprising a repetitive fragment of from 70 to 300
amino acid residues derived from the repetitive fragment of a
spider silk protein; a C-terminal fragment of from 70 to 120 amino
acid residues derived from the C-terminal fragment of a spider silk
protein; and optionally an N-terminal fragment of from 100 to 160
amino acid residues derived from the N-terminal fragment of a
spider silk protein.
2. A method for the preparation of eukaryotic cells, comprising:
applying a sample of eukaryotic cells to a cell scaffold material;
maintaining said cell scaffold material having cells applied
thereto under conditions suitable for cell culture; and preparing a
sample of cells from said cell scaffold material; wherein said cell
scaffold material comprises a polymer of a spider silk protein
consisting of from 140 to 600 amino acid residues and comprising a
repetitive fragment of from 70 to 300 amino acid residues derived
from the repetitive fragment of a spider silk protein; a C-terminal
fragment of from 70 to 120 amino acid residues derived from the
C-terminal fragment of a spider silk protein; and optionally an
N-terminal fragment of from 100 to 160 amino acid residues derived
from the N-terminal fragment of a spider silk protein.
3. The method according to claim 1, wherein said eukaryotic cells
are mammalian cells.
4. The method according to claim 1, wherein said eukaryotic cells
are mammalian cells selected from the group consisting of stem
cells and cells from islets of Langerhans.
5. The method according to claim 1, wherein said spider silk
protein is selected from the group of proteins defined by the
formulas REP-CT and NT-REP-CT, wherein NT is a protein fragment
having from 100 to 160 amino acid residues, which fragment is a
N-terminal fragment derived from a spider silk protein; REP is a
protein fragment having from 70 to 300 amino acid residues, wherein
said fragment is selected from the group consisting of
L(AG).sub.nL, L(AG).sub.nAL, L(GA).sub.nL, and L(GA).sub.nGL,
wherein n is an integer from 2 to 10; each individual A segment is
an amino acid sequence of from 8 to 18 amino acid residues, wherein
from 0 to 3 of the amino acid residues are not Ala, and the
remaining amino acid residues are Ala; each individual G segment is
an amino acid sequence of from 12 to 30 amino acid residues,
wherein at least 40% of the amino acid residues are Gly; and each
individual L segment is a linker amino acid sequence of from 0 to
20 amino acid residues; and CT is a protein fragment having from 70
to 120 amino acid residues, which fragment is a C-terminal fragment
derived from a spider silk protein.
6. The method according to claim 1, wherein said spider silk
protein comprises a cell-binding motif.
7. The method according to claim 6, wherein said cell-binding motif
is an oligopeptide coupled to a remainder of the spider silk
protein via at least one peptide bond.
8. The method according to claim 7, wherein said oligopeptide is
coupled to the N-terminal of the remainder of the spider silk
protein.
9. The method according to claim 7, wherein said oligopeptide
comprises an amino acid sequence selected from the group consisting
of RGD, RGE, IKVAV (SEQ ID NO:23), YIGSR (SEQ ID NO:24), EPDIM (SEQ
ID NO:25) and NKDIL (SEQ ID NO:26).
10. The method according to claim 1, wherein said cell scaffold
material is in a physical form selected from the group consisting
of film, foam, fiber and fiber-mesh.
11. The method according to claim 2, wherein said eukaryotic cells
are mammalian cells.
12. The method according to claim 2, wherein said eukaryotic cells
are mammalian cells selected from the group consisting of stem
cells and cells from islets of Langerhans.
13. The method according to claim 2, wherein said spider silk
protein is selected from the group of proteins defined by the
formulas REP-CT and NT-REP-CT, wherein NT is a protein fragment
having from 100 to 160 amino acid residues, which fragment is a
N-terminal fragment derived from a spider silk protein; REP is a
protein fragment having from 70 to 300 amino acid residues, wherein
said fragment is selected from the group consisting of
L(AG).sub.nL, L(AG).sub.nAL, L(GA).sub.nL, and L(GA).sub.nGL,
wherein n is an integer from 2 to 10; each individual A segment is
an amino acid sequence of from 8 to 18 amino acid residues, wherein
from 0 to 3 of the amino acid residues are not Ala, and the
remaining amino acid residues are Ala; each individual G segment is
an amino acid sequence of from 12 to 30 amino acid residues,
wherein at least 40% of the amino acid residues are Gly; and each
individual L segment is a linker amino acid sequence of from 0 to
20 amino acid residues; and CT is a protein fragment having from 70
to 120 amino acid residues, which fragment is a C-terminal fragment
derived from a spider silk protein.
14. The method according to claim 2, wherein said spider silk
protein comprises a cell-binding motif.
15. The method according to claim 14, wherein said cell-binding
motif is an oligopeptide coupled to a remainder of the spider silk
protein via at least one peptide bond.
16. The method according to claim 15, wherein said oligopeptide is
coupled to the N-terminal of the remainder of the spider silk
protein.
17. The method according to claim 15, wherein said oligopeptide
comprises an amino acid sequence selected from the group consisting
of RGD, RGE, IKVAV (SEQ ID NO:23), YIGSR (SEQ ID NO:24), EPDIM (SEQ
ID NO:25) and NKDIL (SEQ ID NO:26).
18. The method according to claim 2, wherein said cell scaffold
material is in a physical form selected from the group consisting
of film, foam, fiber and fiber-mesh.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 13/639,763, filed Jan. 17, 2013. U.S. application Ser. No.
13/639,763 is the National Phase of PCT/SE2011/050448 filed on Apr.
12, 2011, which claims priority under 35 U.S.C. 119(e) to U.S.
Provisional Application No. 61/323,226 filed on Apr. 12, 2010 and
under 35 U.S.C. 119(a) to Patent Application Nos. 10159694.8 filed
in Europe on Apr. 12, 2010 and 11153543.1 filed in Europe on Feb.
7, 2011, all of which are hereby expressly incorporated by
reference into the present application.
FIELD OF THE INVENTION
[0002] The present invention relates to the fields of eukaryotic
cell culture and tissue engineering, and provides methods and a
combination for eukaryotic cell culture and preparation, wherein a
polymer of a spider silk protein is used as a cell scaffold
material.
BACKGROUND
[0003] Spider silks are nature's high-performance polymers,
obtaining extraordinary toughness due to a combination of strength
and elasticity. Spiders have up to seven different glands which
produce a variety of silk types with different mechanical
properties and functions. Dragline silk, produced by the major
ampullate gland, is the toughest fiber, and on a weight basis it
outperforms man-made materials, such as tensile steel. The
properties of dragline silk are attractive in development of new
materials for medical or technical purposes.
[0004] Dragline silk consists of two main polypeptides, mostly
referred to as major ampullate spidroin (MaSp) 1 and 2, but e.g. as
ADF-3 and ADF-4 in Araneus diadematus. These proteins have
molecular masses in the range of 200-720 kDa. The genes coding for
dragline proteins of Latrodectus hesperus are the only ones that
have been completely characterized, and the MaSp1 and MaSp2 genes
encode 3129 and 3779 amino acids, respectively (Ayoub N A et al.
PLoS ONE 2(6): e514, 2007). The properties of dragline silk
polypeptides are discussed in Huemmerich, D. et al. Curr. Biol. 14,
2070-2074 (2004).
[0005] Spider dragline silk proteins, or MaSps, have a tripartite
composition; a non-repetitive N-terminal domain, a central
repetitive region comprised of many iterated poly-Ala/Gly segments,
and a non-repetitive C-terminal domain. It is generally believed
that the repetitive region forms intermolecular contacts in the
silk fibers, while the precise functions of the terminal domains
are less clear. It is also believed that in association with fiber
formation, the repetitive region undergoes a structural conversion
from random coil and .alpha.-helical conformation to .beta.-sheet
structure. The C-terminal region of spidroins is generally
conserved between spider species and silk types. The N-terminal
domain of spider silks is the most conserved region (Rising, A. et
al. Biomacromolecules 7, 3120-3124 (2006)).
[0006] WO03/057727 discloses expression of soluble recombinant silk
polypeptides in mammalian cell lines and animals. The obtained silk
polypeptides exhibit poor solubility in aqueous media and/or form
precipitates. Since the obtained silk polypeptides do not
polymerise spontaneously, spinning is required to obtain polymers
or fibers.
[0007] WO07/078239 and Stark, M. et al. Biomacromolecules 8,
1695-1701, (2007) disclose a miniature spider silk protein
consisting of a repetitive fragment with a high content of Ala and
Gly and a C-terminal fragment of a protein, as well as soluble
fusion proteins comprising the spider silk protein. Fibers of the
spider silk protein are obtained spontaneously upon liberation of
the spider silk protein from its fusion partner. The small fusion
unit is sufficient and necessary for the fiber formation.
[0008] Hedhammar, M. et al. Biochemistry 47, 3407-3417, (2008)
study the thermal, pH and salt effects on the structure and
aggregation and/or polymerisation of recombinant N- and C-terminal
spidroin domains and a repetitive spidroin domain containing four
poly-Ala and Gly rich co-blocks.
[0009] In vitro studies on the biocompatibility of recombinant
spider silk are so far few, and the materials studied vary a lot in
amino acid sequence, mode of production and format.
DESCRIPTION OF THE INVENTION
[0010] In a first aspect, the invention provides a method for the
cultivation of eukaryotic cells, comprising [0011] providing a
sample of eukaryotic cells to be cultured; [0012] applying said
sample to a cell scaffold material; and [0013] maintaining said
cell scaffold material having cells applied thereto under
conditions suitable for cell culture. The cell scaffold material
comprises a polymer of a spider silk protein.
[0014] In a second aspect, the invention provides a method for the
preparation of eukaryotic cells, comprising: [0015] providing a
sample of eukaryotic cells; [0016] applying said sample to a cell
scaffold material; [0017] maintaining said cell scaffold material
having cells applied thereto under conditions suitable for cell
culture; and [0018] preparing a sample of cells from said cell
scaffold material. The cell scaffold material comprises a polymer
of a spider silk protein.
[0019] It has been found by the present inventors that a cell
scaffold material comprising a polymer of a spider silk protein
provides a beneficial environment for the cultivation of eukaryotic
cells in a variety of different settings. Furthermore, this
environment enables the establishment of cultures of cells that are
otherwise very difficult, very costly or even impossible to culture
in a laboratory, and for the establishment of cell-containing
materials useful for tissue engineering and/or transplantation.
[0020] In some embodiments thereof, the cultivation or preparation
methods may be performed in conditions comprising maintaining the
cell scaffold material having cells applied thereto in a serum-free
medium. The possibility to culture cells in a serum-free medium
affords a cost-efficient and controlled alternative to the use of
serum-containing media and/or media containing specific growth
factors or extracellular matrix (ECM) components. This type of
culture media is often very expensive, sometimes even prohibitively
so, and heterogeneous.
[0021] In a third aspect, the invention provides a combination of
eukaryotic cells and a cell scaffold material comprising a polymer
of a spider silk protein. Such a combination according to the
invention may be presented in a variety of different formats, and
tailored to suit the needs of a specific situation. It is
contemplated, for example, that the inventive combination may be
useful as a cell-containing implant for the replacement of cells in
damaged or diseased tissue.
[0022] In some embodiments of the methods and combination presented
herein, the eukaryotic cells are mammalian cells, for example human
cells. In other embodiments, the eukaryotic cells are non-mammalian
cells, such as insect or yeast cells.
[0023] Non-limiting examples of mammalian cells that may be
cultivated or prepared by the methods or included in the
combination according to the invention are listed in the following
multi-level listing:
Cells of the Integumentary System
[0024] Keratinizing Epithelial Cells [0025] Epidermal keratinocyte
(differentiating epidermal cell) [0026] Epidermal basal cell (stem
cell) [0027] Keratinocyte of fingernails and toenails [0028] Nail
bed basal cell (stem cell) [0029] Medullary hair shaft cell [0030]
Cortical hair shaft cell [0031] Cuticular hair shaft cell [0032]
Cuticular hair root sheath cell [0033] Hair root sheath cell of
Huxley's layer [0034] Hair root sheath cell of Henle's layer [0035]
External hair root sheath cell [0036] Hair matrix cell (stem
cell)
[0037] Wet Stratified Barrier Epithelial Cells [0038] Surface
epithelial cell of stratified squamous epithelium of cornea,
tongue, oral cavity, esophagus, anal canal, distal urethra and
vagina [0039] Basal cell (stem cell) of epithelia of cornea,
tongue, oral cavity, esophagus, anal canal, distal urethra and
vagina Urinary epithelium cell (lining urinary bladder and urinary
ducts)
Gland Cells
[0040] Exocrine Secretory Epithelial Cells [0041] Salivary gland
mucous cell (polysaccharide-rich secretion) [0042] Salivary gland
serous cell (glycoprotein enzyme-rich secretion) [0043] von Ebner's
gland cell in tongue (washes taste buds) [0044] Mammary gland cell
(milk secretion) [0045] Lacrimal gland cell (tear secretion) [0046]
Ceruminous gland cell in ear (wax secretion) [0047] Eccrine sweat
gland dark cell (glycoprotein secretion) [0048] Eccrine sweat gland
clear cell (small molecule secretion) [0049] Apocrine sweat gland
cell (odoriferous secretion, sex-hormone sensitive) [0050] Gland of
Moll cell in eyelid (specialized sweat gland) [0051] Sebaceous
gland cell (lipid-rich sebum secretion) [0052] Bowman's gland cell
in nose (washes olfactory epithelium) [0053] Brunner's gland cell
in duodenum (enzymes and alkaline mucus) [0054] Seminal vesicle
cell (secretes seminal fluid components, including fructose for
swimming sperm) [0055] Prostate gland cell (secretes seminal fluid
components) Bulbourethral gland cell (mucus secretion) [0056]
Bartholin's gland cell (vaginal lubricant secretion) Gland of
Littre cell (mucus secretion) [0057] Uterus endometrium cell
(carbohydrate secretion) [0058] Isolated goblet cell of respiratory
and digestive tracts (mucus secretion) [0059] Stomach lining mucous
cell (mucus secretion) [0060] Gastric gland zymogenic cell
(pepsinogen secretion) [0061] Gastric gland oxyntic cell, parietal
cell (hydrochloric acid secretion) [0062] Enterochromaffin like
(ECL) cells (release histamine) [0063] Pancreatic acinar cell
(bicarbonate and digestive enzyme secretion) [0064] Paneth cell of
small intestine (lysozyme secretion) [0065] Type II pneumocyte of
lung (surfactant secretion) [0066] Clara cell of lung
[0067] Hormone Secreting Cells [0068] Anterior pituitary cells
[0069] Somatotropes [0070] Lactotropes [0071] Thyrotropes [0072]
Gonadotropes [0073] Corticotropes [0074] Intermediate pituitary
cell, secreting melanocyte-stimulating hormone [0075] Magnocellular
neurosecretory cells [0076] secreting oxytocin [0077] secreting
vasopressin [0078] Gut and respiratory tract cells [0079] Cells
included in Islets of Langerhans: [0080] Alpha cells (produce
glucagon), beta cells (insulin producing cells), delta cells
(somatostatin producing cells), pp cells (produce pancreatic
polypeptide), epsilon cells (produce ghrelin) [0081] secreting
serotonin [0082] secreting endorphin [0083] secreting gastrin
[0084] secreting secretin [0085] secreting cholecystokinin [0086]
secreting bombesin [0087] Thyroid gland cells [0088] Thyroid
epithelial cell [0089] Parafollicular cell [0090] Parathyroid gland
cells [0091] Parathyroid chief cell [0092] Oxyphil cell [0093]
Adrenal gland cells [0094] Chromaffin cells [0095] secreting
steroid hormones (mineralcorticoids, androgens and gluco
corticoids) [0096] Leydig cell of testes secreting testosterone
[0097] Theca interna cell of ovarian follicle secreting estrogen
[0098] Corpus luteum cell of ruptured ovarian follicle secreting
progesterone [0099] Granulosa lutein cells [0100] Theca lutein
cells [0101] Juxtaglomerular cell (renin secretion) [0102] Macula
densa cell of kidney [0103] Peripolar cell of kidney [0104]
Mesangial cell of kidney
Metabolism and Storage Cells
[0104] [0105] Hepatocyte (liver cell) [0106] White fat cell
(adipocytes/blasts) [0107] Brown fat cell [0108] Liver lipocyte
Barrier Function Cells (Lung, Gut, Exocrine Glands and Urogenital
Tract)
[0109] Kidney [0110] Kidney glomerulus parietal cell [0111] Kidney
glomerulus podocyte [0112] Kidney proximal tubule brush border cell
[0113] Loop of Henle thin segment cell [0114] Kidney distal tubule
cell [0115] Kidney collecting duct cell
[0116] Other [0117] Type I pneumocyte (lining air space of lung)
[0118] Pancreatic duct cell (centroacinar cell) [0119] Nonstriated
duct cell (of sweat gland, salivary gland, mammary gland, etc.)
[0120] Principal cell [0121] Intercalated cell [0122] Duct cell (of
seminal vesicle, prostate gland, etc.) [0123] Intestinal brush
border cell (with microvilli) [0124] Exocrine gland striated duct
cell [0125] Gall bladder epithelial cell [0126] Ductulus efferens
nonciliated cell [0127] Epididymal principal cell [0128] Epididymal
basal cell
Epithelial Cells Lining Closed Internal Body Cavities
[0128] [0129] Microvascular endothelial cells [0130] Blood vessel
and lymphatic vascular endothelial fenestrated cell [0131] Blood
vessel and lymphatic vascular endothelial continuous cell [0132]
Blood vessel and lymphatic vascular endothelial splenic cell [0133]
Synovial cell (lining joint cavities, hyaluronic acid secretion)
[0134] Serosal cell (lining peritoneal, pleural, and pericardial
cavities) [0135] Squamous cell (lining perilymphatic space of ear)
[0136] Squamous cell (lining endolymphatic space of ear) [0137]
Columnar cell of endolymphatic sac with microvilli (lining
endolymphatic space of ear) [0138] Columnar cell of endolymphatic
sac without microvilli (lining endolymphatic space of ear) [0139]
Dark cell (lining endolymphatic space of ear) [0140] Vestibular
membrane cell (lining endolymphatic space of ear) [0141] Stria
vascularis basal cell (lining endolymphatic space of ear) [0142]
Stria vascularis marginal cell (lining endolymphatic space of ear)
[0143] Cell of Claudius (lining endolymphatic space of ear) [0144]
Cell of Boettcher (lining endolymphatic space of ear) [0145]
Choroid plexus cell (cerebrospinal fluid secretion) [0146]
Pia-arachnoid squamous cell [0147] Pigmented ciliary epithelium
cell of eye [0148] Nonpigmented ciliary epithelium cell of eye
[0149] Corneal endothelial cell [0150] Peg cell (of Fallopian tube)
Ciliated Cells with Propulsive Function [0151] Respiratory tract
ciliated cell [0152] Oviduct ciliated cell (in female) [0153]
Uterine endometrial ciliated cell (in female) [0154] Rete testis
ciliated cell (in male) [0155] Ductulus efferens ciliated cell (in
male) [0156] Ciliated ependymal cell of central nervous system
(lining brain cavities)
Extracellular Matrix Secretion Cells
[0156] [0157] Ameloblast epithelial cell (tooth enamel secretion)
[0158] Planum semilunatum epithelial cell of vestibular apparatus
of ear (proteoglycan secretion) [0159] Organ of Corti interdental
epithelial cell (secreting tectorial membrane covering hair cells)
[0160] Loose connective tissue fibroblasts [0161] Corneal
fibroblasts (corneal keratocytes) [0162] Tendon fibroblasts [0163]
Bone marrow reticular tissue fibroblasts [0164] Other nonepithelial
fibroblasts [0165] Pericyte [0166] Nucleus pulposus cell of
intervertebral disc [0167] Cementoblast/cementocyte (tooth root
bonelike cementum secretion) [0168] Odontoblast/odontocyte (tooth
dentin secretion) [0169] Hyaline cartilage chondrocyte [0170]
Fibrocartilage chondrocyte [0171] Elastic cartilage chondrocyte
[0172] Osteoblast/osteocyte [0173] Osteoprogenitor cell (stem cell
of osteoblasts) [0174] Hyalocyte of vitreous body of eye [0175]
Stellate cell of perilymphatic space of ear [0176] Hepatic stellate
cell (Ito cell) [0177] Pancreatic stellate cell
Contractile Cells
[0177] [0178] Skeletal muscle cells [0179] Red skeletal muscle cell
(slow) [0180] White skeletal muscle cell (fast) [0181] Intermediate
skeletal muscle cell [0182] Nuclear bag cell of muscle spindle
[0183] Nuclear chain cell of muscle spindle [0184] Satellite cell
(stem cell) [0185] Heart muscle cells [0186] Ordinary heart muscle
cell [0187] Nodal heart muscle cell [0188] Purkinje fiber cell
[0189] Smooth muscle cell (various types) [0190] Myoepithelial cell
of iris [0191] Myoepithelial cell of exocrine glands
Blood and Immune System Cells
[0191] [0192] Megakaryocyte (platelet precursor) [0193] Monocyte
[0194] Connective tissue macrophage (various types) [0195]
Epidermal Langerhans cell [0196] Osteoclast (in bone) [0197]
Dendritic cell (in lymphoid tissues) [0198] Microglial cell (in
central nervous system) [0199] Neutrophil granulocyte [0200]
Eosinophil granulocyte [0201] Basophil granulocyte [0202] Mast cell
[0203] Helper T cell [0204] Suppressor T cell [0205] Cytotoxic T
cell [0206] Natural Killer T cell [0207] B cell [0208] Natural
killer cell [0209] Reticulocyte [0210] Committed progenitors for
the blood and immune system (various types, e.g. megakaryocyte,
myeloblast)
Cells of the Nervous System
[0211] Sensory Transducer Cells [0212] Auditory inner hair cell of
organ of Corti [0213] Auditory outer hair cell of organ of Corti
[0214] Basal cell of olfactory epithelium (stem cell for olfactory
neurons) [0215] Cold-sensitive primary sensory neurons [0216]
Heat-sensitive primary sensory neurons [0217] Merkel cell of
epidermis (touch sensor) [0218] Olfactory receptor neuron
[0219] Pain-sensitive primary sensory neurons (various types)
[0220] Photoreceptor cells of retina in eye: [0221] Photoreceptor
rod cells [0222] Photoreceptor blue-sensitive cone cell of eye
[0223] Photoreceptor green-sensitive cone cell of eye [0224]
Photoreceptor red-sensitive cone cell of eye [0225] Proprioceptive
primary sensory neurons (various types) [0226] Touch-sensitive
primary sensory neurons (various types)
[0227] Type I carotid body cell (blood pH sensor) [0228] Type II
carotid body cell (blood pH sensor) [0229] Type I hair cell of
vestibular apparatus of ear (acceleration and gravity) [0230] Type
II hair cell of vestibular apparatus of ear (acceleration and
gravity) [0231] Type I taste bud cell
[0232] Autonomic Neuron Cells [0233] Cholinergic neural cell
(various types) [0234] Adrenergic neural cell (various types)
[0235] Peptidergic neural cell (various types)
[0236] Sense Organ and Peripheral Neuron Supporting Cells [0237]
Inner pillar cell of organ of Corti [0238] Outer pillar cell of
organ of Corti [0239] Inner phalangeal cell of organ of Corti
[0240] Outer phalangeal cell of organ of Corti [0241] Border cell
of organ of Corti [0242] Hensen cell of organ of Corti [0243]
Vestibular apparatus supporting cell [0244] Taste bud supporting
cell [0245] Olfactory epithelium supporting cell [0246] Schwann
cell [0247] Satellite cell (encapsulating peripheral nerve cell
bodies) [0248] Enteric glial cell
[0249] Central Nervous System Neurons and Glial Cells [0250]
Astrocyte (various types) [0251] Neuron cells (large variety of
types, still poorly classified) [0252] Oligodendrocyte [0253]
Spindle neuron [0254] Pineocyte (produce melatonin)
[0255] Lens Cells [0256] Anterior lens epithelial cell [0257]
Crystallin-containing lens fiber cell
Pigment Cells
[0257] [0258] Melanocyte [0259] Retinal pigmented epithelial
cell
Germ Cells
[0259] [0260] Oogonium/Oocyte [0261] Spermatid [0262] Spermatocyte
[0263] Spermatogonium cell (stem cell for spermatocyte) [0264]
Spermatozoon
Nurse Cells
[0264] [0265] Ovarian follicle cell [0266] Sertoli cell (in testis)
[0267] Thymus epithelial cell [0268] Stem Cells and Progenitor
Cells
[0269] Embryonic stem cells [0270] Adult stem cells (e.g.,
hematopoietic stem cells, endothelial stem cells, epithelial stem
cells, neural stem cells, mesenchymal stem cells) [0271] Progenitor
cells (neural progenitor cells, lymphoid progenitor cells,
satellite cells, endothelial progenitor cells, periosteal
progenitor, pancreatic progenitor cells, satellite cells in
muscles, hematopoietic progenitor cells) [0272] Amniotic stem cells
(multipotent and can differentiate to cells of adipogenic,
osteogenic, myogenic, endothelial, hepatic and also neuronal lines)
[0273] Induced pluripotent stem cells
[0274] In organs, there is usually a main tissue and sporadic
tissues. The main tissue is the one that is unique for the specific
organ. In an embodiment of the invention, it is contemplated that
the cells for use in the methods or combination disclosed herein
are main tissue cells, i.e. cells that contribute to the function
of organs in their natural environment. Furthermore, in an
embodiment, cells forming sporadic tissue, in particular connective
tissue, are not included, since the role of connective tissue is
considered to be fulfilled by the spider silk protein in this
embodiment.
[0275] For example, the main tissue of the heart is the myocardium,
while sporadic are the nerves, blood, connective etc. Below follows
a non-limiting listing of examples of organ systems, whose main
tissue cells may be useful in the methods or combination disclosed
herein.
[0276] Circulatory system: pumping and channeling blood to and from
the body and lungs with heart, blood and blood vessels.
[0277] Digestive system: digestion and processing food with
salivary glands, esophagus, stomach, liver, gallbladder, pancreas,
intestines, rectum and anus.
[0278] Endocrine system: communication within the body using
hormones made by endocrine glands such as the hypothalamus,
pituitary or pituitary gland, pineal body or pineal gland,
pancreas, thyroid, parathyroids and adrenals, i.e., adrenal
glands.
[0279] Excretory system: kidneys, ureters, bladder and urethra
involved in fluid balance, electrolyte balance and excretion of
urine.
[0280] Integumentary system: skin, hair and nails.
[0281] Lymphatic system: structures involved in the transfer of
lymph between tissues and the blood stream, the lymph and the nodes
and vessels that transport it including the endothelium.
[0282] Immune system: defending against disease-causing agents with
leukocytes, tonsils, adenoids, thymus and spleen.
[0283] Muscular system: movement with muscles.
[0284] Nervous system: collecting, transferring and processing
information with brain, spinal cord, peripheral nerves and
nerves.
[0285] Reproductive system: the sex organs, such as ovaries,
fallopian tubes, uterus, vagina, mammary glands, testes, vas
deferens, seminal vesicles, prostate and penis.
[0286] Respiratory system: the organs used for breathing, the
pharynx, larynx, trachea, bronchi, lungs and diaphragm.
[0287] Skeletal system: structural support and protection with
bones, cartilage, ligaments and tendons.
[0288] Various different embodiments of the methods or combination
disclosed herein may employ any sub-group or sub-listing of cells,
or even individual cell types, from the above general listings of
cell types and organ and tissue systems.
[0289] In a more specific embodiment, said mammalian cells are
selected from the group consisting of stem cells and cells from
islets of Langerhans (e.g. beta cells).
[0290] In a more specific embodiment, said cells are selected from
embryonic stem cells, adult stem cells, induced pluripotent stem
cells, amniotic stem cells and progenitor cells, and may in
particular be selected from embryonic stem cells, adult stem cells
and induced pluripotent stem cells.
[0291] In yet a specific embodiment, said cells are embryonic stem
cells.
[0292] In yet a specific embodiment, said cells are adult stem
cells selected from the group consisting of hematopoietic, neural,
mesenchymal, mammary, endothelial, epithelial and olfactory stem
cells, in particular selected from the group consisting of
hematopoietic, neural and mesenchymal stem cells.
[0293] In yet a specific embodiment, said cells are progenitor
cells selected from the group consisting of neural progenitor
cells, mesenchymal progenitor cells and hematopoietic progenitor
cells.
[0294] In yet a specific embodiment, the mammalian cells are neural
stem cells (interchangeably denoted neural cortical stem cells),
which may be provided as single cells or in the form of at least
one neurosphere.
[0295] In yet a specific embodiment, the mammalian cells are
insulin-producing beta cells, which may be provided as single cells
or in the form of at least one islet of Langerhans.
[0296] In yet a specific embodiment, the mammalian cells are
somatic cells, for example selected from the group consisting of
hepatocytes, fibroblasts, keratinocytes and endothelial cells.
[0297] The cell scaffold material used in the context of the
present disclosure comprises a polymer of a spider silk protein or
polypeptide, also denoted "spidroin".
[0298] In one of the embodiments of the cell scaffold material,
said spidroin consists of from 140 to 600 amino acid residues and
comprises
[0299] a repetitive fragment of from 70 to 300 amino acid residues
derived from the repetitive fragment of a spider silk protein;
[0300] a C-terminal fragment of from 70 to 120 amino acid residues
derived from the C-terminal fragment of a spider silk protein; and
optionally
[0301] an N-terminal fragment of from 100 to 160 amino acid
residues derived from the N-terminal fragment of a spider silk
protein.
[0302] The spidroin consists of from 140 to 600 amino acid
residues, preferably from 280 to 600 amino acid residues, such as
from 300 to 400 amino acid residues, more preferably from 340 to
380 amino acid residues. The small size is advantageous because
longer spider silk proteins tend to form amorphous aggregates,
which require use of harsh solvents for solubilisation and
polymerisation. The protein fragments are covalently coupled,
typically via a peptide bond.
[0303] In specific preferred embodiments, the spidroin for use in
the cell scaffold material is selected from the group of proteins
defined by the formulas NT-REP-CT and REP-CT.
[0304] The (optional) NT fragment has a high degree of similarity
to the N-terminal amino acid sequence of spider silk proteins. As
shown in FIG. 1, this amino acid sequence is well conserved among
various species and spider silk proteins, including MaSp1 and
MaSp2. See also the following Table 1:
TABLE-US-00001 TABLE 1 Spidroin NT fragments GenBank Code Species
and spidroin acc. no. Ea MaSp1 Euprosthenops australis MaSp 1
AM259067 Lg MaSp1 Latrodectus geometricus MaSp 1 ABY67420 Lh MaSp1
Latrodectus hesperus MaSp 1 ABY67414 Nc MaSp1 Nephila clavipes MaSp
1 ACF19411 At MaSp2 Argiope trifasciata MaSp 2 AAZ15371 Lg MaSp2
Latrodectus geometricus MaSp 2 ABY67417 Lh MaSp2 Latrodectus
hesperus MaSp 2 ABR68855 Nim MaSp2 Nephila inaurata
madagascariensis MaSp 2 AAZ15322 Nc MaSp2 Nephila clavipes MaSp 2
ACF19413 Ab CySp1 Argiope bruennichi cylindriform spidroin 1
BAE86855 Ncl CySp1 Nephila clavata cylindriform spidroin 1 BAE54451
Lh TuSp1 Latrodectus hesperus tubuliform spidroin ABD24296 Nc Flag
Nephila clavipes flagelliform silk protein AF027972 Nim Flag
Nephila inaurata madagascariensis AF218623 flagelliform silk
protein (translated)
[0305] It is not critical which, if any, specific NT fragment is
present in the spidroin of the cell scaffold material disclosed
herein. Thus, the NT fragment according to the invention can be
selected from any of the amino acid sequences shown in FIG. 1 or
sequences with a high degree of similarity. A wide variety of
N-terminal sequences can be used as spidroin in the cell scaffold
material disclosed herein. Based on the homologous sequences of
FIG. 1, the following sequence constitutes a consensus NT amino
acid sequence:
TABLE-US-00002 (SEQ ID NO: 8)
QANTPWSSPNLADAFINSF(M/L)SA(A/I)SSSGAFSADQLDDMSTIG
(D/N/Q)TLMSAMD(N/S/K)MGRSG(K/R)STKSKLQALNMAFASSMA
EIAAAESGG(G/Q)SVGVKTNAISDALSSAFYQTTGSVNPQFV(N/S)
EIRSLI(G/N)M(F/L)(A/S)QASANEV.
[0306] The sequence of the NT fragment has at least 50% identity,
preferably at least 60% identity, to the consensus amino acid
sequence SEQ ID NO:8, which is based on the amino acid sequences of
FIG. 1. In a preferred embodiment, the sequence of the NT fragment
has at least 65% identity, preferably at least 70% identity, to the
consensus amino acid sequence SEQ ID NO:8. In preferred
embodiments, the NT fragment has furthermore 70%, preferably 80%,
similarity to the consensus amino acid sequence SEQ ID NO:8.
[0307] A representative NT fragment in a protein for use in the
cell scaffold material disclosed herein is the Euprosthenops
australis sequence SEQ ID NO:6. According to an embodiment, the NT
fragment has at least 80% identity to SEQ ID NO:6 or any individual
amino acid sequence in FIG. 1. For example, the NT fragment has at
least 90%, such as at least 95% identity, to SEQ ID NO:6 or any
individual amino acid sequence in FIG. 1. The NT fragment may be
identical to SEQ ID NO:6 or any individual amino acid sequence in
FIG. 1, in particular to Ea MaSp1.
[0308] The NT fragment contains from 100 to 160 amino acid
residues. It is preferred that the NT fragment contains at least
100, or more than 110, preferably more than 120, amino acid
residues. It is also preferred that the NT fragment contains at
most 160, or less than 140 amino acid residues. A typical NT
fragment contains approximately 130-140 amino acid residues.
[0309] The REP fragment has a repetitive character, alternating
between alanine-rich stretches and glycine-rich stretches. The REP
fragment generally contains more than 70, such as more than 140,
and less than 300, preferably less than 240, such as less than 200,
amino acid residues, and can itself be divided into several L
(linker) segments, A (alanine-rich) segments and G (glycine-rich)
segments, as will be explained in more detail below. Typically,
said linker segments, which are optional, are located at the REP
fragment terminals, while the remaining segments are in turn
alanine-rich and glycine-rich. Thus, the REP fragment can generally
have either of the following structures, wherein n is an
integer:
[0310] L(AG).sub.nL, such as
LA.sub.1G.sub.1A.sub.2G.sub.2A.sub.3G.sub.3A.sub.4G.sub.4A.sub.5G.sub.5L;
[0311] L(AG).sub.nAL, such as
LA.sub.1G.sub.1A.sub.2G.sub.2A.sub.3G.sub.3A.sub.4G.sub.4A.sub.5G.sub.5A.-
sub.6L;
[0312] L(GA).sub.nL, such as
LG.sub.1A.sub.1G.sub.2A.sub.2G.sub.3A.sub.3G.sub.4A.sub.4G.sub.5A.sub.5L;
or
[0313] L(GA).sub.nGL, such as
LG.sub.1A.sub.1G.sub.2A.sub.2G.sub.3A.sub.3G.sub.4A.sub.4G.sub.5A.sub.5G.-
sub.6L.
[0314] It follows that it is not critical whether an alanine-rich
or a glycine-rich segment is adjacent to the N-terminal or
C-terminal linker segments. It is preferred that n is an integer
from 2 to 10, preferably from 2 to 8, also preferably from 4 to 8,
more preferred from 4 to 6, i.e. n=4, n=5 or n=6.
[0315] In some embodiments, the alanine content of the REP fragment
is above 20%, preferably above 25%, more preferably above 30%, and
below 50%, preferably below 40%, more preferably below 35%. It is
contemplated that a higher alanine content provides a stiffer
and/or stronger and/or less extendible fiber.
[0316] In certain embodiments, the REP fragment is void of proline
residues, i.e. there are no Pro residues in the REP fragment.
[0317] Turning now to the segments that constitute the REP
fragment, it is emphasized that each segment is individual, i.e.
any two A segments, any two G segments or any two L segments of a
specific REP fragment may be identical or may not be identical.
Thus, it is not a general feature of the spidroin that each type of
segment is identical within a specific REP fragment. Rather, the
following disclosure provides the skilled person with guidelines
how to design individual segments and gather them into a REP
fragment, which is a part of a functional spider silk protein
useful in a cell scaffold material.
[0318] Each individual A segment is an amino acid sequence having
from 8 to 18 amino acid residues. It is preferred that each
individual A segment contains from 13 to 15 amino acid residues. It
is also possible that a majority, or more than two, of the A
segments contain from 13 to 15 amino acid residues, and that a
minority, such as one or two, of the A segments contain from 8 to
18 amino acid residues, such as 8-12 or 16-18 amino acid residues.
A vast majority of these amino acid residues are alanine residues.
More specifically, from 0 to 3 of the amino acid residues are not
alanine residues, and the remaining amino acid residues are alanine
residues. Thus, all amino acid residues in each individual A
segment are alanine residues, with no exception or with the
exception of one, two or three amino acid residues, which can be
any amino acid. It is preferred that the alanine-replacing amino
acid(s) is (are) natural amino acids, preferably individually
selected from the group of serine, glutamic acid, cysteine and
glycine, more preferably serine. Of course, it is possible that one
or more of the A segments are all-alanine segments, while the
remaining A segments contain 1-3 non-alanine residues, such as
serine, glutamic acid, cysteine or glycine.
[0319] In an embodiment, each A segment contains 13-15 amino acid
residues, including 10-15 alanine residues and 0-3 non-alanine
residues as described above. In a more preferred embodiment, each A
segment contains 13-15 amino acid residues, including 12-15 alanine
residues and 0-1 non-alanine residues as described above.
[0320] It is preferred that each individual A segment has at least
80%, preferably at least 90%, more preferably 95%, most preferably
100% identity to an amino acid sequence selected from the group of
amino acid residues 7-19, 43-56, 71-83, 107-120, 135-147, 171-183,
198-211, 235-248, 266-279, 294-306, 330-342, 357-370, 394-406,
421-434, 458-470, 489-502, 517-529, 553-566, 581-594, 618-630,
648-661, 676-688, 712-725, 740-752, 776-789, 804-816, 840-853,
868-880, 904-917, 932-945, 969-981, 999-1013, 1028-1042 and
1060-1073 of SEQ ID NO:10. Each sequence of this group corresponds
to a segment of the naturally occurring sequence of Euprosthenops
australis MaSp1 protein, which is deduced from cloning of the
corresponding cDNA, see WO2007/078239. Alternatively, each
individual A segment has at least 80%, preferably at least 90%,
more preferably 95%, most preferably 100% identity to an amino acid
sequence selected from the group of amino acid residues 143-152,
174-186, 204-218, 233-247 and 265-278 of SEQ ID NO:3. Each sequence
of this group corresponds to a segment of expressed, non-natural
spider silk proteins, which proteins have the capacity to form silk
fibers under appropriate conditions. Thus, in certain embodiments
of the spidroin, each individual A segment is identical to an amino
acid sequence selected from the above-mentioned amino acid
segments. Without wishing to be bound by any particular theory, it
is envisaged that A segments according to the invention form
helical structures or beta sheets.
[0321] Furthermore, it has been concluded from experimental data
that each individual G segment is an amino acid sequence of from 12
to 30 amino acid residues. It is preferred that each individual G
segment consists of from 14 to 23 amino acid residues. At least 40%
of the amino acid residues of each G segment are glycine residues.
Typically the glycine content of each individual G segment is in
the range of 40-60%.
[0322] It is preferred that each individual G segment has at least
80%, preferably at least 90%, more preferably 95%, most preferably
100% identity to an amino acid sequence selected from the group of
amino acid residues 20-42, 57-70, 84-106, 121-134, 148-170,
184-197, 212-234, 249-265, 280-293, 307-329, 343-356, 371-393,
407-420, 435-457, 471-488, 503-516, 530-552, 567-580, 595-617,
631-647, 662-675, 689-711, 726-739, 753-775, 790-803, 817-839,
854-867, 881-903, 918-931, 946-968, 982-998, 1014-1027, 1043-1059
and 1074-1092 of SEQ ID NO:10. Each sequence of this group
corresponds to a segment of the naturally occurring sequence of
Euprosthenops australis MaSp1 protein, which is deduced from
cloning of the corresponding cDNA, see WO2007/078239.
Alternatively, each individual G segment has at least 80%,
preferably at least 90%, more preferably 95%, most preferably 100%
identity to an amino acid sequence selected from the group of amino
acid residues 153-173, 187-203, 219-232, 248-264 and 279-296 of SEQ
ID NO:3. Each sequence of this group corresponds to a segment of
expressed, non-natural spider silk proteins, which proteins have
the capacity to form silk fibers under appropriate conditions.
Thus, in certain embodiments of the spidroin in the cell scaffold
material, each individual G segment is identical to an amino acid
sequence selected from the above-mentioned amino acid segments.
[0323] In certain embodiments, the first two amino acid residues of
each G segment are not -Gln-Gln-.
[0324] There are the three subtypes of the G segment. This
classification is based upon careful analysis of the Euprosthenops
australis MaSp1 protein sequence (see WO2007/078239), and the
information has been employed and verified in the construction of
novel, non-natural spider silk proteins.
[0325] The first subtype of the G segment is represented by the
amino acid one letter consensus sequence GQG(G/S)QGG(Q/Y)GG
(L/Q)GQGGYGQGA GSS (SEQ ID NO:11). This first, and generally the
longest, G segment subtype typically contains 23 amino acid
residues, but may contain as little as 17 amino acid residues, and
lacks charged residues or contain one charged residue. Thus, it is
preferred that this first G segment subtype contains 17-23 amino
acid residues, but it is contemplated that it may contain as few as
12 or as many as 30 amino acid residues. Without wishing to be
bound by any particular theory, it is envisaged that this subtype
forms coil structures or 3.sub.1-helix structures. Representative G
segments of this first subtype are amino acid residues 20-42,
84-106, 148-170, 212-234, 307-329, 371-393, 435-457, 530-552,
595-617, 689-711, 753-775, 817-839, 881-903, 946-968, 1043-1059 and
1074-1092 of SEQ ID NO:10. In certain embodiments, the first two
amino acid residues of each G segment of this first subtype
according to the invention are not -Gln-Gln-.
[0326] The second subtype of the G segment is represented by the
amino acid one letter consensus sequence GQGGQGQG(G/R)Y
GQG(A/S)G(S/G)S (SEQ ID NO:12). This second, generally mid-sized, G
segment subtype typically contains 17 amino acid residues and lacks
charged residues or contain one charged residue. It is preferred
that this second G segment subtype contains 14-20 amino acid
residues, but it is contemplated that it may contain as few as 12
or as many as 30 amino acid residues. Without wishing to be bound
by any particular theory, it is envisaged that this subtype forms
coil structures. Representative G segments of this second subtype
are amino acid residues 249-265, 471-488, 631-647 and 982-998 of
SEQ ID NO:10; and amino acid residues 187-203 of SEQ ID NO:3.
[0327] The third subtype of the G segment is represented by the
amino acid one letter consensus sequence G(R/Q)GQG(G/R)YGQG
(A/S/V)GGN (SEQ ID NO:13). This third G segment subtype typically
contains 14 amino acid residues, and is generally the shortest of
the G segment subtypes. It is preferred that this third G segment
subtype contains 12-17 amino acid residues, but it is contemplated
that it may contain as many as 23 amino acid residues. Without
wishing to be bound by any particular theory, it is envisaged that
this subtype forms turn structures. Representative G segments of
this third subtype are amino acid residues 57-70, 121-134, 184-197,
280-293, 343-356, 407-420, 503-516, 567-580, 662-675, 726-739,
790-803, 854-867, 918-931, 1014-1027 of SEQ ID NO:10; and amino
acid residues 219-232 of SEQ ID NO:3.
[0328] Thus, in preferred embodiments of the spidroin in the cell
scaffold material, each individual G segment has at least 80%,
preferably 90%, more preferably 95%, identity to an amino acid
sequence selected from SEQ ID NO:11, SEQ ID NO:12 and SEQ ID
NO:13.
[0329] In an embodiment of the alternating sequence of A and G
segments of the REP fragment, every second G segment is of the
first subtype, while the remaining G segments are of the third
subtype, e.g. . . .
A.sub.1G.sub.shortA.sub.2G.sub.longA.sub.3G.sub.shortA.sub.4G.sub.longA.s-
ub.5G.sub.short . . . . In another embodiment of the REP fragment,
one G segment of the second subtype interrupts the G segment
regularity via an insertion, e.g. . . .
A.sub.1G.sub.shortA.sub.2G.sub.longA.sub.3G.sub.midA.sub.4G.sub.shortA.su-
b.5G.sub.long . . . .
[0330] Each individual L segment represents an optional linker
amino acid sequence, which may contain from 0 to 20 amino acid
residues, such as from 0 to 10 amino acid residues. While this
segment is optional and not critical for the function of the spider
silk protein, its presence still allows for fully functional spider
silk proteins and polymers thereof which form fibers, films, foams
and other structures. There are also linker amino acid sequences
present in the repetitive part (SEQ ID NO:10) of the deduced amino
acid sequence of the MaSp1 protein from Euprosthenops australis. In
particular, the amino acid sequence of a linker segment may
resemble any of the described A or G segments, but usually not
sufficiently to meet their criteria as defined herein.
[0331] As shown in WO2007/078239, a linker segment arranged at the
C-terminal part of the REP fragment can be represented by the amino
acid one letter consensus sequences ASASAAASAA STVANSVS and
ASAASAAA, which are rich in alanine. In fact, the second sequence
can be considered to be an A segment according to the definition
herein, whereas the first sequence has a high degree of similarity
to A segments according to this definition. Another example of a
linker segment has the one letter amino acid sequence GSAMGQGS,
which is rich in glycine and has a high degree of similarity to G
segments according to the definition herein. Another example of a
linker segment is SASAG.
[0332] Representative L segments are amino acid residues 1-6 and
1093-1110 of SEQ ID NO:10; and amino acid residues 138-142 of SEQ
ID NO:3, but the skilled person will readily recognize that there
are many suitable alternative amino acid sequences for these
segments. In one embodiment of the REP fragment, one of the L
segments contains 0 amino acids, i.e. one of the L segments is
void. In another embodiment of the REP fragment, both L segments
contain 0 amino acids, i.e. both L segments are void. Thus, these
embodiments of the REP fragments according to the invention may be
schematically represented as follows: (AG).sub.nL, (AG).sub.nAL,
(GA).sub.nL, (GA).sub.nGL; L(AG).sub.n, L(AG).sub.nA, L(GA).sub.n,
L(GA).sub.nG; and (AG).sub.n, (AG).sub.nA, (GA).sub.n, (GA).sub.nG.
Any of these REP fragments are suitable for use with any CT
fragment as defined below.
[0333] The CT fragment of the spidroin in the cell scaffold
material has a high degree of similarity to the C-terminal amino
acid sequence of spider silk proteins. As shown in WO2007/078239,
this amino acid sequence is well conserved among various species
and spider silk proteins, including MaSp1 and MaSp2. A consensus
sequence of the C-terminal regions of MaSp1 and MaSp2 is provided
as SEQ ID NO:9. In FIG. 2, the following MaSp proteins are aligned,
denoted with GenBank accession entries where applicable:
TABLE-US-00003 TABLE 2 Spidroin CT fragments Species and spidroin
Entry Euprosthenops sp MaSp1 Cthyb_Esp (Pouchkina-Stantcheva, N N
& McQueen-Mason, S J, ibid) Euprosthenops australis MaSp1
CTnat_Eau Argiope trifasciata MaSp1 AF350266_At1 Cyrtophora
moluccensis Sp1 AY666062_Cm1 Latrodectus geometricus MaSp1
AF350273_Lg1 Latrodectus hesperus MaSp1 AY953074_Lh1 Macrothele
holsti Sp1 AY666068_Mh1 Nephila clavipes MaSp1 U20329_Nc1 Nephila
pilipes MaSp1 AY666076_Np1 Nephila madagascariensis MaSp1
AF350277_Nm1 Nephila senegalensis MaSp1 AF350279_Ns1 Octonoba
varians Sp1 AY666057_Ov1 Psechrus sinensis Sp1 AY666064_Ps1
Tetragnatha kauaiensis MaSp1 AF350285_Tk1 Tetragnatha versicolor
MaSp1 AF350286_Tv1 Araneus bicentenarius Sp2 ABU20328_Ab2 Argiope
amoena MaSp2 AY365016_Aam2 Argiope aurantia MaSp2 AF350263_Aau2
Argiope trifasciata MaSp2 AF350267_At2 Gasteracantha mammosa MaSp2
AF350272_Gm2 Latrodectus geometricus MaSp2 AF350275_Lg2 Latrodectus
hesperus MaSp2 AY953075_Lh2 Nephila clavipes MaSp2 AY654293_Nc2
Nephila madagascariensis MaSp2 AF350278_Nm2 Nephila senegalensis
MaSp2 AF350280_Ns2 Dolomedes tenebrosus Fb1 AF350269_DtFb1
Dolomedes tenebrosus Fb2 AF350270_DtFb2 Araneus diadematus ADF-1
U47853_ADF1 Araneus diadematus ADF-2 U47854_ADF2 Araneus diadematus
ADF-3 U47855_ADF3 Araneus diadematus ADF-4 U47856_ADF4
[0334] It is not critical which specific CT fragment is present in
the spider silk protein in the cell scaffold material. Thus, the CT
fragment can be selected from any of the amino acid sequences shown
in FIG. 2 and Table 2 or sequences with a high degree of
similarity. A wide variety of C-terminal sequences can be used in
the spider silk protein.
[0335] The sequence of the CT fragment has at least 50% identity,
preferably at least 60%, more preferably at least 65% identity, or
even at least 70% identity, to the consensus amino acid sequence
SEQ ID NO:9, which is based on the amino acid sequences of FIG.
2.
[0336] A representative CT fragment is the Euprosthenops australis
sequence SEQ ID NO:7. Thus, in one embodiment, the CT fragment has
at least 80%, preferably at least 90%, such as at least 95%,
identity to SEQ ID NO:7 or any individual amino acid sequence of
FIG. 2 and Table 2. For example, the CT fragment may be identical
to SEQ ID NO:7 or any individual amino acid sequence of FIG. 2 and
Table 2.
[0337] The CT fragment typically consists of from 70 to 120 amino
acid residues. It is preferred that the CT fragment contains at
least 70, or more than 80, preferably more than 90, amino acid
residues. It is also preferred that the CT fragment contains at
most 120, or less than 110 amino acid residues. A typical CT
fragment contains approximately 100 amino acid residues.
[0338] The term "% identity", as used herein, is calculated as
follows. The query sequence is aligned to the target sequence using
the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research,
22:4673-4680 (1994)). A comparison is made over the window
corresponding to the shortest of the aligned sequences. The amino
acid residues at each position are compared, and the percentage of
positions in the query sequence that have identical correspondences
in the target sequence is reported as % identity.
[0339] The term "% similarity", as used herein, is calculated as
described above for "% identity", with the exception that the
hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile, Trp, Met and Cys
are similar; the basic residues Lys, Arg and His are similar; the
acidic residues Glu and Asp are similar; and the hydrophilic,
uncharged residues Gln, Asn, Ser, Thr and Tyr are similar. The
remaining natural amino acid Gly is not similar to any other amino
acid in this context.
[0340] Throughout this description, alternative embodiments
according to the invention fulfill, instead of the specified
percentage of identity, the corresponding percentage of similarity.
Other alternative embodiments fulfill the specified percentage of
identity as well as another, higher percentage of similarity,
selected from the group of preferred percentages of identity for
each sequence. For example, a sequence may be 70% similar to
another sequence; or it may be 70% identical to another sequence;
or it may be 70% identical and 90% similar to another sequence.
[0341] In some more specific embodiments of the methods or
combination disclosed herein, the spider silk protein of the cell
scaffold material comprises a spidroin selected from the group
consisting of 4RepCT, RGD-4RepCT, RGE-4RepCT, IKVAV-4RepCT,
YIGSR-4RepCT, NT4RepCTHis, 5RepCT, 8RepCT, 4RepCTMa2, NT8RepCT and
NTNT8RepCT (see list of appended sequences below). In an even more
specific embodiment, the spidroin is 4RepCT (SEQ ID NO:2)
LIST OF APPENDED SEQUENCES
[0342] SEQ ID NO: [0343] 1 4Rep [0344] 2 4RepCT [0345] 3 NT4Rep
[0346] 4 NT5Rep [0347] 5 NT4RepCTHis [0348] 6 NT [0349] 7 CT [0350]
8 consensus NT sequence [0351] 9 consensus CT sequence [0352] 10
repetitive sequence from Euprosthenops australis MaSp1 [0353] 11
consensus G segment sequence 1 [0354] 12 consensus G segment
sequence 2 [0355] 13 consensus G segment sequence 3 [0356] 14
5RepCT [0357] 15 8RepCT [0358] 16 4RepCTMa2 [0359] 17 NT8RepCT
[0360] 18 NTNT8RepCT [0361] 19 RGD-4RepCT [0362] 20 RGE-4RepCT
[0363] 21 I KVAV-4RepCT [0364] 22 YIGSR-4RepCT
[0365] In one embodiment of the methods or combination as disclosed
herein, said spider silk protein comprises a cell-binding motif. In
connection with the cultivation of certain cells in certain
situations, the presence of a cell-binding motif has been observed
to improve or maintain cell viability, and the inclusion of such a
motif into the cell scaffold material as a part of the spider silk
protein is thought to provide additional benefits.
[0366] In some embodiments, the cell-binding motif is an
oligopeptide coupled to the rest of the spider silk protein via at
least one peptide bond. For example, it may be coupled to the
N-terminal or the C-terminal of the rest of the spider silk
protein, or at any position within the amino acid sequence of the
rest of the spider silk protein. With regard to the selection of
oligopeptidic cell-binding motifs, the skilled person is aware of
several alternatives. Said oligopeptide may for example comprise an
amino acid sequence selected from the group consisting of RGD, RGE,
IKVAV (SEQ ID NO:23), YIGSR (SEQ ID NO:24), EPDIM (SEQ ID NO:25)
and NKDIL (SEQ ID NO:26). RGD, IKVAV and YIGSR are general
cell-binding motifs, whereas EPDIM and NKDIL are known as
keratinocyte-specific motifs that may be particularly useful in the
context of cultivation of keratinocytes. The coupling of an
oligopeptide cell-binding motif to the rest of the spider silk
protein is readily accomplished by the skilled person using
standard genetic engineering or chemical coupling techniques. Thus,
in some embodiments, the cell-binding motif is introduced via
genetic engineering, i.e. forming part of a genetic fusion between
nucleic acid encoding the "wild-type" spider silk protein and the
cell-binding motif. As an additional beneficial characteristic of
such embodiments, the cell-binding motif will be present in a 1:1
ratio to the monomers of spider silk protein in the polymer making
up the cell scaffold material.
[0367] The polymer in the cell scaffold material used in the
methods or combination described herein may adopt a variety of
physical forms, and use of a specific physical form may offer
additional advantages in different specific situations. For
example, in an embodiment of the methods or combination, said cell
scaffold material is in a physical form selected from the group
consisting of film, foam, fiber and fiber-mesh.
[0368] In the context of the present invention, the terms
"cultivation" of cells, "cell culture" etc are to be interpreted
broadly, such that they encompass for example situations in which
cells divide and/or proliferate, situations in which cells are
maintained in a differentiated state with retention of at least one
functional characteristic exhibited by the cell type when present
in its natural environment, and situations in which stem cells are
maintained in an undifferentiated state.
[0369] Furthermore, as is evident from the above disclosure, it is
contemplated that cells may be provided in the form of single
cells, or as part of a cellular structure or "micro-organ".
Cultivation of cells in the form of a cellular structure or
"micro-organ" may entail maintenance of the entire structure in
combination with the cell scaffold material.
Itemized Listing of Embodiments
[0370] 1. Method for the cultivation of eukaryotic cells,
comprising [0371] providing a sample of eukaryotic cells to be
cultured; [0372] applying said sample to a cell scaffold material;
and [0373] maintaining said cell scaffold material having cells
applied thereto under conditions suitable for cell culture;
characterized in that
[0374] said cell scaffold material comprises a polymer of a spider
silk protein consisting of from 140 to 600 amino acid residues and
comprising
[0375] a repetitive fragment of from 70 to 300 amino acid residues
derived from the repetitive fragment of a spider silk protein;
[0376] a C-terminal fragment of from 70 to 120 amino acid residues
derived from the C-terminal fragment of a spider silk protein; and
optionally
[0377] an N-terminal fragment of from 100 to 160 amino acid
residues derived from the N-terminal fragment of a spider silk
protein.
[0378] 2. Method for the preparation of eukaryotic cells,
comprising: [0379] providing a sample of eukaryotic cells; [0380]
applying said sample to a cell scaffold material; [0381]
maintaining said cell scaffold material having cells applied
thereto under conditions suitable for cell culture; and [0382]
preparing a sample of cells from said cell scaffold material;
characterized in that
[0383] said cell scaffold material comprises a polymer of a spider
silk protein consisting of from 140 to 600 amino acid residues and
comprising
[0384] a repetitive fragment of from 70 to 300 amino acid residues
derived from the repetitive fragment of a spider silk protein;
[0385] a C-terminal fragment of from 70 to 120 amino acid residues
derived from the C-terminal fragment of a spider silk protein; and
optionally
[0386] an N-terminal fragment of from 100 to 160 amino acid
residues derived from the N-terminal fragment of a spider silk
protein.
[0387] 3. Method according to item 1 or 2, wherein said conditions
comprise maintaining the cell scaffold material having cells
applied thereto in a serum-free medium.
[0388] 4. Method according to any preceding item, wherein said
eukaryotic cells are mammalian cells.
[0389] 5. Method according to item 4, wherein said mammalian cells
are derived from organ main tissue.
[0390] 6. Method according to item 4 or 5, wherein said mammalian
cells are selected from the group consisting of stem cells and
cells from islets of Langerhans including beta cells.
[0391] 7. Method according to item 6, wherein said cells are stem
cells selected from embryonic stem cells, adult stem cells, induced
pluripotent stem cells, amniotic stem cells and progenitor cells,
in particular selected from embryonic stem cells, adult stem cells
and induced pluripotent stem cells.
[0392] 8. Method according to item 7, wherein said cells are
embryonic stem cells.
[0393] 9. Method according to item 7, wherein said cells are
progenitor cells selected from the group consisting of neural
progenitor cells, mesenchymal progenitor cells and hematopoietic
progenitor cells.
[0394] 10. Method according to item 7, wherein said cells are adult
stem cells selected from the group consisting of hematopoietic,
neural, mesenchymal, mammary, endothelial, epithelial and olfactory
stem cells, in particular selected from the group consisting of
hematopoietic, neural and mesenchymal stem cells.
[0395] 11. Method according to item 6, wherein said cells are cells
from islets of Langerhans, for example beta cells.
[0396] 12. Method according to any preceding item, wherein said
eukaryotic cells are provided as single cells.
[0397] 13. Method according to item 10, wherein said mammalian
cells are neural stem cells provided as at least one
neurosphere.
[0398] 14. Method according to item 11, wherein said mammalian
cells are beta cells provided as at least one islet of
Langerhans.
[0399] 15. Method according to any one of items 1-5, wherein said
mammalian cells are somatic cells, for example selected from the
group consisting of hepatocytes, fibroblasts, keratinocytes and
endothelial cells.
[0400] 16. Method according to any preceding item, wherein said
cells are human cells.
[0401] 17. Method according to any preceding item, wherein said
spider silk protein is selected from the group of proteins defined
by the formulas REP-CT and NT-REP-CT, wherein
[0402] NT is a protein fragment having from 100 to 160 amino acid
residues, which fragment is a N-terminal fragment derived from a
spider silk protein;
[0403] REP is a protein fragment having from 70 to 300 amino acid
residues, wherein said fragment is selected from the group
consisting of L(AG).sub.nL, L(AG).sub.nAL, L(GA).sub.nL, and
L(GA).sub.nGL, wherein [0404] n is an integer from 2 to 10; [0405]
each individual A segment is an amino acid sequence of from 8 to 18
amino acid residues, wherein from 0 to 3 of the amino acid residues
are not Ala, and the remaining amino acid residues are Ala; [0406]
each individual G segment is an amino acid sequence of from 12 to
30 amino acid residues, wherein at least 40% of the amino acid
residues are Gly; and [0407] each individual L segment is a linker
amino acid sequence of from 0 to 20 amino acid residues; and
[0408] CT is a protein fragment having from 70 to 120 amino acid
residues, which fragment is a C-terminal fragment derived from a
spider silk protein.
[0409] 18. Method according to item 17, wherein said spider silk
protein is selected from the group consisting of 4RepCT,
NT4RepCTHis, 5RepCT, 8RepCT, 4RepCTMa2, NT8RepCT and
NTNT8RepCT.
[0410] 19. Method according to any preceding item, wherein said
spider silk protein comprises a cell-binding motif.
[0411] 20. Method according to item 19, wherein said cell-binding
motif is an oligopeptide coupled to the remainder of the spider
silk protein via at least one peptide bond.
[0412] 21. Method according to item 20, wherein said oligopeptide
is coupled to the N-terminal of the remainder of the spider silk
protein.
[0413] 22. Method according to any one of items 20-21, wherein said
oligopeptide comprises an amino acid sequence selected from the
group consisting of RGD, RGE, IKVAV, YIGSR, EPDIM and NKDIL.
[0414] 23. Method according to any preceding item, wherein said
cell scaffold material is in a physical form selected from the
group consisting of film, foam, fiber and fiber-mesh.
[0415] 24. Combination of [0416] eukaryotic cells; and [0417] a
cell scaffold material; characterized in that
[0418] said cell scaffold material comprises a polymer of a spider
silk protein consisting of from 140 to 600 amino acid residues and
comprising
[0419] a repetitive fragment of from 70 to 300 amino acid residues
derived from the repetitive fragment of a spider silk protein;
[0420] a C-terminal fragment of from 70 to 120 amino acid residues
derived from the C-terminal fragment of a spider silk protein; and
optionally
[0421] an N-terminal fragment of from 100 to 160 amino acid
residues derived from the N-terminal fragment of a spider silk
protein.
[0422] 25. Combination according item 24, wherein said eukaryotic
cells are mammalian cells.
[0423] 26. Combination according to item 25, wherein said mammalian
cells are derived from organ main tissue.
[0424] 27. Combination according to item 25 or 26, wherein said
mammalian cells are selected from the group consisting of stem
cells and cells from islets of Langerhans including beta cells.
[0425] 28. Combination according to item 27, wherein said cells are
stem cells selected from embryonic stem cells, adult stem cells,
induced pluripotent stem cells, amniotic stem cells and progenitor
cells, in particular selected from embryonic stem cells, adult stem
cells and induced pluripotent stem cells.
[0426] 29. Combination according to item 28, wherein said cells are
embryonic stem cells.
[0427] 30. Combination according to item 28, wherein said cells are
progenitor cells selected from the group consisting of neural
progenitor cells, mesenchymal progenitor cells and hematopoietic
progenitor cells.
[0428] 31. Combination according to item 28, wherein said cells are
adult stem cells selected from the group consisting of
hematopoietic, neural, mesenchymal, mammary, endothelial,
epithelial and olfactory stem cells, in particular selected from
the group consisting of hematopoietic, neural and mesenchymal stem
cells.
[0429] 32. Combination according to item 27, wherein said cells are
cells from islets of Langerhans, for example beta cells.
[0430] 33. Combination according to any preceding item, wherein
said eukaryotic cells are provided as single cells.
[0431] 34. Combination according to item 31, wherein said mammalian
cells are neural stem cells provided as at least one
neurosphere.
[0432] 35. Combination according to item 30, wherein said mammalian
cells are beta cells provided as at least one islet of
Langerhans.
[0433] 36. Combination according to any one of items 24-26, wherein
said mammalian cells are somatic cells, for example selected from
the group consisting of hepatocytes, fibroblasts, keratinocytes and
endothelial cells.
[0434] 37. Combination according to any one of items 24-36, wherein
said cells are human cells.
[0435] 38. Combination according to any one of items 24-37, wherein
said spider silk protein is selected from the group of proteins
defined by the formulas REP-CT and NT-REP-CT, wherein
[0436] NT is a protein fragment having from 100 to 160 amino acid
residues, which fragment is a N-terminal fragment derived from a
spider silk protein;
[0437] REP is a protein fragment having from 70 to 300 amino acid
residues, wherein said fragment is selected from the group
consisting of L(AG).sub.nL, L(AG).sub.nAL, L(GA).sub.nL, and
L(GA).sub.nGL, wherein [0438] n is an integer from 2 to 10; [0439]
each individual A segment is an amino acid sequence of from 8 to 18
amino acid residues, wherein from 0 to 3 of the amino acid residues
are not Ala, and the remaining amino acid residues are Ala; [0440]
each individual G segment is an amino acid sequence of from 12 to
30 amino acid residues, wherein at least 40% of the amino acid
residues are Gly; and [0441] each individual L segment is a linker
amino acid sequence of from 0 to 20 amino acid residues; and
[0442] CT is a protein fragment having from 70 to 120 amino acid
residues, which fragment is a C-terminal fragment derived from a
spider silk protein.
[0443] 39. Combination according to item 38, wherein said spider
silk protein is selected from the group consisting of 4RepCT,
NT4RepCTHis, 5RepCT, 8RepCT, 4RepCTMa2, NT8RepCT and
NTNT8RepCT.
[0444] 40. Combination according to any one of items 24-39, wherein
said spider silk protein comprises a cell-binding motif.
[0445] 41. Combination according to item 40, wherein said
cell-binding motif is an oligopeptide coupled to the remainder of
the spider silk protein via at least one peptide bond.
[0446] 42. Combination according to item 41, wherein said
oligopeptide is coupled to the N-terminal of the remainder of the
spider silk protein.
[0447] 43. Combination according to any one of items 41-42, wherein
said oligopeptide comprises an amino acid sequence selected from
the group consisting of RGD, RGE, IKVAV, YIGSR, EPDIM and
NKDIL.
[0448] 44. Combination according to any one of items 24-43, wherein
said cell scaffold material is in a physical form selected from the
group consisting of film, foam, fiber and fiber-mesh.
[0449] 45. Method or combination according to any preceding item,
in which said spider silk protein is selected from the group
consisting of 4RepCT, NT4RepCTHis, RGD-4RepCT, RGE-4RepCT,
IKVAV-4RepCT and YIGSR-4RepCT.
BRIEF DESCRIPTION OF THE DRAWINGS
[0450] FIG. 1 shows a sequence alignment of spidroin N-terminal
domains.
[0451] FIG. 2 shows a sequence alignment of spidroin C-terminal
domains.
[0452] FIG. 3 is a series of photographs showing murine mesenchymal
stem cells cultured on 4RepCT fibers.
[0453] FIG. 4 is a series of photographs showing human mesenchymal
stem cells cultured on 4RepCT fibers.
[0454] FIG. 5 is a series of photographs showing differentiation of
human mesenchymal stem cells cultured on 4RepCT scaffolds.
[0455] FIG. 6 is a series of photographs showing results of
experiments on adipogenic differentiation of human mesenchymal stem
cells cultured on 4RepCT scaffolds.
[0456] FIGS. 7A-B are series of photographs showing results of
experiments on osteogenic differentiation of human mesenchymal stem
cells cultured on 4RepCT scaffolds.
[0457] FIG. 8 is a pair of photographs at A) 4.times. and B)
10.times. magnification of the RCM-1 hESC cells prior to the
experiments described in Example 2.
[0458] FIG. 9 is a pair of photographs at 10.times. magnification
showing RCM-1 hESC cultures after culturing for 48 hours on A)
CELLstart.TM. CTS.TM. and B) RGD-4RepCT.
[0459] FIG. 10 is a pair of photographs at 10.times. magnification
showing RCM-1 hESC cultures after culturing for 144 hours on A)
CELLstart.TM. CTS.TM. and B) RGD-4RepCT.
[0460] FIG. 11 is a pair of photographs at 10.times. magnification
showing the result of alkaline phosphatase staining of RCM-1 hESC
cultures on A) CELLstart.TM. CTS.TM., and B) RGD-4RepCT as
described in Example 2.
[0461] FIG. 12 is a pair of photographs at 10.times. magnification
showing A) RCM-1 hESC after culturing for 240 hours on RGD-4RepCT,
and B) alkaline phosphatase staining of RCM-1 hESC after culturing
for 240 hours on RGD-4RepCT.
[0462] FIG. 13 is a series of pair-wise photographs showing R1
mESCs cultured for three passages on 4RepCT (WT) foam and fiber
mesh, RGD-4RepCT (RGD) foam and fiber mesh, on MEFs (control) or
gelatin, as indicated.
[0463] FIG. 14 shows NSCs cultured on 4RepCT film and control
(PORN) as indicated, maintained undifferentiated (upper row) and
subjected to neuronal differentiation (lower row). Cells have been
stained with nestin, (upper row) and TuJ1 (lower row).
[0464] FIG. 15 shows NSCs cultured on 4RepCT film and control
(PORN) as indicated, maintained undifferentiated and subjected to
oligodendrocyte (upper row) and astrocyte (lower row)
differentiation. Cells have been stained with MBP (upper row) and
GFAP (lower row). Also, the appearance of NSCs maintained
undifferentiated on 4RepCT foam and subjected to astrocyte
differentiation is shown.
[0465] FIG. 16 shows results from the EdU-assay of NSCs growing on
4RepCT film (at 48 h post seeding), as described in Example 4.
[0466] FIG. 17 shows Live/Dead staining of NSCs growing on 4RepCT
film on the left (at 72 h post seeding) and PORN on the right (at
48 h post seeding). Each column of pictures represents the same
area, showing live and dead cells respectively.
[0467] FIG. 18 is a series of photographs showing that human (A)
and mouse (B) islets of Langerhans adhered to 4RepCT fiber, foam
and film, respectively, after 5-7 days of culture.
[0468] FIG. 19 is a diagram showing that mouse islets showed
significantly higher adherence to the 4RepCT foam structure at all
time points as compared to corresponding fiber and film, and to
control (n=16, ***P=0.001).
[0469] FIG. 20 is a diagram showing the number of human islets
adhered to foam of 4RepCT without (WT) and with different peptide
motifs as indicated after culture for 1-5 days (n=3.+-.SEM; n=2 for
RGE.+-.StdDev).
[0470] FIG. 21 is a pair of diagrams showing islet adherence to the
different scaffolds; A: Mouse islets adherence to foam of 4RepCT
without (WT) and with different peptide motifs as indicated after
culture of 1-5 days (n=4, .+-.SEM, *=p<0.05, **=p<0.01,
***=p<0.001); B: Mouse islets adherence to NT4RepCT foam
compared to RGD-4RepCT and control after culture of 2 days (n=1,
experiment done in triplicates, .+-.SEM)
[0471] FIG. 22A-F is a series of diagrams showing insulin release
upon glucose stimulation of islets cultured for 5 days in wells
with 4RepCT without (WT) and with different peptide motifs as
indicated; A: Insulin release from all mouse islets cultured with
4RepCT without (WT) and with different peptide motifs as indicated,
for 5 days; B: Stimulation index of mouse islets cultured with
4RepCT without (WT) and with different peptide motifs for 5 days;
C: Insulin release from adhered mouse islets cultured with 4RepCT
without (WT) and with different peptide motifs for 5 days (n=2,
experiments done in duplicate); D: Insulin release from adhered
mouse islets cultured with NT4RepCT scaffolds (NT) for 2 days; E:
Insulin release from all human islets cultured with 4RepCT without
(WT) and with different peptide motifs for 5 days; F: Insulin
release from adhered human islets cultured with 4RepCT without (WT)
and with different peptide motifs for 5 days (n=1, experiments done
in triplicates).
[0472] FIG. 23 is a diagram which illustrates the [Ca.sup.2+].sub.i
response after high glucose and KCl stimuli of one islet cultured
for 1 day on RGD-4RepCT.
[0473] FIG. 24 is a pair of photographs, in which morphologic
analysis of human islet viability shows more dispersed islets in
the control group (A) compared to the more intact adhered islets on
RGD-4RepCT foam (B).
[0474] FIG. 25 is a photograph enabling morphologic analysis of
human islets after long-term culture (30 days) on RGD-4RepCT. An
islet can be seen above the star.
[0475] FIG. 26 is a pair of diagrams showing insulin release per
islet after long-term culture of human islets with 4RepCT without
(WT) and with different peptide motifs as indicated. The insulin
release (pmol/l) on day 5 (A) and after 4 weeks (B). Low glucose (3
mM) stimulation gave the basal level of insulin release (white) and
high glucose (16.7 mM) gave the stimulated insulin release (black)
(n=1; experiments done in triplicates).
[0476] FIG. 27 is a pair of photographs at the indicated
magnification, showing positive staining of insulin-producing cells
(white, pointed to by arrows) in human islets and islet-like
cluster after long-term culture (78 days) on RGD-4RepCT foam (light
grey).
[0477] FIG. 28A-C is a series of photographs showing cluster
formation after culture of single islets cells (mouse) on the
control tissue culture plate (A) and on RGD-4RepCT (B). C:
enlargements (63.times.) of insulin-positive clusters (bright;
pointed to by arrows) in foam of 4RepCT (left), RGD-4RepCT (middle)
and IKVAV-4RepCT (right) in close contact with the scaffold.
[0478] FIG. 29 is a pair of photographs at the indicated
magnification, showing adherence and growth of mesenchymal stem
cells (MSC) on foam scaffolds 4RepCT (WT) and RGD-4RepCT (RGD) as
indicated. MSC (gray) could readily adhere to the foam structure
(light gray, exemplified by arrow) and continued to proliferate
over time thereon (day 7, n=2).
[0479] FIG. 30 is a pair of photographs showing co-culture of islet
beta cells, endothelial cells and mesenchymal stem cells ("BEM")
visualized by cell tracking dyes (beta cells: bright white;
endothelial cells:light grey; mesenchymal cells: grey). The
co-cultured BEMs form a cluster on 4RepCT foam. Clusters of BEM
were found adhered to 4RepCT foam (left) and RGD-4RepCT foam
(right, n=2, experiment done in duplicates).
[0480] FIGS. 31A-E are photographs showing cultured endothelial
cells on different 4RepCT scaffolds in the form of film or foam
with different cell-binding motifs, and control plates (cell
culture glass); A: film, left panel 4RepCT (wild-type), right panel
RGD-4RepCT; B: film, left panel IKVAV-4RepCT, right panel
YIGSR-4RepCT; C: foam, left panel 4RepCT (wild-type), right panel
RGD-4RepCT; D: foam, left panel IKVAV-4RepCT, right panel
YIGSR-4RepCT; E: control.
[0481] FIG. 32 is a diagram showing the endothelial cell density
ratio of total area analyzed within a 96-well culture plate coated
with different 4RepCT scaffolds with different cell-binding motifs
as indicated.
[0482] FIG. 33 is a series of photographs of cell scaffold
materials prepared from 4RepCT. The upper panel shows wells in
96-well plate at approx 25.times. magnification. The lower panel
shows scaffolds viewed in an inverted light microscope at
200.times. magnification.
[0483] FIGS. 34 and 35 show diagrams of fibroblast growth on 4RepCT
scaffolds and controls as indicated, measured with Alamar blue
viability assay. Error bars show standard deviation of
hexaplicates.
[0484] FIG. 36 is a series of photographs of HDFn cultured 4 days
on 4RepCT scaffolds and control as indicated, at a seeding density
of 15000 cells/cm.sup.2, and stained with Live/dead for detection
of living and dead cells.
[0485] FIG. 37 shows a diagram of SF-HDF growth on 4RepCT film
under serum-free conditions. The seeding densities (cells/cm.sup.2)
were as indicated. After day 6, the number of cells continued to
increase, thereby exceeding the highest standard and preventing
recalculation of data for plotting. The number of viable cells was
measured with Alamar blue. Error bars show standard deviation of
hexaplicates.
[0486] FIG. 38 shows photographs of HDFn attaching to 4RepCT fiber
(left panel) and film (right panel) as visualized by staining
filamentous actin green with ALEXA FLUOR.TM.488-Phallodin (appears
as grey strands in photos). Nuclei were stained red (appears as
bright white) with EthD-1.
[0487] FIGS. 39A-D show the production of collagen type I by cells
growing on different 4RepCT scaffolds as indicated. A is a diagram
showing the concentration of C peptide in the cell culture medium
secreted during the first 5 days of culture (not accumulated). B is
a diagram showing the amount of C-peptide secreted/cell growing on
the different scaffolds during the same culture period. C-peptide
concentration was determined by EIA. Error bars show standard
deviation of duplicates. C and D are photographs showing
intracellular collagen type I (appears as white dots) present in
cells growing on film (C) and fiber-mesh (D), stained with
immunofluorescence. Nuclei appear as light grey.
[0488] FIG. 40 shows the production and secretion of collagen by
fibroblasts cultured on different 4RepCT scaffolds as indicated for
14 days and then reseeded onto tissue culture treated (TCT) plates
or chamber slides for analysis of procollagen type I C peptide
(upper panel) and intracellular collagen type I production (lower
panel) respectively.
[0489] FIGS. 41A-C are diagrams showing the number of live HDFn
growing on 4RepCT scaffolds of fiber-mesh (A) and film (B-C) with
or without the integrin binding motif RGD as indicated. Assayed
with Alamar blue. Error bars show standard deviation of
hexaplicates.
[0490] FIG. 42 is a diagram showing the number of SF-HDF growing on
wild-type 4RepCT (wt, open bars) or RGD-4RepCT (filled bars) film
at day 3. Seeding densities are given in cells/cm.sup.2. The number
of viable cells is measured with Alamar blue. Error bars show
standard deviation of hexaplicates.
[0491] FIG. 43 is a series of photographs showing fibroblasts grown
on 4RepCT film with various cell binding motifs as indicated.
Fibroblasts exhibit focal adhesions on all film variants after only
3 h, indicating integrin-mediated adhesion to the material. The
focal adhesions appear as bright elongated spots. The cells are
cultured without any serum added. WT: 4RepCT; NRC: NT4RepCTHis.
[0492] FIG. 44 is a diagram showing growth of human primary
keratinocytes on 4RepCT film with or without different cell binding
motifs as indicated, and on NT4RepCTHis (NRC). Controls used were
untreated cell plastic (HP) and Pluronic-coated cell plastic (to
prevent adhesion). Live cells were detected with Alamar blue at day
1 and day 4 after seeding.
[0493] FIG. 45 is a series of photographs showing human primary
keratinocytes in passage 4 after 3 days of culture on 4RepCT film
with various cell binding motifs as indicated. WT: 4RepCT; NRC:
NT4RepCTHis; CTRL: cell culture glass.
[0494] FIG. 46 is a series of photographs showing keratinocytes on
day 4 after seeding on the indicated material. Cells were stained
for vinculin to visualize focal adhesions, which are shown as
bright, elongated spots close to the cell membrane and indicated by
white arrows. WT: 4RepCT, RGD: RGD-4RepCT, IKVAV: IKVAV-4RepCT,
CTRL: cell culture glass.
[0495] FIG. 47 is a set of photographs showing adherence of
hepatocytes to 4RepCT film and fiber (panels A and B,
respectively). Close interaction between hepatocyte and the fiber
scaffold can be seen in C and D (n=1, experiment done in
hexaplicates).
[0496] FIG. 48 is a pair of photographs showing the appearance of
hESC grown on RGD-4RepCT film (RGD) for 16 days (left) and hESCs
grown on control coating (Cellstart) for 192 hours (right). The
cells are in the first passage of the experiment described in
Example 12.
[0497] FIG. 49 is a pair of photographs showing the appearance of
hESC 24 hours post seeding in the second passage of the experiment
described in Example 12 on respective coating. Left: Cellstart
coating (control); Right: RGD-4RepCT film (RGD).
[0498] FIG. 50 is a pair of photographs showing the appearance of
AP-stained hESC 24 hours post seeding in the third passage of the
experiment described in Example 12 on respective coating. Left:
Cellstart coating (control); Right: YIGSR-4RepCT film (Y). Positive
AP staining appears in brown color (dark grey in picture).
[0499] FIG. 51 is a pair of photographs showing the appearance of
AP-stained hESC 24 hours post seeding in the third passage of the
experiment described in Example 12 on respective coating. Left:
cells grown on RGD-4repCT film (RGD); Right: cells grown on
RGE-4RepCT film (RGE). Positive AP staining appears in brown color
(dark grey in picture).
[0500] FIG. 52 is a pair of photographs showing the appearance of
AP-stained hESC 24 hours post seeding in the third passage of the
experiment described in Example 12 on respective coating. Left:
cells grown on IKVAV-4repCT film (IKVAV); Right: cells grown on
NT4RepCTHis film (NRC). Positive AP staining appears in brown color
(dark grey in picture).
EXAMPLES
Example 1
Hematopoietic and Mesenchymal Stem Cells on Recombinant Spider
Silk
[0501] Hematopoietic stem cells (HSC), which are known to be
extremely sensitive to unfavorable influences from their direct
microenvironment, and mesenchymal stem cells (MSC), which have been
shown to adhere and grow on a variety of biodegradable natural and
synthetic scaffolds, were cultured on recombinant spider silk
matrices comprising 4RepCT as described above. HSC could be
cultured on 4RepCT foam and maintained their ability to
differentiate as well as their phenotype when compared to HSC:s
cultured on Falcon 1008 plastic and retronectin-coated plates. MSC
showed similar cell count and differentiation as compared to
controls when grown on 4RepCT fibers, and retained their ability to
differentiate into cells of mesodermal lineage, such as bone,
cartilage and fatty cells, when grown on 4RepCT films and
foams.
Materials and Methods
Expression of Recombinant Spider Silk Proteins
[0502] The recombinant spider silk protein 4RepCT (SEQ ID NO:2) was
produced as previously described (Hedhammar et al (2008), supra).
Briefly, Escherichia coli BL21(DE3) cells (Merck Biosciences) with
the vector for 4RepCT expression were grown at 30.degree. C. in
Luria-Bertani medium containing kanamycin to an OD.sub.600 of 0.8-1
and then induced with isopropyl 13-D-thiogalactopyranoside and
further incubated for up to 3 h at room temperature. Thereafter,
cells were harvested and resuspended in 20 mM Tris-HCl (pH 8.0)
supplemented with lysozyme and DNasel. After complete lysis, the
supernatants from centrifugation at 15,000 g were loaded onto a
column packed with Ni SEPHAROSE.TM. (GE Healthcare, Uppsala,
Sweden). The column was washed extensively before elution of bound
proteins with 300 mM imidazole. Fractions containing the target
proteins were pooled and dialyzed against 20 mM Tris-HCl (pH 8.0).
4RepCT was released from the tags by proteolytic cleavage using a
thrombin:fusion protein ratio of 1:1000 (w/w) at room temperature
for 1-2 h. To remove the released HisTrxHis tag, the cleavage
mixture was loaded onto a second Ni SEPHAROSE.TM. column and the
flowthrough was collected. The protein content was determined from
the absorbance at 280 nm.
Scaffold Preparation
[0503] Purified 4RepCT proteins were allowed to self-assemble into
fibers as described in Stark et al (2007), supra. The fibers were
then cut into smaller pieces before being used for culturing in
non-tissue culture treated Falcon dishes (Falcon 1008). Films were
prepared by coating of Falcon 1008 dishes with 0.5-2.0 ml protein
solution and air drying to allow formation of a thin layer at the
bottom of the dishes. In addition, some Falcon 1008 dishes were
coated with foam, obtained after vigorous mixing of the protein
solution, and allowed to air dry for 24 hours to form a 3D matrix
on the bottom of the dishes. Dishes with fibers, films or foams
were sterilized by exposure to 280 Gy .gamma.-radiation, delivered
by a .sup.137Cs source (Gammacell, Atomic Energy of Canada, Ottawa,
Canada).
Hematopoietic Stem Cell Isolation and Culture
[0504] Healthy Balb/c mice were killed and femurs removed. Bone
marrow (BM) cells were flushed from femora with Hanks' balanced
salt solution buffered with 10 mM HEPES buffer (HH; GIBCO). BM
cells were either used directly or after lineage depletion with a
lineage cell depletion kit (Miltenyi Biotec) according to the
manufacturer's instructions and cultured for 4 days, at
3.times.10.sup.5 and 5.times.10.sup.4 cells/dish, respectively, in
serum-free DMEM (Wognum et al (2000), Hum Gene Ther 11:2129-2141),
supplemented with murine stem cell factor (mSCF, 100 ng/ml;
Immunex, Seattle, Wash.) and mIL-3 (30 ng/ml; Genentech, San
Francisco, Calif.).
Measurement of Surface Antigens
[0505] Murine bone marrow cells, before and after culture in the
presence of 4RepCT, were collected for phenotypic analysis.
Briefly, cells were washed twice with Hanks' buffered HEPES
solution (HHBS) containing 0.5% (vol/vol) bovine serum albumin
(BSA; Sigma, St Louis, Mo.), 0.05% (wt/vol) sodium azide, and 0.45%
(wt/vol) glucose (Merck, Darmstadt, Germany) (HBN) and resuspended
in 50 .mu.l HBN containing 2% (vol/vol) normal, heat-inactivated
mouse serum to prevent nonspecific binding of the monoclonal
antibodies (MoAbs) and subsequently incubated for 30 minutes with
MoAbs raised against the following surface markers: c-kit, sca-1,
CD4, CD8, CD11b and B220 (BD Biosciences, San Jose, Calif.). Cells
were washed twice in HBN and dead cells were excluded from analysis
based on propidium iodine (PI, Sigma) staining. Cell samples were
measured using a FACSCalibur flow cytometer, and 10,000 list mode
events were collected and analyzed using the Cellquest software (BD
Biosciences, San Jose, Calif.).
In Vitro Clonogenic Progenitor Assays
[0506] 5.times.10.sup.4 murine BM cells or 1.times.10.sup.3 lineage
depleted BM cells (lin.sup.-/-) were plated in Falcon 1008 (35 mm
diameter) Petri dishes in 1 ml of serum-free semi-solid
methylcellulose culture medium containing 0.8% (wt/vol)
methylcellulose (METHOCEL.TM. A4M Premium grade, Dow Chemical Co,
Barendrecht, The Netherlands) in enriched DMEM, 1% (wt/vol) BSA,
0.3 mg/ml human transferrin, 0.1 pmol/l sodium selenite, 1 mg/I
nucleosides (cytidine, adenosine, uridine, guanosine,
2'-deoxycytidine, 2'-deoxyadenosine, thymidine and
2'-deoxyguanosine; Sigma), 0.1 mmol/l .beta.-mercaptoethanol, 15
pmol/l linoleic acid, 15 pmol/l cholesterol, 10 .mu.g/ml insulin,
100 U/ml penicillin, and 100 .mu.g/ml streptomycin.
Granulocyte/macrophage colony formation (CFU-GM) was stimulated by
addition of 10 ng/ml mIL-3, 100 ng/ml mSCF, and 20 ng/ml GM-CSF and
scored at day 8-10 of culture. Burst-forming erythroid (BFU-E)
growth was induced by 100 ng/ml mSCF and 4 U/ml human
erythropoietin (hEPO; Behringwerke, Marburg, Germany) and counted
after 8-10 days, whereas colony-forming unit erythroid (CFU-E)
growth was stimulated with hEPO alone and counted after 2 days.
Megakaryocyte progenitor cells (CFU-Meg) were stimulated in 0.275%
agar cultures supplemented with 100 ng/ml mSCF, 10 ng/ml mIL-3m,
and 10 ng/ml mTPO (Genentech, San Francisco, Calif.). Colonies were
dried after 10 days and stained for acetyl cholinesterase positive
cells, and enumerated.
Mesenchymal Stem Cell Isolation and Culture
[0507] Human mesenchymal stem cells (hMSC) were purchased from
Lonza (Verviers, Belgium). Cells were cultured in complete medium-1
(CM-1) consisting of 54% DMEM-LG, 36% MCDB-201, 10% FCS, 1 mM
L-Glutamin and 1% penicillin/streptomycin (Reyes et al (2001),
Blood 98:2615-2625). Cells were trypsinized and subcultured when
confluence reached 80-90%, and medium was refreshed every 3-4
days.
[0508] Murine mesenchymal stem cells (mMSC) were obtained by
flushing the femurs of Balb/c mice with HH. Full BM cells were
cultured in the presence of DMEM-LG supplemented with 10% FCS, 1 mM
L-Glutamin and 1% penicillin/streptomycin (CM-2). Adherent cells
were subcultured and passaged once weekly. Medium was changed every
3-4 days.
Differentiation Assays
[0509] For adipogenic differentiation, MSC:s were cultured in the
presence of adipogenic medium consisting of DMEM-LG, 10% FCS, 1
.mu.M dexamethasone, 60 .mu.M indomethacine, 500 .mu.M
isobutylmethylxanthine (IBMX) and 5 .mu.g/ml insulin (Sigma, St.
Louis, USA) for 21 days and stained with Oil Red 0 (Sigma, St
Louis, USA). For osteogenic differentiation, MSC:s were maintained
for 21 days in osteogenic medium consisting of DMEM-LG, 10% FCS,
100 nM dexamethasone, 10 mM .beta.-glycerophosphate and 0.2 mM
ascorbic acid (Sigma, St. Louis, USA). Cells were stained with
Alizarin Red S (Sigma, St. Louis, USA) to confirm presence of
calcium phosphate deposits. For chondrogenic differentiation,
2.5.times.10.sup.5 cells were spun down in a 15 ml polypropylene
tube and the spontaneously formed three-dimensional pellet was
cultured for 21 days in chondrogenic medium consisting of DMEM-HG
(Gibco) with 100 nM dexamethasone, 10 ng/ml TGF.beta.3 (Peprotech,
USA), 50 .mu.g/ml ascorbic acid, 50 mg/ml ITS+Premix (Becton
Dickinson, USA). Sections of the pellet were prepared for
histological studies and stained with Alcian Blue for confirmation
of chondrocytic lineage.
Results
Stem Cell Expansion and Differentiation in the Presence of 4RepCT
Fibers
[0510] In three separate experiments, performed in duplicate, the
expansion of murine bone marrow cells in presence or absence of
small pieces of 4RepCT was investigated. Results are displayed in
Table 3, which shows the effect of 4 day culture in the presence of
4RepCT on expansion of murine bone marrow (BM) cells in serum-free
medium supplemented with 100 ng/ml mSCF and 30 ng/ml mIL-3. Cells
counted were colony forming units-erythrocyte (CFU-E);
burst-forming unit-erythrocyte (BFU-E); colony forming
unit-granulocyte/macrophage (CFU-GM); and colony forming
unit-megakaryocyte (CFU-Meg). No significant differences were found
between cell numbers and amount of colonies formed after culture
for 4 days in the presence of 4RepCT fibers in comparison to
control wells.
TABLE-US-00004 TABLE 3 Day 4 Day 4 BM- Day 0 BM control BM S.I.
4RepCT S.I. CFU-E.sup.1 507.0 .+-. 98.1 2599.7 .+-. 375.3 5.1
2296.7 .+-. 1413.0 4.5 BFU-E.sup.1 106.7 .+-. 8.5 173.5 .+-. 61.5
1.6 282.0 .+-. 183.8 2.6 CFU- 210.3 .+-. 50.4 1935.7 .+-. 663.4 9.2
1495.3 .+-. 187.6 7.1 GM.sup.1 CFU- 40.3 .+-. 13.6 163.0 .+-. 17.6
4.0 91.0 .+-. 78.9 2.3 Meg.sup.1 Cells.sup.2 3.0 .+-. 0.0 8.2 .+-.
3.6 2.7 5.5 .+-. 2.6 1.8 Results are expressed as the average of
three separate experiments .+-. standard deviation. All experiments
were done in duplicates. .sup.1Colonies per 1 .times. 10.sup.5 BM
cells .sup.2Cells .times. 10.sup.5 S.I.: stimulation index in
comparison to day 0 values
[0511] Murine MSC:s were maintained in culture medium until
subconfluence and then trypsinized. 1.times.10.sup.5 mMSC were
cultured in 6-well plates in presence or absence of 1 cm pieces of
4RepCT fibers for 7 days and then trypsinized and counted. Control
wells showed a 5.7-fold expansion after 7 days, whereas cells
cultured in the presence of 4RepCT expanded 4.0-fold. However, only
cells remaining in the culture dishes were trypsinized, and cells
growing on the fibers were not included in the total cell count,
thus leading to an underestimation of the actual number of cells
present in each well. To prevent excretion of inhibitory signals
due to contact inhibition in dense near-confluent cultures, after 7
days, 14 days and 21 days, the fibers were carefully removed from
the culture dishes, rinsed once with PBS and transferred to a fresh
well containing culture medium only. Cells arising in these culture
dishes were thus all derived from a single source, i.e. the
recombinant spider silk threads (FIG. 3).
[0512] Human MSC:s were maintained in culture medium under similar
conditions as the mMSC:s. Fibers were transferred to fresh culture
dishes once weekly (FIG. 4). At day 21 of culture, 4RepCT fibers
were removed. The remaining hMSC:s, which covered the surface of
the culture wells and were derived from the recombinant spider silk
fibers, were trypsinized and tested for their capacity to
differentiate into adipogenic, osteogenic and chondrogenic lineage
(FIG. 5). All tests were done in triplicate. Cells derived from the
4RepCT fibers displayed a comparable differentiation capacity
compared with hMSC:s from the same passage cultured in the absence
of 4RepCT.
Expansion and Differentiation on 4RepCT-Coated Tissue Culture
Plates
[0513] Murine bone marrow cells were lineage depleted (lin.sup.-/-)
and cultured on culture dishes covered with 4RepCT foam in
serum-free medium containing mSCF and mIL-3. Expansion of
lin.sup.-/- BM cells and colony forming unit numbers, after 4 days
of culture, were compared with cultures of lineage negative cells
cultured on non-tissue culture treated dishes (35 mm, Falcon 1008)
and Falcon 1008 dishes coated with recombinant fibronectin fragment
CH-296 (Retronectin: RN; Takara Shuzo, Otzu, Japan) at a
concentration of 10 .mu.g/cm.sup.2. Results are shown in Table 4,
for the number of in vitro burst-forming unit-erythrocyte (BFU-E);
colony forming unit-granulocyte/-macrophage (CFU-GM); and colony
forming unit-megakaryocyte (CFU-Meg).
TABLE-US-00005 TABLE 4 Day 4 Day 4 lin.sup.-/- Day 4 lin.sup.-/-
Day 0 Falcon lin.sup.-/- 4RepCT lin.sup.-/- 1008 S.I. retronectin
S.I. foam S.I. BFU-E.sup.1 340 70 0.2 35 0.1 0 0 CFU-GM.sup.1 1385
2555 1.8 1720 1.2 1310 0.9 CFU- 50 10 0.2 15 0.3 35 0.7 Meg.sup.1
Cells.sup.2 4.0 57 14.3 62 15.5 63 15.8 Results of one experiment
performed in duplicate are shown .sup.1Colonies per 1 .times.
10.sup.5 BM cells .sup.2Cells .times. 10.sup.5 S.I.: stimulation
index in comparison to day 0 values
[0514] In addition, expression of cell surface markers was measured
before and after culture on differentially coated dishes (Table
5).
TABLE-US-00006 TABLE 5 Day 4 Day 4 lin.sup.-/- Day 4 lin.sup.-/-
Day 0 Falcon lin.sup.-/- 4RepCT lin.sup.-/- 1008 S.I. retronectin
S.I. foam S.I. CD4 0.1 0.5 5.0 0.6 6.0 0.8 8.0 CD8 0.1 0.1 1.0 0.2
2.0 0.1 1.0 CD11b 0.2 8.6 43.0 12.1 60.5 20.0 100.0 B220 0.1 0.8
8.0 0.3 3.0 0.3 3.0 Sca-1 0.1 25.6 256.0 20.6 206 4.5 45.0 c-Kit
0.5 1.1 2.2 1.1 2.2 1.7 3.4 Results of one experiment performed in
duplicate are shown. Data are expressed as absolute cell numbers
.times. 10.sup.5. S.I.: stimulation index in comparison to day 0
values
[0515] 1.times.10.sup.5 senescent hMSC:s were plated onto
non-tissue culture treated dishes (35 mm, Falcon 1008), tissue
culture treated dishes (35 mm, Falcon 3001), tissue culture treated
6-well plates (Falcon) and dishes coated with 4RepCT film or foam
(35 mm, Falcon 1008), and cultured until almost confluent. Cells
were then maintained for 21 days in control medium, adipogenic
differentiation medium or osteogenic differentiation medium.
Results are displayed in FIGS. 6 and 7 for adipogenic
differentiation and osteogenic differentiation, respectively. Cells
cultured in the presence of adipogenic medium showed, independently
of the coating of the dishes, a similar degree of adipogenic
differentiation, having rounded cells containing multiple lipid
vesicles divided equally over the surface of the dishes. In
contrast, the senescent hMSC:s were no longer able to differentiate
into osteogenic lineage despite regular medium changes with
osteogenic differentiation medium, when cultured under any of the
control conditions (FIG. 7A), but interestingly showed some patchy
Alizarin Red S staining in the film and foam coated plates (FIG.
7B, upper left and right, respectively). This staining could not be
attributed to staining of the 4RepCT itself, as plates coated with
either 4RepCT film or foam, containing no cells, did not show any
positive Alizarin Red S areas (FIG. 7B, lower left and right,
respectively).
Example 2
Human Embryonic Stem Cells on Recombinant Spider Silk
[0516] The experiment shows the feasibility of culturing human
embryonic stem cells on a recombinant spider silk material.
Materials and Methods
[0517] Standard six-well tissue culture plates were prepared. In
the plates, three wells contained RGD-4RepCT film and the remaining
three wells were empty. The 4RepCT film with the cell-binding motif
RGD was prepared essentially as described in Example 1. The tissue
culture plates were kept dry at room temperature until further
use.
[0518] In preparation for the experiment, the tissue culture plates
were UV irradiated for 30 minutes in a Class II microbiological
safety cabinet. The three empty wells in each plate were then
coated with CELLstart.TM. CTS.TM. (Invitrogen; cat no A10142-01),
as per manufacturer's protocol.
[0519] Human embryonic stem cells (RCM-1, De Sousa et al, Stem Cell
Res 2:188-197; Roslin Cells, Edinburgh, UK) were cultured on
CELLstart.TM. with the serum and feeder free medium STEM PRO.RTM.
hESC SFM--Human Embryonic Stem Cell Culture Medium (Invitrogen; cat
no A1000701), as per manufacturer's protocol. The cells were
passaged at a ratio of 1:6 from a 90% confluent well using
STEMPRO.RTM. EZPassage.TM.--Disposable Stem Cell Passaging Tool
(Invitrogen; cat no 23181-010) as per manufacturer's protocol.
[0520] Wells were washed with PBS, and medium was placed in all
wells and preincubated prior to cell seeding. Cells at passage 64
were seeded into all wells of all plates at the recommended
density, and care was taken not to disturb cells after seeding. All
wells were cultured with STEMPRO.RTM. hESC SFM as per
manufacturer's protocol. Incubation was done in a standard culture
incubator at 37.degree. C., 5% CO.sub.2 in air, at 95% humidity.
Cells were observed daily and 100% medium exchanged every 48 hours.
All medium was pre-equilibrated to incubator conditions for two
hours prior to exchange feeding.
[0521] At the end of study, wells were stained with Sigma-Aldrich:
Alkaline Phosphatase (AP), Leukocyte (Sigma-Aldrich; Ref 86R-1 KT,
Lot 019K4349) as per manufacturer's protocol.
Results
[0522] Selected images from the cell culture experiments are
presented as FIGS. 8-12.
[0523] All control wells showed the characteristics and morphology
as would be expected with this hESC line under the control culture
conditions employed.
[0524] The RGD-4RepCT films were successful in sustaining cell
attachment and expansion. Even though the growth was much slower
than that seen in the control wells, this is thought to be a
consequence of the cell line adapting to the new matrix. Cells
showed, on initial cell plating, some adherence to the matrix, but
non-adherent cells took on the appearance of Embryoid Body (EB)
structures, which are seen when hESC:s are placed into non-adherent
or low cluster plates to specifically derive EB structures for
lineage and differentiation analysis. However, these EB like masses
of cells did eventually adhere to the matrix, and from these
"clumps", cells were seen to grow and expand, suggesting adaptation
to the new matrix.
[0525] The cells of this study are continually grown and fed, and
are expected to continue to expand. It is furthermore expected that
the cells will be capable of passaging and expansion and can then
be subsequently assessed for their pluripotency and differentiation
status. The positive alkaline phosphatase staining indicates that
the cells still exhibit undifferentiated stem cell
characteristics.
Example 3
Mouse Embryonic Stem Cells on Recombinant Spider Silk
Background
[0526] Mouse embryonic stem cells (mESCs) can be either
feeder-dependent (i.e. they need to be cultured on a layer of mouse
embryonic fibroblasts, MEFs) or feeder-independent (usually
cultured on gelatin). mESCs need to be cultured with LIF (leukemia
inhibitory factor) present in the culture medium in order to remain
undifferentiated. Their differentiation status (or maintenance of
"sternness") can be determined by staining of alkaline phosphatase
activity (AP), since pluripotent cells express AP and thus stain
positive, or by determination of the transcription of a set of
differentiation markers (genes).
Procedure
[0527] Feeder-dependent mESCs (R1) in passage 17 were thawed and
plated onto a layer of MEFs (irradiated, 1 day culture) in a 60 mm
Petri dish in DMEM with glutamax (Invitrogen, cat no 31966-021)
supplemented with 20% heat inactivated, ES-cell qualified FBS
(Invitrogen, cat no 16141-079) and 5.times.10.sup.5 units/ml of LIF
(Chemicon, ESG 1107). After 2 days, cells were harvested and seeded
onto different 4RepCT scaffolds prepared as described in the
Examples herein (foam and fiber mesh of 4RepCT and foam and fiber
mesh of RGD-4RepCT), as well as onto controls (MEFs or gelatin) in
12-well plates. The split ratio was 1:7. Medium was changed every
day, and cells were split every second day (MEFs or gelatin) or
every fourth day (4RepCT scaffolds). Longer split intervals were
used for cells grown on scaffolds since they proliferated slower.
To evaluate whether cells had maintained pluripotency or not, cells
were fixed with 4% paraformaldehyde for 1 min and then stained for
alkaline phosphatase activity (kit from VECTOR Laboratories) at the
end of each passage (p19, p20 and p21), or after 3 days for cells
on scaffolds in passage 21 (2 days for cells on gelatin or MEFs).
Thus, cells that were maintained on scaffolds had been cultured for
4+4+3=11 days in total at the end of p21, whereas cells maintained
on gelatin or MEFs had been cultured for 2+2+2=6 days in the end of
p21. Micrographs were taken in an inverted microscope.
Results
[0528] mESCs grown on gelatin started to differentiate and lose
morphology, whereas cells grown on MEFs showed maintained rounded
colony morphology and stemness also after 3 passages. mESCs
exhibited a lower degree of binding to both foam and fiber mesh
compared to the binding to MEFs, as indicated by a lower number of
colonies on all scaffold types compared to MEFs. The number of
colonies growing on the scaffolds was approximately the same as the
number of colonies growing on gelatin.
[0529] Once bound to the scaffold material, cells grew and formed
colonies on foam and fiber mesh, although some colonies tended to
show a less smooth shape as compared to MEFs. Colonies looked
similar to those on gelatin. After 4 days without split on foam and
fiber mesh, colonies were very large and had partly started to
differentiate (i.e. lose their AP positivity). In the third passage
(p21), cells were allowed to grow on respective scaffold material
for 3 days. Colonies maintained their AP positivity on foam, but
showed signs of differentiation on fiber mesh. On 4RepCT foam,
colonies were both more numerous and larger, as compared to on
RGD-4RepCT foam (FIG. 13).
[0530] With reference to FIG. 13, cells were stained for AP
activity at day 3 (foam and mesh) and at day 2 (control and
gelatin) of the 3rd passage on respective substrate (cell passage
21). Cells growing on 4RepCT (WT) foam showed a maintained
stemness, as indicated by AP staining (dark grey colonies) similar
to that seen for mESCs on MEFs (control). On the contrary, mESCs
growing on fiber mesh showed a weaker staining, which is a sign of
differentiation. Colonies on RGD4RepCT foam were smaller but AP
positive, indicating less proliferation but maintained
stemness.
[0531] The differentiation seen in colonies on foam and fiber mesh
after 4 days culture without split could be the result of colonies
being overgrown and too large, but it could also be due to lack of
factors secreted by MEFs, which the cells are dependent on to
maintain their pluripotency. This is supported by the observation
that cells maintained on gelatin without MEFs had also started to
differentiate (already after 2 days). On fiber mesh, the cells
started to differentiate after 3 days, at a point when colonies are
still of sizes comparable to colonies on MEFs after 2 days.
Conclusion
[0532] mESCs show binding and proliferation on 4RepCT and
RGD-4RepCT foam and fiber mesh. After 4 days, the mESCs on 4RepCT
and RGD4RepCT foam and fiber mesh started to differentiate,
possibly due to the large size of the colonies. However, when these
cells were re-seeded onto newly prepared scaffolds and grown for 3
days, they maintained their stemness on 4RepCT and RGD-4RepCT foam,
and the size of the colonies were similar to those seen on MEFs
after two days. Slower growth was observed on RGD-4RepCT foam
compared to 4RepCT foam. Cells maintained for 2 days on gelatin
started to differentiate and mESCs on MEFs will also start to
differentiate after 4 days due to large (overgrown) colonies. On
4RepCT foam, attachment, growth and maintained stemness were
improved compared to gelatin.
[0533] The results are surprising, since mESCs are normally
dependent on factors provided by the MEFs to keep their
stemness
Example 4
Neural Stem Cells (NSCs) on Recombinant Spider Silk
Materials and Methods
Preparation of Wells Containing Scaffolds and Positive Control
Wells
[0534] 4RepCT, IKVAV-4RepCT and RGD-4RepCT were recombinantly
produced and purified in analogy to the description in Hedhammar et
al (2008), supra. One fraction of the protein solutions obtained
was purified from lipopolysaccharides (Ips) as described in
Hedhammar et al (2010), Biomacromolecules 11:953-959. The protein
solutions were sterile filtered (0.22 .mu.m) before being used to
prepare scaffolds (film, foam or fibers) as described in Example 1.
Half of the scaffolds were made from protein solutions depleted of
lipopolysaccharides. Fibers were sterilized through autoclaving for
15 minutes at 121.degree. C. in distilled water at 2.8 bar before
being put into the cell culture plates. Scaffolds were prepared in
hydrophobic 6-well cell culture plates (Sarstedt). As positive
control, wells coated with poly-L-ornitine and fibronectin (PORN)
were used. Representative schematics of the 6-well plates with
"wild-type" 4RepCT are given below:
TABLE-US-00007 Film, 4RepCT Film, 4RepCT Film, 4RepCT Coated,
positive control Film, 4RepCT, lps Film, 4RepCT, lps Film, 4RepCT,
lps depleted depleted depleted Coated, positive control Foam,
4RepCT Foam, 4RepCT Foam, 4RepCT Coated, positive control Foam,
4RepCT, lps Foam, 4RepCT, lps Foam, 4RepCT, lps depleted depleted
depleted Coated, positive control Fiber-mesh, 4RepCT Fiber-mesh,
4RepCT Fiber-mesh, 4RepCT Coated, positive control Fiber-mesh,
4RepCT, Fiber-mesh, 4RepCT, Fiber-mesh, 4RepCT, lps depleted lps
depleted lps depleted Coated, positive control
[0535] In addition, similar plates were prepared by exchanging the
"wild-type" 4RepCT scaffold with scaffolds made from IKVAV-4RepCT
and RGD-4RepCT, respectively, making a total of 9 experimental
plates.
[0536] Two wells of a tenth six-well plate to serve as control
plate were prepared using the following protocol: [0537] Addition
of 2 ml of poly-L-ornithine solution to each well [0538] Incubation
over night in cell culture incubator at 37.degree. C. [0539]
Removal of poly-L-ornithine solution by aspiration [0540] Washing
twice with PBS 1.times. [0541] Addition of 2 ml fibronectin
solution per well [0542] Incubation for 2-4 h in cell culture
incubator [0543] Washing twice with PBS 1.times.
[0544] The remaining four wells of the plate were not coated, i.e.
cells were seeded directly onto the polystyrene plastic surface.
Thus, the control plate can be schematically represented as:
TABLE-US-00008 Coated, Empty Empty positive control Coated Empty
Empty positive control
The above described material was used for culturing of NSCs in an
undifferentiated state for 48 hours, whereupon the cells were
differentiated into astrocytes. In addition, for a detailed
investigation of neural stem cell (NSC) characteristics, a series
of cell culture plates with 4RepCT films was prepared as follows:
[0545] 3 six-well polystyrene plates (Sarstedt) with 4RepCT film in
five of the six wells in each plate. The empty well and one
additional well were coated with fibronectin and poly-L-ornithine
according to the above. These plates were used for growing the NSCs
in an undifferentiated state for 48-96 hours, after which the cells
were differentiated into astrocytes, oligodendrocytes and neurons
(cf below). [0546] Control plate: 1 six-well polystyrene plate
(Sarstedt). The first row was coated with fibronectin and
poly-L-ornithine according to the above, the second row was coated
with BSA (bovine serum albumin), and the third row was left
uncoated. [0547] Seven 35-mm polystyrene plates (Sarstedt) with
4RepCT film are prepared to specifically and accurately analyze the
proliferation (cell division) rate and proportion of cell
death.
Solutions
Medium:
[0548] N2 medium (500 ml)
[0549] DMEM:F12 (1:1)+L-glutamine (500 ml bottle; Gibco
11320-074)
[0550] 1 ml of 50 mg/ml transferrin (Sigma T-1147; diluted in
DMEM:F12)
[0551] 100 .mu.l of 100 .mu.M progesterone
[0552] 50 .mu.l of 1 M putrescine solution
[0553] 30 .mu.l of 500 .mu.M sodium selenite
[0554] 1 ml of 12.5 mg/ml insulin
[0555] 5 ml Pen/Strep (100.times.) solution
[0556] N2 medium is a standard medium for the culture of primary
(tissue-derived, non-cell-line) cells.
Buffers:
HANKS (500 ml)
[0557] 50 ml of 10.times.HBSS (Gibco 14170), 1.85 g NaHCO.sub.3 and
1.95 g HEPES dissolved in ddH.sub.2O and adjusted to pH 7.2. Filter
sterilized.
Working Solutions:
[0558] Poly-L-ornithine (15 .mu.g/ml) in ddH.sub.2O, filter
sterilized (Sigma P-3655) Fibronectin (1 .mu.g/ml) in ddH.sub.2O,
filter sterilized (Sigma F-1141)
NaOH (10 mM)
Stock Solutions:
[0559] Putrescine (1 M) in ddH.sub.2O (Sigma P-5780)
Progesterone (100 .mu.M) in EtOH (Sigma P-8783)
[0560] Sodium selenite (500 .mu.M) in ddH.sub.2O (Sigma S-5261) FGF
(10 .mu.g/ml) in PBS (R&D Systems, rhFGF-basic) Insulin (12.5
mg/ml) in 0.02 M HCl, sterile filtered (Sigma 1-6634) Transferrin
(50 mg/ml) in DMEM:F12, sterile filtered (Sigma T-1147)
Cortical Neural Stem Cell Cultures
[0561] Neural stem cells (NSCs) were obtained from the dissociated
cerebral cortices of timed pregnant Sprague Dawley E15.5 rat
embryos. NSC:s were cultured in 1 ml/962 mm.sup.2 of serum-free
DMEM:F12 medium, enriched with N2 supplement and grown on
poly-L-ornithine/fibronectin coated cell culture dishes. Cells were
maintained in a proliferative state using 10 ng/ml FGF2 until
reaching 80% of confluence, and passaged twice before use in
experiments. After the second passage, cells were plated at 150000
cells/cm.sup.2 and allowed to proliferate for 48 h. To determine if
the cells had remained undifferentiated, they were stained with
nestin.
[0562] To induce differentiation of NSCs, FGF2 was withdrawn from
the cultures and fresh medium added along with either specific
recombinant growth factors or small molecules to induce specific
differentiation.
[0563] For astrocytic differentiation, 10 ng/ml recombinant CNTF
(ciliary neurotrophic factor) was added. CNTF and other factors of
the interleukin-6 family (e.g., CT-1, LIF) induce a rapid (within
48 hours) and efficient (>>50%) differentiation of cortical
NSCs into cells with an astrocytic morphology and positive for the
archetypical astrocyte marker GFAP (Hermanson et al (2002), Nature
419:934-939), and it has recently been shown by calcium imaging
techniques that these cells are functional astrocytes (Andersson et
al (2011), Mol Cell Neurosci, Epub 14 Jan. 2011).
[0564] For neuronal differentiation, either 0.5-1.0 mM valproic
acid (VPA) or 10 ng/ml recombinant BMP4 (bone morphogenetic protein
4) and 10 ng/ml Wnt3a were administered after FGF2 withdrawal. VPA
induces a rapid (within 72 hours) differentiation of NSCs into
10-30% of early, electrophysiologically non-responsive cells with
neuronal morphology, which are positive for the neuronal marker
antibody TuJ1. Gene expression analysis of VPA-induced
differentiated cells suggests that they initiate a differentiation
program towards inhibitory (GABAergic) neurons. BMP4+Wnt3a induce a
slow (5-14 days; herein 7 days) differentiation of NSCs into 10-30%
of mature, electrophysiologically active cells with neuronal
morphology, which are positive for all pan-neuronal markers,
including TuJ1 (Leao et al (2010), PLoS One 5:e13833). Gene
expression and physiological analysis of BMP4+Wnt3a-induced
differentiated cells suggest that they initiate a differentiation
program towards excitatory (glutamatergic) neurons. BMP4+Wnt3a
treatment also results in increased astrocytic differentiation
contributing to the mature phenotype of the neuronal cells, as
compared to cells differentiated using VPA.
[0565] For oligodendrocytic differentiation, 50 ng/ml of thyroid
hormone (T3) was added. T3 induces an enhanced differentiation
(1-20%) into cells with oligodendrocyte morphology, which are
positive for most archetypical oligodendrocyte characteristic
proteins (e.g. MBP) within 4-7 days. Although the differentiation
is less efficient than that resulting from other protocols, it
should be noted that the number of oligodendrocytic cells in
control cultures (only FGF2 withdrawal) is very low, such as
<<1%.
Immunocytochemistry
[0566] For immunocytochemistry, cultures were washed once with PBS
and fixed using 10% formalin (Sigma) for 20 min. Next, cells were
washed 3.times.5 min with PBS+0.1% Triton X100. The primary
antibody was incubated over night at 4.degree. C., (antibody
dilution 1:500 in PBS+0.1% Triton X100+BSA 0.1%).
[0567] Next, plates were washed 6.times.5 min with PBS+0.1% Triton
X100, and incubated for 1 h at room temperature with secondary
antibody.
[0568] The following antibodies were used: mouse monoclonal
anti-smooth muscle actin (SMA) from Sigma (1:1000); rabbit
polyclonal anti-glial fibrillary acidic protein (GFAP) from DAKO
(1:500); mouse TuJ1 (to detect neurons) from CoVance (1:500), and
rat anti-MBP (MAB386) from Chemicon (1:250) followed by appropriate
species specific Alexa-488 and Alexa-594 conjugated secondary
antibodies (Molecular Probes; 1:500). Nuclei were visualized using
Vectashield containing DAPI (Vector Laboratories, Inc.).
Fluorescent and brightfield images were acquired using a Zeiss
Axioskop 2 mot plus/Axiocam MRm camera with Axiovision
software.
Proliferation Assay
[0569] Cells were fixed with 10% formalin 15 min after the addition
of 50 .mu.M 5-ethynyl-2'-deoxyuridine (EdU; Invitrogen) followed by
immunocytochemistry according to the supplier's
recommendations.
Cell Death Assay
[0570] A Live/Dead kit (Invitrogen) was used to differentiate
between living and dead cells attached to the scaffolds. Plates
were rinsed twice with pre-warmed PBS (37.degree. C.) before
proceeding with the assay according to the manufacturer's
recommendations. The assay enabled identification of live cells
(green color) and dead cells (red color).
Results
[0571] Experiments on Proliferation and Viability of Neural Stem
Cells when Grown and Expanded on Fiber-Mesh, Foam or Film, and
Potential to Differentiate into Astrocytes, Neurons and
Oligodendrocytes. Unless Otherwise Stated, all Experiments were
Performed in Triplicates (n=3).
[0572] After 48-72 h, NSCs proliferated normally on foam and film.
In wells were a part of the plastic was exposed next to the film
and foam, cells only grew on the films and foams. The morphology
was indistinguishable from control cultures (poly-L-ornithin and
fibronectin). When stained with nestin (48 h post seeding), the
appearance of NSCs on 4RepCT film was indistinguishable from that
of cells growing on poly-L-ornithin and fibronectin (FIG. 14).
[0573] In wells with silk scaffolds that had been coated with
fibronectin and poly-L-ornithine, the cells grew all over the plate
wells, which is expected since also the plastic surface of the well
is coated. No obvious or significant differences were seen at any
stage beyond 48 hours in proliferation or cell death between NSCs
grown on 4RepCT films compared to control. Also, no obvious
difference was seen between NSCs grown on 4RepCT foam structures
compared to control (n=1). In wells with BSA coating, the cells had
not attached and were dying within 5 days, as expected.
[0574] NSCs remained morphologically indistinguishable from stem
cells in control cultures in the presence of FGF2 when grown on
4RepCT film and foam structures. They were viable (85% as
determined by Live/Dead staining), proliferating (28% as determined
by the EdU assay, controls 20-30%) and remained in an
undifferentiated state, and no significant differences compared to
control cultures (using poly-L-ornithine and fibronectin) were
detected (FIGS. 14, 16 and 17).
[0575] To test whether the NSCs remained multipotent with regard to
differentiation capacity, a series of protocols to test
differentiation into various neural lineages (e.g., neurons,
astrocytes, oligodendrocytes) were applied as described in detail
in the Methods section above.
[0576] When NSCs grown on 4RepCT films were treated with CNTF as
described in Methods, they differentiated rapidly and efficiently
into cells with astrocytic morphology expressing the archetypical
marker protein GFAP with no significant difference in efficiency,
proliferation or cell death compared to control cultures
(poly-L-ornithine and fibronectin) (FIG. 15)
[0577] When NSCs grown on 4RepCT films were treated with BMP4+Wnt3a
as described in Methods, they differentiated into cells with
neuronal morphology positively stained with the archetypical
antibody TuJ1 with no significant difference in efficiency,
proliferation or cell death compared to control cultures
(poly-L-ornithine and fibronectin) (FIG. 14).
[0578] When NSCs grown on 4RepCT films were treated with VPA as
described in Methods, they differentiated into cells with neuronal
morphology positively stained with the archetypical antibody TuJ1.
Whereas no significant difference in proliferation or cell death
compared to control cultures were detected, a slightly lower
efficiency in differentiation was observed (around 10-50%
differentiated cells compared to control numbers). It has
previously been observed that VPA-mediated differentiation is
affected by substrate, likely due to the fact that the small
molecule VPA, which, in contrast to recombinant growth factors,
acts intracellularly, gets attached to and degrades on the
substrate. Nevertheless, VPA-mediated neuronal differentiation of
NSCs was indeed observed on 4RepCT films.
[0579] When NSCs grown on 4RepCT films were treated with T3 as
described in Methods, they differentiated efficiently into cells
with oligodendrocyte morphology expressing the archetypical marker
protein MBP with no significant difference in efficiency,
proliferation or cell death compared to control cultures (n=2). It
was observed that the MBP staining suggested a possibly less mature
morphology than the cells differentiated under control conditions.
It should be noted, however, that oligodendrocyte maturation is
complex and that such an observation thus needs careful and
extended analysis before becoming conclusive (FIG. 15).
[0580] No significant differences in morphology, proliferation,
viability, or differentiation capacity in any experiment were
observed upon comparison between scaffolds made from the 4RepCT
protein which had and had not been Ips depleted. Further, no
obvious negative differences in morphology, proliferation, or
viability were seen in NSCs grown on IKVAV-4RepCT or RGD-4RepCT
(n=1).
Example 5
Islets of Langerhans (A) and Single Beta Cells, Alone (B) or in
Combination with Other Cells (C), on Recombinant Spider Silk
Scaffolds
Background
[0581] Transplantation of the islets of Langerhans is one of the
most promising approaches to finding a widely applicable treatment
of severe type 1 diabetes. Unfortunately, currently available
procedures suffer from low efficacy due to loss of function and
survival of the pancreatic cells (Alejandro et al (2008),
Transplantation 86:1783-1788). The low success rates are
incompletely understood, but prior to transplantation, during islet
isolation, the environment surrounding the cells is disrupted,
which leads to a loss of vascularization and innervations and to
altered interactions with the extracellular matrix. This has been
implicated as a major cause of the limited survival and function
(van der Windt et al (2007), Xenotransplantation 14:288-297;
Kilkenny and Rocheleau (2008), Mol Endocrinol 22:196-205). The
endocrine parts of the pancreas, unlike the exocrine part, do not
produce a basement membrane of their own but rather depend on their
surrounding environment, indicating again that the right niche
might be of importance (Otonkoski et al (2008), Diabetes Obes Metab
10 Suppl 4:119-127).
[0582] Another major obstacle to islet transplantation is the
limited availability of beta cells due to shortage of donors. Beta
cells, unlike many other cell types, have so far not been possible
to propagate in vitro, since efforts to expand them result in
dedifferentiation (Beck et al (2007), Tissue Eng 13:589-599). Thus,
the establishment of an environment which is optimized for islets
and beta cells is necessary, both for the study and propagation of
pancreatic islets and islet-cells (beta cells), and for the design
of an artificial islet/beta cell carrier for transplantation. In
order to accomplish this, a highly versatile biomaterial is needed
as a scaffold.
[0583] Recently, success in producing a recombinant spider silk
protein under physiological conditions was reported by the
inventors' research group. Polymers of the protein can yield a
strong and highly versatile material that can adopt various
physical forms, e.g. three-dimensional fiber-meshes, foams or
films. The stability of such "scaffolds" allows retrieval of cells
for subsequent transplantation. Moreover, the scaffolds can be
functionalized with specific cell binding motifs, suitable for
adherence of e.g. beta cells. These properties make the protein an
excellent candidate for the production of scaffolds that mimic the
natural cell environment and thus provide support for islets of
Langerhans and individual beta cells after isolation.
[0584] Subsequent to transplantation, it is important that the
islets are well adopted into the host environment, for example with
proper vascularization. At the same time, negative host immune
responses, e.g. instant inflammatory reaction, should be avoided.
The formation of new capillaries requires endothelial cells, and of
course these cells readily tolerate contact with blood. Mesenchymal
stem cells can up-regulate the expression of important growth
factors in endothelial cells and also produce proteases, and can
thereby create pathways for new capillaries (Zacharek, A. et al.
Neurosci Lett 404, 28-32 (2006)). The experiments described herein
indicate that a personalized islet environment can be built up by
using a functionalized silk scaffold, which allows adherence and
combined growth of islet cells, endothelial cells (cf Example 6)
and mesenchymal stem cells (cf Example 1).
Experimental Material
[0585] Recombinant spider silk protein, 4RepCT (SEQ ID NO:2),
prepared essentially as described in Hedhammar et al (2008), supra,
in the form of scaffold structures prepared as described in Example
1. The material was used in the original form 4RepCT, or in the
form of variants modified by the incorporation of different
cell-binding motifs related to the extracellular matrix, for
example RGD, IKVAV, and YIGSR, or by the incorporation of the
tripeptide RGE. In other variants, the spidroin N-terminal domain
(NT) and a C-terminal His-tag were included, yielding a protein
designated NT4RepCTHis (SEQ ID NO:5). [0586] Islets of Langerhans
(human and rodent) [0587] Cells from human and mouse islets, e.g
beta cells [0588] Endothelial cells [0589] Mesenchymal stem
cells
Experimental Methods
Cell Isolation
[0590] Human islets of Langerhans were isolated at the Division of
Clinical Immunology at Uppsala University, Sweden, by using a
modified semi-automated digestion-filtration method and were
thereafter cultured in CMRL-1066 medium with supplements and 10%
human serum (Johansson et al (2008), Diabetes 57:2393-2401).
[0591] Rodent islets of Langerhans were isolated by collagenase
treated pancreata, digested by a continuous mechanic shaking,
separated from exocrine tissue, and thereafter cultured in
RPMI-1640 medium with supplements and 10% FBS (Nyqvist et al
(2005), Diabetes 54:2287-2293).
[0592] Single cells were prepared from islets from 10 months old
obese mice or from islets from human donors. The single cells were
isolated according to an accutase digestion protocol. In brief, 200
islets were pooled and washed twice with 1.times.PBS. Thereafter, 1
ml accutase was added to the islets. The islet-accutase suspension
was incubated in 37.degree. C. for 10-15 min with two steps of mild
shaking, after which islet single cells were washed twice with
1.times.PBS and then plated and cultured on different variants of
4RepCT scaffolds in various physical forms, and without and with
various peptide motifs.
Cell Culture
[0593] Human islets (20 islets/well) in combination with 4RepCT
scaffolds were cultured in CMRL-1066 with supplement (Johansson et
al, supra).
[0594] Rodent islets (10 islets/well) and single islet cells in
combination with 4RepCT scaffolds were cultured in supplemented
RPMI-1640 medium (Nyqvist et al, supra).
[0595] Commercially available, human microvascular endothelial
cells were obtained and maintained in culture according to the
provider's instructions in a well-known manner.
[0596] Commercially available, human mesenchymal stem cells derived
from bone marrow were obtained and maintained in culture according
to the provider's instructions in a well-known manner.
[0597] Cell culture plates (hydrophobic plastic) were used as
control for islets (as islets usually are cultured floating free in
these plates). Tissue-culture treated plastic was used as control
for growth of single cells.
Adherence Assay
[0598] A number (10-25) of islets were plated onto normal culture
plastics as control and onto different variants of 4RepCT scaffolds
and cell-binding motifs. Specific islet medium was used, and 4RepCT
and islets were cultured for 5 days. During this time, adherent
islets were counted every day. Medium change was performed on day 2
and insulin release was studied on day 5. Thereafter, the islets
within 4RepCT scaffolds were cultured for up to 2 weeks, whereupon
islet survival was analyzed. In one experiment using islets from a
human donor, the islets were cultured for 12 weeks.
Assessment of Function and Survival of Islets and Islet Cells
[0599] Signal transduction, both in the individual islets and the
islets' cells, both human and rodent, was studied subsequent to
culture of the intact islets or islet cells under different growth,
function and survival promoting conditions, and for different
periods of time, in the various 4RepCT-based scaffolds. Islet cell
growth, function and survival were monitored with the help of
various fluorescent dyes and biosensors for specific steps in the
signal transduction pathway. Several key events were tested:
glucose metabolism, cytoplasmic concentration of free Ca.sup.2+
([Ca.sup.2].sub.i), proliferation, and apoptosis/necrosis. It is
also possible to test for example ATP production, exocytosis, and
stimulus-induced insulin gene transcription.
In Vivo Transplantation
[0600] Islet and 4RepCT transplantation (e.g. into the anterior
chamber of the eye) is done according to the method developed in P.
O. Berggren's laboratory (Speier et al (2008), Nat Protoc
3:1278-1286; Speier et al (2008), Nat Med 14:574-578). In this way,
the cornea is used as a window to study cell survival, function and
integration in a living organism, under both physiological and
diabetic conditions.
Results
[0601] A) Culturing of Islets
[0602] The use of 4RepCT, both the wild type and variants modified
by incorporation of different extracellular matrix related
cell-binding motifs (e.g. RGD, RGE, IKVAV and YIGSR), may define an
optimal environment for maintaining pancreatic islet function and
survival after isolation.
[0603] Islets were isolated from human pancreata (N=7) and mouse
pancreata (N=10).
[0604] The islets were cultured for from 3 h to 3 days in their
specific medium and serum. Thereafter, they were plated onto
different variants of 4RepCT (fiber, foam and film with
incorporated peptidic motifs; none (wild-type; WT), RGD, RGE, IKVAV
and YIGSR). Islets were also plated on NT4RepCTHis.
[0605] Islets adhered spontaneously to the 4RepCT variants, as
shown in FIG. 18A (human) and FIG. 18B (mouse). Empty culture
plates (hydrophobic plastic) were used as control. From these
results, a preferential adherence to the foam scaffold structure
was observed, as compared to film and fiber (FIG. 19). Therefore,
all tests of adherence and function were continued on foam
scaffolds.
[0606] Adhered islets were counted every day after plating until
day 5. Islets adhered in different numbers, as shown in FIG. 20
(human islets, n=3 except for RGE where n=2; all experiments done
in triplicates) and FIGS. 21A-B (mouse islets, n=4 (A) and n=1 (B);
experiments done in triplicates). A comparison between different
motifs was also performed, and a significant increase in adhesion
was seen for 4RepCT comprising the motif RGD on days 2, 4 and 5
(FIG. 21A). Islets also adhered to NT4RepCTHis in an increased
manner compared to the control (FIG. 21B).
[0607] Islets in contact with 4RepCT were glucose challenged, and
insulin release was measured on day 5. The results are shown in
FIGS. 22A-F. Insulin was released from mouse islets cultured on
4RepCT (FIG. 22A-B; n=6; experiments done in duplicates). Mouse
islets also released insulin when cultured on NT4RepCTHis (FIG.
22D; n=1; experiment done in triplicates). Human islets released
insulin after glucose stimulation when cultured on 4RepCT (FIG.
22E; n=3; experiments done in triplicates). Not all islets adhered
to the foam scaffolds, and therefore the adhered islets' insulin
release function on days 2 and 5 was tested in separate experiments
(FIG. 22C (mouse), FIG. 22F (human)).
[0608] The glucose stimulated insulin release by the islets is
similar after culturing in the wells coated with the 4RepCT
scaffolds, with or without peptide motifs, and the control wells.
This indicates that islets cultured with the 4RepCT scaffolds
maintain their function. Also, islets cultured on NT4RepCTHis
exhibited maintained function (FIG. 22D).
[0609] The measured insulin release at the initial basal glucose
(black bars) is higher in a few mouse experiments on 4RepCT
scaffolds with or without peptide motifs, and this is believed to
be a result of exposure to the initial culture medium, which is
difficult to wash away from the scaffolds, and of a high
variability between these experimental islets. The stimulation
index (Stimulated insulin/Basal insulin) is measured separately in
each well and shows that all islets on scaffolds responded to
glucose by releasing insulin equally well as the control (FIG.
22B).
[0610] The human islets cultured on 4RepCT scaffolds (with and
without peptide motifs) showed a low basal insulin level regardless
of cell-binding motif, and exhibited a satisfactory stimulated
insulin release after high glucose challenge.
[0611] The cytoplasmic concentration of free Ca.sup.2+
([Ca.sup.2].sub.i) was measured for islets cultured on scaffolds
bearing different motifs. The islets responded to high glucose and
showed an increase in Ca.sup.2+ as evidenced by ratiometric imaging
analysis (FIG. 23). No difference between control islets and islets
cultured on scaffolds was observed.
[0612] Islets were cultured (medium change every second day) for 2
weeks. Thereafter, islets were counted and scanned for viability
(e.g. necrosis). The islet morphology of islets on scaffolds was
preserved after 2 weeks, whereas control islets exhibited islet
degradation as visualized by a more irregular shape and suspended
single cells (FIG. 24). Necrotic bodies were analyzed by light
microscopy. More necrotic bodies were seen in the control islets,
whereas islets cultured on 4RepCT scaffolds (with and without
peptide motifs), both mouse and human, were more intact and
viable.
[0613] Islets from a young human donor were long-term cultured and
tested for islet-like cluster formation and insulin release at day
5 and after 4 weeks and 12 weeks. Islets on the foam scaffold with
the RGD motif exhibited an increased islet-like cluster formation
after 4 weeks (Table 6).
TABLE-US-00009 TABLE 6 Scaffold Number of islets and islet-like
clusters 4RepCT 3 .+-. 1 RGD-4RepCT 71 .+-. 16 IKVAV-4RepCT 4 .+-.
1 YIGSR-4RepCT 6 .+-. 1 RGE-4RepCT 4 .+-. 1 Control 3 .+-. 1
[0614] These clusters adhered to the scaffold, and cells between
the islets and islet-like clusters grew along the RGD-4RepCT
scaffold structure (FIG. 25). Such cell growth between islets and
along the foam structures was also seen on scaffolds having the
YIGSR motif. Insulin release increased over time for scaffolds with
RGD and was maintained in scaffolds with the YIGSR motif, and these
results demonstrate satisfactory function compared to control
islets after such a long time of culture (FIG. 26). Staining showed
insulin positive cells in the islets and islet-like clusters (FIG.
27).
[0615] B) Culturing of Beta Cells Alone
[0616] Single cells (beta cells in majority) were isolated from
islets of Langerhans from obese mouse pancreata (n=3) and from
human islets (n=1). The single cells were plated and cultured for
3-7 days on different variants of 4RepCT (fiber, foam and film with
the incorporated motifs: none (wild-type; WT), RGD, RGE, IKVAV and
YIGSR).
[0617] Morphology analysis showed that the single cells adhered in
different manners onto different variants of 4RepCT.
[0618] Long-term culture (over 2 weeks) of single cells was
analyzed. Tissue-culture treated plastic served as control (FIG.
28A) and was compared to wells coated with film and foam of 4RepCT
with incorporated cell-binding motifs, e.g. RGD (FIG. 28B). In all
the different wells, single cells formed clusters of cells, as
shown in FIG. 28. These clusters of cells differed in morphology,
exhibiting scattered clusters e.g. in the control (FIG. 28A), and
round clusters e.g. in 4RepCT with the RGD motif (FIG. 28B). The
clusters were counted after two weeks and then saved for
histological analysis, such as insulin staining. The results showed
that the amount of round clusters found was enhanced in 4RepCT foam
with RGD, and that these round clusters were insulin positive, as
shown in FIG. 28C. These results indicate that 4RepCT can maintain
islet beta cell function and enhance growth of round cell clusters
that are insulin positive, compared to single cells cultured on
normal cell-culture plastic.
[0619] C) Culturing of Beta Cells in Combination with Other
Cells
[0620] Single mouse beta cells, human endothelial cells and human
mesenchymal stem cells were plated alone or together and cultured
on 4RepCT foam scaffolds with the incorporated motifs: none
(wild-type; WT), RGD, RGE, IKVAV and YIGSR. The mesenchymal stem
cells, when in culture alone, adhered to and grew along the
structure of the scaffolds (FIG. 29).
[0621] Single beta cells, endothelial cells and mesenchymal stem
cells grew on scaffolds of foam, and exhibited increased cluster
formation on 4RepCT and RGD-4RepCT (FIG. 30).
[0622] Transplantation of 4RepCT scaffold in combination with
islets of Langerhans or islet cells is expected to show engraftment
support.
Conclusions
[0623] Culturing of islets with the various 4RepCT scaffolds
(having different formats and peptide motifs) allowed basic
research regarding the development of potential treatment
strategies of diabetes at the cellular level.
[0624] Culturing of islets and islet single cells, such as beta
cells, within a 4RepCT scaffold in different variants helps to
maintain and increase their function in vitro.
[0625] Culturing of composite cell populations (comprising islet
beta cells, endothelial cells and mesenchymal stem cells) within
4RepCT scaffolds has a potential as an insulin-making device if
transplanted to a recipient using the recipients own endothelial
cells and mesenchymal stem cells.
[0626] 4RepCT with islets, transplanted together into the anterior
chamber of the eye of a mouse using a technique recently developed
at Prof Berggren's laboratory is expected to improve islet
engraftment and survival. Transplantation of a 4RepCT scaffold in
combination with islets of Langerhans or islet cells is expected to
exhibit engraftment support.
[0627] The work described in this example accomplished the
pre-requisites for the development of a transplantable, artificial
insulin-producing device based on a 4RepCT scaffold (with or
without peptide motifs).
Example 6
Endothelial Cells on Recombinant Spider Silk
[0628] The growth of blood vessels is essential for tissue
engineering in regenerative medicine. Endothelial cells are
responsible for vessel growth, and this process is triggered during
certain circumstances, such as wound healing. In the body, there
are many different kinds of endothelial cells depending on organ
and tissue. The endothelial cells that are in close contact with
the tissue are known as microvascular endothelial cells.
Experimental Material
[0629] Commercially available, microvascular endothelial cells were
obtained and maintained in culture according to the provider's
instructions in a well-known manner.
[0630] Cell scaffold material comprising polymers of different
variants of the 4RepCT protein was prepared into different physical
forms as described in Example 1. The 4RepCT protein was used in
unmodified, "wild-type" form without any additional cell-binding
motif, as well as modified with the peptidic cell-binding motifs
RGD, IKVAV or YIGSR.
Experimental Methods
[0631] Cells were added to the different scaffold materials and
studied. A variety of assays were carried out on the cells,
including a proliferation assay and a BD Pathway analysis (BD
Biosciences). It is also possible to carry out for example cell
function analysis, Live/Dead assay of apoptosis and necrosis, and
histology analysis. Tissue-culture treated plastic plates were used
as control.
Results
[0632] Results are presented in FIGS. 31 and 32. Endothelial cells
adhered and grew in culture together with the different 4RepCT
scaffolds. After 3 days of culture, their morphology was analyzed
and the amounts of cells were counted. The morphological analysis
showed that endothelial cells on 4RepCT scaffolds in different
physical forms, with and without cell-binding motifs, adhered and
were viable (FIGS. 31 and 32).
Conclusions
[0633] Endothelial cells were viable and could grow on 4RepCT
scaffolds, as well as showing a proliferative capacity thereon.
Example 7
Fibroblasts on Recombinant Spider Silk
[0634] Experiments were done showing that scaffolds prepared from
4RepCT support the growth of anchorage dependent primary
fibroblasts, that the cells survive, attach to the material and
maintain one of their main functions, i.e. to secrete collagen type
I. 4RepCT scaffolds show increased capacity to support cell growth
compared to tissue culture treated plastics. Introducing the
integrin binding motif RGD further improves this cell supportive
capacity. Both growth on wildtype 4RepCT and RGD-4RepCT was shown
to be independent of serum proteins.
Materials and Methods
Cell Culture
[0635] Primary human dermal fibroblasts of neonatal origin, HDFn
(ECACC/HPA) were cultured in Dulbecco's modified Eagle's medium
nutrient mixture F12 HAM (Sigma) supplemented with 5% foetal bovine
serum (Gibco), penicillin and streptomycin (National Veterinary
Institute, SVA, Sweden) (FIGS. 34-36 and 38-41). In parallel
experiments, (FIGS. 37 and 42), primary human fibroblasts (SF-HDF,
PromoCell) were cultured in serum-free cell culture medium PC-1
(Lonza, Belgium) supplemented with 25 .mu.g/ml ascorbic acid
(Sigma), and 5 mM L-glutamine (SVA). Cells were seeded onto 4RepCT
scaffolds at the densities 3000 cells/cm.sup.2 or 15000
cells/cm.sup.2. All experiments were performed in passage 8 (with
the exception of FIGS. 36 and 41, which were performed in passage
4) at 37.degree. C. with 5% CO.sub.2 and 95% humidity.
Cell Culture Scaffolds
[0636] After purification, recombinantly expressed 4RepCT
(Hedhammar et al (2008), supra) with and without additional
N-terminal cell binding motifs RGD, IKVAV and YIGSR, or the
tripeptide RGE, was concentrated by centrifugal filtration (Amicon
Ultra, Millipore) and sterile filtered (0.22 .mu.m) before
preparation of scaffolds in accordance with the description in
Example 1. Likewise, recombinant NT4RepCTHis was prepared. Fibers
were sterilized through autoclaving for 15 minutes at 121.degree.
C. in distilled water, 2.8 bar. Scaffolds were prepared in
hydrophobic 96-well cell culture plates (Sarstedt) or cell culture
chamber glass slides (LabTek). Wells without scaffolds were used as
negative control (HP), and tissue culture treated plates as a
positive control (TCT).
[0637] The plates and chamber slides were allowed to dry over night
at room temperature under sterile conditions with full speed fan.
Scaffolds were washed twice with sterile PBS and pre-incubated with
complete cell culture medium 1 h at 37.degree. C. with 5% CO.sub.2
before cell seeding.
Enumeration of Cells with Alamar Blue
[0638] Cell growth on 96-well plate scaffolds was monitored with
Alamar Blue cell viability assay (Molecular Probes) every second
day during the culture period. After 4 h incubation with Alamar
blue diluted 1:10 in cell culture medium, fluorescence intensity at
excitation 544/emission 595 was measured in 100 .mu.l supernatants
from the cultures with a fluorescence plate reader (FarCyte,
TECAN). A standard curve ranging from 50-64000 cells/well was
established to enable recalculation of fluorescence intensity to
cell numbers (live cells). Each scaffold type was analyzed in
hexaplicate.
Cellular Stainings
[0639] Cells cultured on scaffolds in chamber slides were stained
for either viable/dead cells, filamentous actin or collagen type I
every third day during the culture period. The stainings were
viewed under a confocal microscope (Leica), green fluorescence:
excitation at 488 nm/detection at 500-530 nm, red fluorescence:
excitation at 543 nm/detection at 620-660), and pictures were taken
with Leica confocal software (LCS).
[0640] Live/Dead Staining:
[0641] Live/dead viability assay (Molecular Probes) was used to
visualize living and dead cells growing on scaffolds. The cultures
were washed twice with pre-warmed phosphate buffered saline (PBS)
and stained with Calcein-AM and ethidium homodimer-1 (EthD-1) for
30 minutes at room temperature.
[0642] Filamentous Actin:
[0643] Scaffolds were washed twice and cells were fixed with 4%
paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and
blocked with 1% bovine serum albumin (BSA, AppliChem) in PBS,
before staining with ALEXA FLUOR.TM.488-Phalloidin (Invitrogen),
1:40 in 1% BSA in PBS. EthD-1 was used as nuclear staining. Slides
were mounted in Fluorescence mounting medium (Dako,
Copenhagen).
[0644] Vinculin:
[0645] Scaffolds were washed twice and cells were fixed with 4%
paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and
blocked with 1% bovine serum albumin (BSA, AppliChem) in PBS,
before staining with mouse anti human vinculin (Sigma V9131) at 9.5
.mu.g/ml in 1% BSA, followed by ALEXA FLUOR.TM.488 goat anti mouse
IgG (H+L), cross adsorbed (Invitrogen) and ALEXA
FLUOR.TM.594-Phalloidin (Invitrogen), 1:40 in 1% BSA in PBS. DAPI
was used as nuclear staining. Slides were mounted in Fluorescence
mounting medium (Dako, Copenhagen).
[0646] Collagen Type I:
[0647] Scaffolds were washed twice and cells were fixed with
acetone at -20.degree. C. and blocked with 1% BSA in PBS before
staining with mouse anti collagen type I (clone COL-1,
Sigma-Aldrich) at 3.5 .mu.g/ml in 1% BSA, followed by ALEXA
FLUOR.TM.488 goat anti mouse IgG (H+L), cross adsorbed
(Invitrogen). EthD-1 was used as nuclear staining. Slides were
mounted in Fluorescence mounting medium. Mouse IgG1 (clone B-Z1,
BioSite) was used as Isotype control.
Quantitative Determination of Secreted Collagen Type I
[0648] Supernatants were collected from cultures every second day,
diluted 1:10 and analysed for C-peptide, which is cleaved off from
the procollagen type I molecule during its secretion into the cell
culture medium, using the Procollagen Type I C-Peptide EIA Kit
(TAKARA) according to the instructions from the manufacturer.
OD.sub.450 was measured with Sunrise plate reader (TECAN), and data
management with MAGELLAN.TM. software was used to achieve the
concentration on C-peptide in the original samples. Data was
recalculated to .mu.g C-peptide secreted/cell.
Results
[0649] Cell Culture Scaffolds Prepared from 4RepCT
[0650] Film, foam, fiber and fiber-mesh scaffolds were successfully
prepared from 4RepCT protein solution (FIG. 33).
Growth of Fibroblasts on 4RepCT Scaffolds
[0651] 4RepCT scaffolds showed an increased capacity to support
growth and expansion of primary fibroblasts compared to the TCT, as
shown by the higher number of live cells present in scaffold wells
after the initial phase of culture (i.e. from day 7 at the lower
seeding density and from day 5 at the higher seeding density, FIG.
34). In the initial phase, however, the cells seem to grow somewhat
slower on the scaffolds compared to the TCT. The number of living
cells on the foam scaffolds is consistently lower than film and
fiber-based scaffolds. However, the accessible scaffold area of the
foam scaffolds is difficult to estimate, thus direct comparison to
the other scaffold formats is not possible. By adding 4RepCT film
under the fiber or fiber-mesh scaffolds, the supportive capacity
increased even more (FIG. 35).
[0652] We also demonstrated that the fibroblasts exhibited high
live/dead ratios on the scaffolds, where film scaffolds showed
close to identical growth pattern, cell density and live/dead
ratios to cell culture treated glass slides (FIG. 36, right
panels). Since the foam and fiber-based scaffolds stained red in
this assay, we were able to see that the shape of living cells
followed the morphology of the material.
Serum-Free Culture of Human Fibroblasts on 4RepCT Scaffolds
[0653] We have also shown that SF-HDFs (primary human fibroblasts
expanded under serum-free conditions) are able to grow on 4RepCT
film scaffolds with no serum present in the cultures (FIG. 37).
This proves that the interaction of cells with the material is not
dependent on serum proteins that have bound non-specifically to the
surface, but that the scaffolds themselves present a hospitable
surface for the cells to grow on.
Attachment of Cells to Scaffolds
[0654] During attachment of cells to the surrounding matrix,
filamentous actin is linked to the membrane-bound receptors that
mediate the binding. By staining intracellular filamentous actin,
the binding points between cells and the underlying material can be
indirectly visualized. With this method, we have been able to
demonstrate that the HDFn actually binds to fiber (FIG. 38, left
panel), fiber-mesh, film (FIG. 38, right panel) and foam scaffolds
both during early and late culture (day 1, 4, 7, 10). Furthermore,
through staining for vinculin in combination with filamentous
actin, focal adhesions could be detected after 3 h culture of
SF-HDF on wild-type (WT) film, RGD, IKVAV, YIGSR and NRC
(NT4RepCTHis), (FIG. 43), under serum free conditions. These
results indicate integrin-mediated adhesion to the different
substrates.
Collagen Type I Secretion by Cells Growing on 4RepCT Scaffolds
[0655] The cells produced collagen type I when growing on all the
different formats of 4RepCT, i.e. film, foam, fiber and fiber-mesh
scaffolds. Thus, the cells maintain one of their important
functions during culture in vitro on the 4RepCT scaffold material.
The levels of secreted collagen (as measured by the C-peptide
cleaved off during secretion) increased in the culture medium
during the first 5 days of culture (FIG. 39, left upper panel).
However, the amount of collagen produced per cell reached a maximum
at 24-72 h post seeding, and was at this time point higher for
cells growing on any of the 4RepCT scaffolds compared to the TCT
(FIG. 39, right upper panel). Intracellular production of collagen
was demonstrated to a similar extent on all scaffold types at day
1, 4, 7 and 10 post seeding (examples shown in FIG. 39, lower
panel).
Maintained Fibroblast Phenotype after Culture on 4RepCT
Scaffolds
[0656] To verify that fibroblasts maintained their phenotype after
culture on 4RepCT scaffolds, cells were harvested from scaffolds
after 14 days culture and reseeded onto either TCT for evaluation
of collagen type I secretion, or onto glass for intracellular
staining of collagen type I. Bo doing this it was demonstrated that
the fibroblasts still produce (FIG. 40, lower panel) and secrete
(FIG. 40, upper panel) collagen type I, and thereby do not lose
what is one of their most important functions even after a
relatively long-term culture on 4RepCT scaffolds.
Fibroblasts Grown on Functionalized 4RepCT Scaffolds
[0657] Preliminary data from Alamar blue viability experiments show
that the introduction of RGD into 4RepCT increases the total number
of living cells growing on the film and fiber-mesh scaffolds from
day 3 and 1 respectively (FIG. 41). Over time, the number of cells
also exceeds the cell counts present in TCT wells, i.e. from day 7.
In a parallel set up, a serum free cell culture system were used,
showing that even in the absence of serum, enhanced growth of
primary fibroblasts was achieved on the RGD-4RepCT film, compared
to 4RepCT without RGD (FIG. 42). Thus, introduction of RGD can
improve the capacity of the scaffolds to support the growth of
anchorage dependent cells markedly, and the effect is not dependent
on serum components present in the cell culture medium. The results
also indicate that the RGD motifs are properly exposed on the
surface of the scaffolds.
Example 8
Keratinocytes on Recombinant Spider Silk
[0658] Experiments were performed to show that film scaffolds
prepared from 4RepCT support the growth of primary keratinocytes,
and that the cells adhere to the material and maintain their
characteristic morphology. Both growth on wild-type 4RepCT and
functionalized 4RepCT was shown to be independent of serum
proteins.
Materials and Methods
[0659] Keratinocytes isolated from human skin were cultured in
Keratinocyte SFM with supplement (Gibco), which is a serum-free
set-up designed for the growth of keratinocytes. Traditionally, 10%
fetal bovine serum is added to inactivate trypsin after harvesting
the cells, to ensure binding upon re-seeding. In the current
set-up, parallel experiments with and without this serum addition
were performed. Keratinocytes were seeded at passage 4 (10000
cells/cm.sup.2) onto wild type and functionalized film scaffolds of
4RepCT as described in previous Examples. Functionalizations that
were tested included the general cell-binding motifs RGD and IKVAV,
but could also have included the general cell-binding motif YIGSR
and/or the keratinocyte-specific motifs EPDIM and NKDIL.
Keratinocyte-specific markers may furthermore be used to ensure
maintained phenotype at the end of culture, and to determine
differentiation status, e.g. keratin (K1, K5, K10, K6/K16/K17),
filaggrin, Tob, G6K12, gp80 and MRP-8.
[0660] Immunofluorescence staining for detection of vinculin (clone
V9131, Sigma-Aldrich) in combination with filamentous actin
(Phalloidin, Invitrogen) was used to identify focal adhesions, i.e.
actual contact spots between the cells and the material, as
described in Example 7 above. Effect on apoptosis will be evaluated
with EnzChek Caspase3 Assay (Molecular Probes). Living cells were
detected with Alamar blue (Molecular Probes) as described in
Example 7 above.
Results
[0661] Primary human keratinocytes were cultured for 4 days on
4RepCT film with or without cell binding motifs. The cells survived
and increased in cell numbers between day 1 and 4 (FIG. 44). They
showed the characteristic morphology of keratinocytes on all
materials during the full culture period (FIG. 45). Focal adhesions
were detected on wild type, RGD and IKVAV films (analysed at day
4), indicating integrin-mediated adhesion to the tested materials
(FIG. 46). The results were independent of addition of serum before
seeding, thus, the cells bound to the material under strictly
serum-free conditions.
Example 9
Primary Hepatocytes on Recombinant Spider Silk
[0662] The liver is an essential organ with unique functions.
Currently, cases of acute liver failure, e.g. liver-based metabolic
diseases and chronic liver disease, are rescued by either liver
transplantation or liver cell therapy. Unfortunately, currently
available hepatocyte therapy is not optimal, for example because
many of the cells are lost and not engrafted during transplantation
of hepatocytes. A scaffold, which serves as a host for hepatocytes
and keeps them in place, could pre-engraft these cells prior to
transplantation. This opens a new way of transplanting
hepatocytes.
Experimental Material
[0663] Rodent hepatocytes were isolated by digestion of collagenase
treated liver by continuous mechanic shaking, separated and
cultured in RPMI-1640 medium supplemented with 10% FBS.
[0664] The recombinant spider silk protein 4RepCT described in
previous Examples was used, both as wild type and in the form of
variants modified by incorporation of different extracellular
matrix related cell-binding motifs (e.g. RGD, IKVAV, YIGSR and
RGE), and was prepared essentially as described in Hedhammar et al
(2008), supra, in the form of scaffold structures prepared as
described in Example 1.
[0665] Cell Culture:
[0666] Rodent hepatocytes in combination with 4RepCT scaffolds were
cultured in supplemented RPM I-1640 medium.
[0667] Assessment of Survival of Hepatocytes:
[0668] The cells were plated on fiber and film scaffolds with
incorporation of different extracellular matrix related
cell-binding motifs (e.g. RGD, IKVAV, YIGSR and RGE). Cell
morphology and survival was assessed over time in culture.
Results
[0669] Hepatocytes adhered to fiber and film scaffolds (FIG.
47).
[0670] The hepatocytes were also able to survive in culture with
4RepCT.
Example 10
Recombinant Spider Silk Protein Scaffolds for Tissue
Engineering
[0671] The experiment is expected to show that 4RepCT scaffolds
enable regeneration of axons in spinal cord injuries in vivo. Both
4RepCT scaffolds without seeded cells and 4RepCT scaffolds combined
with the engraftment of human neural cells and human
oligodendrocyte progenitor cells are employed. Histological and
behavioral analysis is used to document the results.
Materials and Methods
Animal Surgery
[0672] The weight of rats is 170-200 g at the time of surgery.
Animals are injected with Atropin (0.05 mg/kg i.p., NM Pharma AB)
30 minutes before surgery. Rats are anesthetized with a mixture of
Hypnorm (fentanyl citrate, 0.22 mg/kg, and fluanisome, 6.8 mg/kg,
Janssen Pharmaceutical) and Dormicum (midazolam, 3.4 mg/kg,
Hoffman-La Roche). Body temperature of the rats is monitored and
kept at 38.degree. C. throughout the surgical procedure.
[0673] Lumbar spinal cords are surgically exposed by partial
laminectomy and treated with a few drops of Xylocain (lidocain
hydrochloride, 20 mg/ml, AstraZeneca Sweden AB) on the exposed
spinal cord surface prior to the spinal cord injury.
[0674] Transection of the cord is performed by cutting the spinal
cord with a scalpel. Through careful visual inspection, the surgeon
assures that the rostral and caudal ends are completely separated,
and slightly retracted. After lesioning the cord, a layer of
LYOPLANT.TM. (B/Braun Aesculap AG) is placed on the spinal cord as
dura substitution before the wound is sutured. The rats are
subcutaneously injected twice with 3 ml Ringer/glucose (2.5%)
before and after surgery. After surgery, the rats are given
intramuscular injections of Temgesic (buprenorphin, 7 .mu.g/kg,
Reckitt & Colman) twice a day for four days. The urinary
bladders are emptied manually twice daily, and Borgal (trimetoprim
sulfa, 15 mg/kg s.c., Intervet International B.V.) is given if
signs of urinary infection appear.
[0675] A 2-3 mm long bundle of 4RepCT fibers, for example prepared
as described in the previous Examples (with or without additional
peptide motifs such as RGD, RGE, IKVAV and YIGSR or using another
variant of a spidroin protein, e.g. NT4RepCTHis), is placed in the
gap of the spinal cord, either at the time of injury or one week
after injury. In the acute implantation paradigm, a bundle of
4RepCT fibers is cut to fit the gap of the cut spinal cord, and
gently placed in contact with the spinal cord stumps rostral and
caudal to the implant, with the 4RepCT fibers in the same axis as
the spinal cord. The LYOPLANT.TM. dura substitution is thereafter
placed over the 4RepCT implant.
[0676] In case the 4RepCT fiber bundle is implanted one week
post-injury, the animal is anesthetized, the wound reopened and the
spinal cord exposed. After trimming the rostral and caudal stumps
of the cord, the fiber bundle, cut to fit the gap, is placed in the
same axis as the spinal cord to ascertain contact between the
fibers and the spinal cord tissue rostral and caudal to the
implant. The wound is closed as described above.
Co-Implantation of Human Immature Cells
[0677] In a similar experiment, implantation of 4RepCT fibers as
described above is combined with engraftment of human neural stem
cells and human oligodendrocyte progenitor cells. The cells are
derived from human routine abortions, according to procedures
approved by the Regional Ethical Committee, and after written
consent from the abortion-seeking woman.
[0678] After in vitro culture of the appropriate cells according to
proprietary procedures, a suspension of 100,000-500,000 cells in a
minimal volume of DMEM-F12 without growth factors is injected into
the 4RepCT fiber bundle, which is then used as described in the
previous section.
Behavioral Assessment
[0679] To study hind-limb motor function, rats are allowed to move
in an open field (150.times.64 cm) and observed for at least four
minutes. The function of the hind-limbs, movement of joints,
positioning of paws, weight bearing, coordination and toe clearance
is evaluated and rated according to the Basso, Beattie, Bresnahan
(BBB) Locomotor Rating Scale (Basso et al (1995), J Neurotrauma
12:1-21, for review see Basso (2004), J Neurotrauma 21:395-404).
The tests are video recorded, and at least two independent
observers blinded to the treatment evaluate each rat. Animals are
evaluated preoperatively and 1, 2, 6, 12 and 18 weeks post
surgery.
Tracing of Neuronal Outgrowth
[0680] Anterograde tracing is performed by injecting biotinylated
dextrane amine (BDA, Neurotrace) at multiple sites in the parietal
cortex for one week according to the manufacturer's description.
Retrograde tracing is accomplished by injection of Fluoro-gold in
the spinal cord 3-5 mm caudal of the lesion. BDA is visualized in
the spinal cord caudal of the injury, to visualize cortico-spinal
descending axons crossing the spinal lesion, while
Fluoro-gold-labeled neurons are screened for in nucleus ruber of
the brainstem, as evidence for regrowth of descending rubro-spinal
neurons.
Morphological Analysis
[0681] After the last behavioral assessment at 18 weeks
post-lesion, the rats are given a lethal dose of intravenous
sedatives and perfused through the ascending aorta with 100 ml
Ca.sup.2+-free Tyrode's solution, followed by 400 ml of
phosphate-buffered 4% paraformaldehyde (PFA, Merck). The entire
brain stem and 4-5 cm of the vertebral column including the lesion
are dissected out, post-fixed for 2 hours in the same fixative and
cryo-preserved in 10% sucrose at 4.degree. C. for at least 24 h.
The spinal cord is then carefully dissected out of the vertebral
column, the cord and the brainstem embedded in Tissue-Tek, frozen,
sectioned at 10 .mu.m in a cryostat and mounted on gelatin-coated
slides.
[0682] For immunohistochemical analysis, the following primary
antibodies are used: human-specific rabbit anti-heat shock protein
27, rabbit anti-nestin, rabbit anti-.beta.-tubulin type II, mouse
monoclonal human-specific anti-glial fibrillary acidic protein,
rabbit anti microtubule-associated protein 2. Primary antibodies
are diluted in 0.1 M phosphate buffer with 0.3% Triton X-100
(TPBS). The secondary antibodies used are conjugated to Cy3
(Jackson ImmunoResearch Laboratories Lab. Inc) or Alexa 488
(Molecular Probes). Sections are treated with 1.5% normal goat
serum (Sigma) at room temperature for 30 minutes, incubated with
primary antibodies at 4.degree. C. over night followed by rinsing
and two hour incubation with secondary antibodies at room
temperature. All sections are counterstained with nuclear marker
Hoechst 33342 (30 .mu.g/ml, Molecular Probes), and mounted with
poly vinyl-alcohol (0.1 mg/ml, Sigma) in DABCO
(1,4-diazabicyclo[2.2.2]octane, 0.03 mg/ml, Sigma). The
immunolabeled tissue sections are studied in a fluorescence
microscope (Zeiss Axiophot), for quantitative analyses, images are
captured using a CCD camera (Hamamatsu ORCA-ER) and the OPENLAB.TM.
software for Macintosh (Improvision).
Example 11
Recombinant Spider Silk Protein Scaffolds for Support of the
Regeneration of Axons in Organotypic Cultures
[0683] This experiment will show that 4RepCT scaffolds can support
and guide the regeneration of axons. Pieces of the spinal cord and
brainstem are maintained in cultures ex vivo. By applying a
scaffold of 4RepCT that connects two such tissue pieces,
regenerating axons will be provided sufficient support to bridge
the gap. The experiment will give an indication of the usefulness
of 4RepCT scaffolds to support axonal outgrowth and restoration of
damaged tissue in spinal cord injuries in vivo.
[0684] An organotypic culture of the brain stem and the cervical
region of the cord is prepared. Brains and spinal cords are
collected from early pre-natal and post-natal Sprague-Dawley (SD)
rats. Sections in the sagittal plane through the brain stem and
cervical region of the spinal cord are made using a vibratome. The
dissected tissue is placed on membranes (MILLICELL.TM.-CM;
Millipore, Billerica, Mass., USA), in 1 ml of serum-based medium
(50% basal medium Eagle with Earle's Salts (BME; Sigma, St. Louis,
Md., USA), 25% heat inactivated horse serum (Gibco, Grand Island,
N.Y., USA), and 25% Earle's Balanced Salt Solution (EBSS; Sigma), 1
mM L-glutamine and 0.5% d-glucose) in a 6-well tissue culture plate
in a humidified atmosphere with 5% CO.sub.2 at 37.degree. C. The
tissue sections are incubated for 7-14 days, the medium replaced
every 3 days. At the time of injury, a transection of the upper
cervical spinal cord is made with a razor blade. The tissue is
inspected in an inverted microscope to ensure complete separation
of the two parts of the explant.
[0685] A bundle of 4RepCT fibers, prepared as described in previous
Examples (with or without additional peptide motifs such as RGD,
RGE, IKVAV and YIGSR or using another variant of a spidroin
protein, e.g. NT4RepCTHis), is placed in the gap between the tissue
pieces. The cultures are incubated for another 14 days, before
being fixed by immersion in phosphate-buffered 4% paraformaldehyde
(PFA, Merck) for 2 hours and cryo-preserved in 10% sucrose at
4.degree. C. for 24 h.
[0686] For immunohistochemical analysis, the following primary
antibodies are used: human-specific rabbit anti-heat shock protein
27, rabbit anti-nestin, rabbit anti-.beta.-tubulin type II, mouse
monoclonal human-specific anti-glial fibrillary acidic protein,
rabbit anti microtubule-associated protein 2. Primary antibodies
are diluted in 0.1 M phosphate buffer with 0.3% Triton X-100
(TPBS). The secondary antibodies used are conjugated to Cy3
(Jackson ImmunoResearch Laboratories Lab. Inc) or Alexa 488
(Molecular Probes). Fixed cultures are treated with 1.5% normal
goat serum (Sigma) at room temperature for 30 minutes, incubated
with primary antibodies at 4.degree. C. over night followed by
rinsing and two hour incubation with secondary antibodies at room
temperature. All tissues are counterstained with nuclear marker
Hoechst 33342 (30 .mu.g/ml, Molecular Probes), and mounted with
poly vinyl-alcohol (0.1 mg/ml, Sigma) in DABCO
(1,4-diazabicyclo[2.2.2]octane, 0.03 mg/ml, Sigma). The
immunolabeled tissue sections are studied in a fluorescence
microscope (Zeiss Axiophot) for quantitative analyses, images are
captured using a CCD camera (Hamamatsu ORCA-ER) and the OPENLAB.TM.
software for Macintosh (Improvision).
[0687] The results are predicted to indicate that 4RepCT scaffolds
can guide regeneration of injured axons.
Example 12
Human Embryonic Stem Cells on Recombinant Spider Silk
[0688] The experiment explores further the feasibility of culturing
human embryonic stem cells on a recombinant spider silk material,
building on the study reported as Example 2 above.
Materials and Methods
[0689] Standard six-well tissue culture plates were prepared. In
the plates, three wells contained film of one of the following
4RepCT variants: RGD-4RepCT, RGE-4RepCT, IKVAV-4RepCT, YIGSR-4RepCT
and NT4RepCTHis, while the remaining three wells of each plate were
empty. All films of 4RepCT with peptide motifs and of NT4RepCTHis
were prepared essentially as described in Example 1. In total, 15
plates were included in the experiment (representing triplicates of
each 4RepCT variant). The plates were kept dry at room temperature
until further use.
[0690] In preparation for the experiment, the tissue culture plates
were UV irradiated for 30 minutes in a Class II microbiological
safety cabinet. The three empty wells in each plate were then
coated with CELLstart.TM. CTS.TM. (Invitrogen; cat no A10142-01),
as per manufacturer's protocol.
[0691] Human embryonic stem cells (RCM-1, De Sousa et al, Stem Cell
Res 2:188-197; Roslin Cells, Edinburgh, UK) were cultured on
CELLstart.TM. with the serum and feeder free medium STEM PRO.RTM.
hESC SFM--Human Embryonic Stem Cell Culture Medium (Invitrogen; cat
no A1000701), as per manufacturer's protocol. The cells were
passaged at a ratio of 1:6 from a 90% confluent well using
STEMPRO.RTM. EZPassage.TM.--Disposable Stem Cell Passaging Tool
(Invitrogen; cat no 23181-010) as per manufacturer's protocol.
[0692] Wells were washed with PBS, and medium was placed in all
wells and preincubated prior to cell seeding. Cells at passage 60
were seeded into all wells of five six-well plates (representing
the five different variants) at the recommended density, and care
was taken not to disturb cells after seeding. All wells were
cultured with STEMPRO.RTM. hESC SFM as per manufacturer's protocol.
Incubation was performed in a standard culture incubator at
37.degree. C., 5% CO.sub.2 in air, at 95% humidity. Cells were
observed daily and 100% medium exchanged every 48 hours. All medium
was pre-equilibrated to incubator conditions for two hours prior to
exchange feeding.
[0693] Cells were passaged after 16 days, and again after 6 more
days, onto the remaining two six well plates of the respective
variants. The passaging regime for these two time points involved a
method established by Roslin Cellabs for the single cell
dissociation of human embryonic stem cells. Single cell
dissociation was achieved using TrypLE Select (Invitrogen cat. no.
A12177 (100 ml 10.times.)) as per manufacturer's instructions with
a pre-treatment for 1 h using a ROCK inhibitor (ROCK
inhibitor/Y27632; FluoroChem cat. no. 047265) as per the
manufacturer's instructions (Watanabe et al (2007), Nat Biotechnol
25, 681-686). This has been shown to be essential for the
successful growth of embryonic stem cells post enzymatic passaging.
Control and trial cells were treated identically and all were
subsequently seeded at equivalence.
[0694] After 24 h of the third passage of the study, wells were
stained with Sigma-Aldrich: alkaline phosphatase (AP), Leukocyte
(Sigma-Aldrich; Ref 86R-1 KT, Lot 019K4349) as per manufacturer's
protocol.
Results
[0695] Selected images from the cell culture experiments are
presented as FIGS. 48-52.
[0696] No differences between the various 4RepCT variants were
observed. Initially, cells were non-adherent "clumps" with a small
degree of adherence. Growth was much slower than that seen in the
control wells, which could be a consequence of the cell line
adapting to the 4RepCT variants, as seen in Example 2. It is often
the case that embryonic stem cell lines will go through a phase of
transition or lag, prior to establishing again on a new matrix or
indeed in a new medium composition. These Embryonic Body (EB)
structures did eventually adhere to the matrix, and from these EB
masses, cells were seen to grow and expand, which suggests
adaptation to the new matrix as illustrated by photographs of cells
in the first, second and third passage of the experiment (FIGS.
48-52).
[0697] Subsequent passaging resulted in improved morphology and
growth.
[0698] As shown by alkaline phosphatase staining, the cells are,
surprisingly, still exhibiting undifferentiated stem cell
characteristics after 23 days of culture on the respective 4RepCT
variants (FIGS. 50-52).
CONCLUSIONS
[0699] All control wells showed the characteristics and morphology
as would be expected with this hESC line under the control culture
conditions employed. The cell line showed signs of adaptation to
all the 4RepCT variants. Initial adherence, growth and culture
morphology was poor in the first passage on 4RepCT variants.
However, subsequent growth and passaging resulted in colonies
exhibiting the morphology characteristic of an undifferentiated
cell, with positive alkaline phosphatase staining for all
variants.
[0700] This study of hESC culture on 4RepCT variants surprisingly
indicates that the variants allow culturing of hESCs and that they
are suitable for maintaining the cells' stemness, for example with
respect to morphology, growth characteristics and Alkaline
Phosphatase staining.
Sequence CWU 1
1
261149PRTEuprosthenops australis 1Gly Ser Gly Asn Ser Gly Ile Gln
Gly Gln Gly Gly Tyr Gly Gly Leu 1 5 10 15 Gly Gln Gly Gly Tyr Gly
Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala
Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln 35 40 45 Gly Gly
Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala Ala Ala Ala 50 55 60
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr 65
70 75 80 Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala
Ala Ala 85 90 95 Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly
Tyr Gly Arg Gln 100 105 110 Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Ala Ala Gly Ser Gly Gln
Gly Gly Tyr Gly Gln Gly Gln Gly 130 135 140 Gly Tyr Gly Gln Ser 145
2265PRTEuprosthenops australisDOMAIN(1)..(167)REP fragment 2Gly Ser
Gly Asn Ser Gly Ile Gln Gly Gln Gly Gly Tyr Gly Gly Leu 1 5 10 15
Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala 20
25 30 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly
Gln 35 40 45 Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala
Ala Ala Ala 50 55 60 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg
Gly Gln Gly Gly Tyr 65 70 75 80 Gly Gln Gly Ser Gly Gly Asn Ala Ala
Ala Ala Ala Ala Ala Ala Ala 85 90 95 Ala Ala Ala Ala Ala Gly Gln
Gly Gly Gln Gly Gly Tyr Gly Arg Gln 100 105 110 Ser Gln Gly Ala Gly
Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Ala
Ala Gly Ser Gly Gln Gly Gly Tyr Gly Gln Gly Gln Gly 130 135 140 Gly
Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala Ala Ala Ser Ala Ala 145 150
155 160 Ser Thr Val Ala Asn Ser Val Ser Arg Leu Ser Ser Pro Ser Ala
Val 165 170 175 Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser Asn
Gly Gln Val 180 185 190 Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn
Ile Ser Ser Ser Val 195 200 205 Ser Ala Ser Ala Pro Gly Ala Ser Gly
Cys Glu Val Ile Val Gln Ala 210 215 220 Leu Leu Glu Val Ile Thr Ala
Leu Val Gln Ile Val Ser Ser Ser Ser 225 230 235 240 Val Gly Tyr Ile
Asn Pro Ser Ala Val Asn Gln Ile Thr Asn Val Val 245 250 255 Ala Asn
Ala Met Ala Gln Val Met Gly 260 265 3296PRTEuprosthenops
australisDOMAIN(1)..(137)NT fragment 3Gly Ser Gly Asn Ser His Thr
Thr Pro Trp Thr Asn Pro Gly Leu Ala 1 5 10 15 Glu Asn Phe Met Asn
Ser Phe Met Gln Gly Leu Ser Ser Met Pro Gly 20 25 30 Phe Thr Ala
Ser Gln Leu Asp Asp Met Ser Thr Ile Ala Gln Ser Met 35 40 45 Val
Gln Ser Ile Gln Ser Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn 50 55
60 Lys Leu Gln Ala Leu Asn Met Ala Phe Ala Ser Ser Met Ala Glu Ile
65 70 75 80 Ala Ala Ser Glu Glu Gly Gly Gly Ser Leu Ser Thr Lys Thr
Ser Ser 85 90 95 Ile Ala Ser Ala Met Ser Asn Ala Phe Leu Gln Thr
Thr Gly Val Val 100 105 110 Asn Gln Pro Phe Ile Asn Glu Ile Thr Gln
Leu Val Ser Met Phe Ala 115 120 125 Gln Ala Gly Met Asn Asp Val Ser
Ala Ser Ala Ser Ala Gly Ala Ser 130 135 140 Ala Ala Ala Ser Ala Gly
Ala Ala Ser Gly Gln Gly Gly Tyr Gly Gly 145 150 155 160 Leu Gly Gln
Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala 165 170 175 Ala
Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly 180 185
190 Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala Ala Ala
195 200 205 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln
Gly Gly 210 215 220 Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala
Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Ala Ala Ala Ala Gly Gln
Gly Gly Gln Gly Gly Tyr Gly 245 250 255 Arg Gln Ser Gln Gly Ala Gly
Ser Ala Ala Ala Ala Ala Ala Ala Ala 260 265 270 Ala Ala Ala Ala Ala
Ala Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln 275 280 285 Gly Gln Gly
Gly Tyr Gly Gln Ser 290 295 4340PRTEuprosthenops
australisDOMAIN(1)..(137)NT fragment 4Gly Ser Gly Asn Ser His Thr
Thr Pro Trp Thr Asn Pro Gly Leu Ala 1 5 10 15 Glu Asn Phe Met Asn
Ser Phe Met Gln Gly Leu Ser Ser Met Pro Gly 20 25 30 Phe Thr Ala
Ser Gln Leu Asp Asp Met Ser Thr Ile Ala Gln Ser Met 35 40 45 Val
Gln Ser Ile Gln Ser Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn 50 55
60 Lys Leu Gln Ala Leu Asn Met Ala Phe Ala Ser Ser Met Ala Glu Ile
65 70 75 80 Ala Ala Ser Glu Glu Gly Gly Gly Ser Leu Ser Thr Lys Thr
Ser Ser 85 90 95 Ile Ala Ser Ala Met Ser Asn Ala Phe Leu Gln Thr
Thr Gly Val Val 100 105 110 Asn Gln Pro Phe Ile Asn Glu Ile Thr Gln
Leu Val Ser Met Phe Ala 115 120 125 Gln Ala Gly Met Asn Asp Val Ser
Ala Ser Ala Ser Ala Gly Ala Ser 130 135 140 Ala Ala Ala Ser Ala Gly
Ala Pro Gly Tyr Ser Pro Ala Pro Ser Tyr 145 150 155 160 Ser Ser Gly
Gly Tyr Ala Ser Ser Ala Ala Ser Ala Ala Ala Ala Ala 165 170 175 Gly
Gln Gly Gly Pro Gly Gly Tyr Gly Pro Ala Pro Asn Gln Gly Ala 180 185
190 Ser Ser Ala Ala Ala Ala Ala Ala Gly Ser Gly Gln Gly Pro Ser Gly
195 200 205 Pro Tyr Gly Thr Ser Tyr Gln Ile Ser Thr Gln Tyr Thr Gln
Thr Thr 210 215 220 Thr Ser Gln Gly Gln Gly Tyr Gly Ser Ser Ser Ala
Gly Ala Ala Ala 225 230 235 240 Ala Gly Ala Ala Gly Ala Gly Gln Gly
Gly Tyr Gly Gly Gln Gly Gln 245 250 255 Gly Gly Tyr Gly Gln Gly Ala
Gly Gly Ala Ala Ala Ala Ala Ala Ala 260 265 270 Ala Ala Ala Ala Ala
Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Gly 275 280 285 Gly Tyr Gly
Gln Gly Gly Gln Gly Gly Gln Gly Gly Gln Gly Gln Gly 290 295 300 Gly
Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala 305 310
315 320 Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly
Pro 325 330 335 Gly Ser Gly Gly 340 5424PRTEuprosthenops
australisDOMAIN(1)..(136)NT fragment 5Met Lys Ala Ser His Thr Thr
Pro Trp Thr Asn Pro Gly Leu Ala Glu 1 5 10 15 Asn Phe Met Asn Ser
Phe Met Gln Gly Leu Ser Ser Met Pro Gly Phe 20 25 30 Thr Ala Ser
Gln Leu Asp Asp Met Ser Thr Ile Ala Gln Ser Met Val 35 40 45 Gln
Ser Ile Gln Ser Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn Lys 50 55
60 Leu Gln Ala Leu Asn Met Ala Phe Ala Ser Ser Met Ala Glu Ile Ala
65 70 75 80 Ala Ser Glu Glu Gly Gly Gly Ser Leu Ser Thr Lys Thr Ser
Ser Ile 85 90 95 Ala Ser Ala Met Ser Asn Ala Phe Leu Gln Thr Thr
Gly Val Val Asn 100 105 110 Gln Pro Phe Ile Asn Glu Ile Thr Gln Leu
Val Ser Met Phe Ala Gln 115 120 125 Ala Gly Met Asn Asp Val Ser Ala
Ser Ala Ser Ala Gly Ala Ser Ala 130 135 140 Ala Ala Ser Ala Gly Ala
Ala Ser Gly Gln Gly Gly Tyr Gly Gly Leu 145 150 155 160 Gly Gln Gly
Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala 165 170 175 Ala
Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln 180 185
190 Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala Ala Ala Ala
195 200 205 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly
Gly Tyr 210 215 220 Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala
Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Ala Ala Ala Gly Gln Gly
Gly Gln Gly Gly Tyr Gly Arg 245 250 255 Gln Ser Gln Gly Ala Gly Ser
Ala Ala Ala Ala Ala Ala Ala Ala Ala 260 265 270 Ala Ala Ala Ala Ala
Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly 275 280 285 Gln Gly Gly
Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala Ala Ala Ser 290 295 300 Ala
Ala Ser Thr Val Ala Asn Ser Val Ser Arg Leu Ser Ser Pro Ser 305 310
315 320 Ala Val Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser Asn
Gly 325 330 335 Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn
Ile Ser Ser 340 345 350 Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly
Cys Glu Val Ile Val 355 360 365 Gln Ala Leu Leu Glu Val Ile Thr Ala
Leu Val Gln Ile Val Ser Ser 370 375 380 Ser Ser Val Gly Tyr Ile Asn
Pro Ser Ala Val Asn Gln Ile Thr Asn 385 390 395 400 Val Val Ala Asn
Ala Met Ala Gln Val Met Gly Lys Leu Ala Ala Ala 405 410 415 Leu Glu
His His His His His His 420 6137PRTEuprosthenops
australisVARIANT(6)..(6)deletion (deltaHis) 6Gly Ser Gly Asn Ser
His Thr Thr Pro Trp Thr Asn Pro Gly Leu Ala 1 5 10 15 Glu Asn Phe
Met Asn Ser Phe Met Gln Gly Leu Ser Ser Met Pro Gly 20 25 30 Phe
Thr Ala Ser Gln Leu Asp Asp Met Ser Thr Ile Ala Gln Ser Met 35 40
45 Val Gln Ser Ile Gln Ser Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn
50 55 60 Lys Leu Gln Ala Leu Asn Met Ala Phe Ala Ser Ser Met Ala
Glu Ile 65 70 75 80 Ala Ala Ser Glu Glu Gly Gly Gly Ser Leu Ser Thr
Lys Thr Ser Ser 85 90 95 Ile Ala Ser Ala Met Ser Asn Ala Phe Leu
Gln Thr Thr Gly Val Val 100 105 110 Asn Gln Pro Phe Ile Asn Glu Ile
Thr Gln Leu Val Ser Met Phe Ala 115 120 125 Gln Ala Gly Met Asn Asp
Val Ser Ala 130 135 798PRTEuprosthenops australis 7 Ser Arg Leu Ser
Ser Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Ser
Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn 20 25 30
Ile Ile Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala 35
40 45 Ser Gly Cys Glu Val Ile Val Gln Ala Leu Leu Glu Val Ile Thr
Ala 50 55 60 Leu Val Gln Ile Val Ser Ser Ser Ser Val Gly Tyr Ile
Asn Pro Ser 65 70 75 80 Ala Val Asn Gln Ile Thr Asn Val Val Ala Asn
Ala Met Ala Gln Val 85 90 95 Met Gly 8131PRTArtificial
SequenceConsensus sequence derived from spidroin NT fragments 8Gln
Ala Asn Thr Pro Trp Ser Ser Pro Asn Leu Ala Asp Ala Phe Ile 1 5 10
15 Asn Ser Phe Met Ser Ala Ala Ser Ser Ser Gly Ala Phe Ser Ala Asp
20 25 30 Gln Leu Asp Asp Met Ser Thr Ile Gly Asp Thr Leu Met Ser
Ala Met 35 40 45 Asp Asn Met Gly Arg Ser Gly Lys Ser Thr Lys Ser
Lys Leu Gln Ala 50 55 60 Leu Asn Met Ala Phe Ala Ser Ser Met Ala
Glu Ile Ala Ala Ala Glu 65 70 75 80 Ser Gly Gly Gly Ser Val Gly Val
Lys Thr Asn Ala Ile Ser Asp Ala 85 90 95 Leu Ser Ser Ala Phe Tyr
Gln Thr Thr Gly Ser Val Asn Pro Gln Phe 100 105 110 Val Asn Glu Ile
Arg Ser Leu Ile Gly Met Phe Ala Gln Ala Ser Ala 115 120 125 Asn Glu
Val 130 9100PRTArtificial SequenceConsensus sequence derived from
known MaSp1 and MaSp2 proteins 9Ser Arg Leu Ser Ser Pro Gln Ala Ser
Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly
Pro Thr Asn Ser Ala Ala Leu Ser Asn 20 25 30 Thr Ile Ser Asn Val
Val Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu 35 40 45 Ser Gly Cys
Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala 50 55 60 Leu
Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70
75 80 Ser Ala Gly Gln Ala Thr Gln Ile Val Gly Gln Ser Val Ala Gln
Ala 85 90 95 Leu Gly Glu Phe 100 101110PRTEuprosthenops
australisREPEAT(7)..(19)REPEAT(20)..(42)REPEAT(43)..(56)REPEAT(57)..(70)R-
EPEAT(71)..(83)REPEAT(84)..(106)REPEAT(107)..(120)REPEAT(121)..(134)REPEAT-
(135)..(147)REPEAT(148)..(170)REPEAT(171)..(183)REPEAT(184)..(197)REPEAT(1-
98)..(211)REPEAT(212)..(234)REPEAT(235)..(248)REPEAT(249)..(265)REPEAT(266-
)..(279)REPEAT(280)..(293)REPEAT(294)..(306)REPEAT(307)..(329)REPEAT(330).-
.(342)REPEAT(343)..(356)REPEAT(357)..(370)REPEAT(371)..(393)REPEAT(394)..(-
406)REPEAT(407)..(420)REPEAT(421)..(434)REPEAT(435)..(457)REPEAT(458)..(47-
0)REPEAT(471)..(488)REPEAT(489)..(502)REPEAT(503)..(516)REPEAT(517)..(529)-
REPEAT(530)..(552)REPEAT(553)..(566)REPEAT(567)..(580)REPEAT(581)..(594)RE-
PEAT(595)..(617)REPEAT(618)..(630)REPEAT(631)..(647)REPEAT(648)..(661)REPE-
AT(662)..(675)REPEAT(676)..(688)REPEAT(689)..(711)REPEAT(712)..(725)REPEAT-
(726)..(739)REPEAT(740)..(752)REPEAT(753)..(775)REPEAT(776)..(789)REPEAT(7-
90)..(803)REPEAT(804)..(816)REPEAT(817)..(839)REPEAT(840)..(853)REPEAT(854-
)..(867)REPEAT(868)..(880)REPEAT(881)..(903)REPEAT(904)..(917)REPEAT(918).-
.(931)REPEAT(932)..(945)REPEAT(946)..(968)REPEAT(969)..(981)REPEAT(982)..(-
998)REPEAT(999)..(1013)REPEAT(1014)..(1027)REPEAT(1028)..(1042)REPEAT(1043-
)..(1059)REPEAT(1060)..(1073)REPEAT(1074)..(1092) 10Gln Gly Ala Gly
Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 15 Ala Ala
Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln 20 25 30
Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala 35
40 45 Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr
Gly 50 55 60 Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala 65 70 75 80 Ala Ala Ser Gly Gln Gly Gly Gln Gly Gly Gln
Gly Gly Gln Gly Gln 85 90 95 Gly Gly Tyr Gly Gln Gly Ala Gly Ser
Ser Ala Ala Ala Ala Ala Ala 100 105 110 Ala Ala Ala Ala Ala Ala Ala
Ala Gly Gln Gly Gln Gly Arg Tyr Gly 115 120
125 Gln Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
130 135 140 Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly Gln Gly Gly Leu
Gly Gln 145 150 155 160 Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala
Ala Ala Ala Ala Ala 165 170 175 Ser Ala Ala Ala Ala Ala Ala Gly Arg
Gly Gln Gly Gly Tyr Gly Gln 180 185 190 Gly Ala Gly Gly Asn Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala 195 200 205 Ala Ala Ala Gly Gln
Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln 210 215 220 Gly Gly Tyr
Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala 225 230 235 240
Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly 245
250 255 Arg Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala
Ala 260 265 270 Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gln Gly Gly
Tyr Gly Gln 275 280 285 Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala 290 295 300 Ala Ala Gly Gln Gly Gly Gln Gly Gly
Gln Gly Gly Leu Gly Gln Gly 305 310 315 320 Gly Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala 325 330 335 Ala Ala Ala Ala
Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly 340 345 350 Ala Gly
Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Glu Ala Ala 355 360 365
Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln Gly 370
375 380 Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala
Ala 385 390 395 400 Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly
Tyr Gly Gln Gly 405 410 415 Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala 420 425 430 Ala Ala Gly Gln Gly Gly Gln Gly
Gly Tyr Gly Gly Leu Gly Gln Gly 435 440 445 Gly Tyr Gly Gln Gly Ala
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala 450 455 460 Ala Ala Ala Ala
Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly Arg 465 470 475 480 Tyr
Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala 485 490
495 Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly
500 505 510 Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala 515 520 525 Ser Gly Gln Gly Ser Gln Gly Gly Gln Gly Gly Gln
Gly Gln Gly Gly 530 535 540 Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala
Ala Ala Ala Ala Ala Ala 545 550 555 560 Ala Ala Ala Ala Ala Ser Gly
Arg Gly Gln Gly Gly Tyr Gly Gln Gly 565 570 575 Ala Gly Gly Asn Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 580 585 590 Ala Ala Gly
Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln Gly 595 600 605 Gly
Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala 610 615
620 Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr
625 630 635 640 Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala
Ala Ala Ala 645 650 655 Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly
Tyr Gly Gln Gly Ser 660 665 670 Gly Gly Asn Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ser 675 680 685 Gly Gln Gly Gly Gln Gly Gly
Gln Gly Gly Gln Gly Gln Gly Gly Tyr 690 695 700 Gly Gln Gly Ala Gly
Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 705 710 715 720 Ala Ala
Ala Ala Ala Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ala 725 730 735
Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 740
745 750 Gly Gln Gly Gly Gln Gly Gly Gln Gly Gly Leu Gly Gln Gly Gly
Tyr 755 760 765 Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala
Ala Ala Ala 770 775 780 Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly
Tyr Gly Gln Gly Val 785 790 795 800 Gly Gly Asn Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala 805 810 815 Gly Gln Gly Gly Gln Gly
Gly Gln Gly Gly Leu Gly Gln Gly Gly Tyr 820 825 830 Gly Gln Gly Ala
Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 835 840 845 Ala Ala
Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly Ser 850 855 860
Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser 865
870 875 880 Gly Gln Gly Ser Gln Gly Gly Gln Gly Gly Gln Gly Gln Gly
Gly Tyr 885 890 895 Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala
Ala Ala Ala Ala 900 905 910 Ala Ala Ala Ala Ser Gly Arg Gly Gln Gly
Gly Tyr Gly Gln Gly Ala 915 920 925 Gly Gly Asn Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala 930 935 940 Ala Gly Gln Gly Gly Gln
Gly Gly Tyr Gly Gly Leu Gly Gln Gly Gly 945 950 955 960 Tyr Gly Gln
Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala 965 970 975 Ala
Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr Gly 980 985
990 Gln Gly Ser Gly Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
995 1000 1005 Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly
Gln Gly 1010 1015 1020 Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala 1025 1030 1035 Ala Ala Ala Ala Gly Gln Gly Gly Gln
Gly Gly Tyr Gly Arg Gln 1040 1045 1050 Ser Gln Gly Ala Gly Ser Ala
Ala Ala Ala Ala Ala Ala Ala Ala 1055 1060 1065 Ala Ala Ala Ala Ala
Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln 1070 1075 1080 Gly Gln Gly
Gly Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala Ala 1085 1090 1095 Ala
Ser Ala Ala Ser Thr Val Ala Asn Ser Val Ser 1100 1105 1110
1123PRTArtificial SequenceConsensus sequence derived from internal
repeats of Euprosthenops australis MaSp1 11Gly Gln Gly Gly Gln Gly
Gly Gln Gly Gly Leu Gly Gln Gly Gly Tyr 1 5 10 15 Gly Gln Gly Ala
Gly Ser Ser 20 1217PRTArtificial SequenceConsensus sequence derived
from internal repeats of Euprosthenops australis MaSp1 12Gly Gln
Gly Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ser 1 5 10 15
Ser 1314PRTArtificial SequenceConsensus sequence derived from
internal repeats of Euprosthenops australis MaSp1 13Gly Arg Gly Gln
Gly Gly Tyr Gly Gln Gly Ala Gly Gly Asn 1 5 10
14287PRTEuprosthenops australis 14Asn Ser Gly Tyr Gly Gln Gly Ala
Gly Gly Asn Ala Ala Ala Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Ala
Ala Ala Ala Gly Gln Gly Gly Gln Gly Gly 20 25 30 Tyr Gly Gly Leu
Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser 35 40 45 Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Gly 50 55 60
Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala 65
70 75 80 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly
Arg Gly 85 90 95 Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala
Ala Ala Ala Ala 100 105 110 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Gly Gln Gly Gly Gln Gly 115 120 125 Gly Tyr Gly Arg Gln Ser Gln Gly
Ala Gly Ser Ala Ala Ala Ala Ala 130 135 140 Ala Ala Ala Ala Ala Ala
Ala Ala Ala Gly Ser Gly Gln Gly Gly Tyr 145 150 155 160 Gly Gly Gln
Gly Gln Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala Ser 165 170 175 Ala
Ala Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Val Ser Arg Leu 180 185
190 Ser Ser Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val Ser Ser Leu
195 200 205 Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile
Ile Ser 210 215 220 Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly
Ala Ser Gly Cys 225 230 235 240 Glu Val Ile Val Gln Ala Leu Leu Glu
Val Ile Thr Ala Leu Val Gln 245 250 255 Ile Val Ser Ser Ser Ser Val
Gly Tyr Ile Asn Pro Ser Ala Val Asn 260 265 270 Gln Ile Thr Asn Val
Val Ala Asn Ala Met Ala Gln Val Met Gly 275 280 285
15382PRTEuprosthenops australis 15Gly Ser Gly Asn Ser Gly Tyr Gly
Gln Gly Ala Gly Ser Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln 20 25 30 Gly Gly Tyr Gly
Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala 35 40 45 Ala Ala
Ala Ala Ala Ala Ser Gly Gln Gly Ser Gln Gly Gly Gln Gly 50 55 60
Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser Ala Ala 65
70 75 80 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Arg
Gly Gln 85 90 95 Gly Gly Tyr Gly Gln Gly Ala Gly Gly Asn Ala Ala
Ala Ala Ala Ala 100 105 110 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln
Gly Gly Gln Gly Gly Tyr 115 120 125 Gly Gly Leu Gly Gln Gly Gly Tyr
Gly Gln Gly Ala Gly Ser Ser Ala 130 135 140 Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly 145 150 155 160 Gln Gly Gln
Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala 165 170 175 Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln 180 185
190 Gly Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala
195 200 205 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln
Gly Gly 210 215 220 Tyr Gly Arg Gln Ser Gln Gly Ala Gly Ser Ala Ala
Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Ala Ala Ala Ala Ala Gly
Ser Gly Gln Gly Gly Tyr Gly 245 250 255 Gly Gln Gly Gln Gly Gly Tyr
Gly Gln Ser Ser Ala Ser Ala Ser Ala 260 265 270 Ala Ala Ser Ala Ala
Ser Thr Val Ala Asn Ser Val Ser Arg Leu Ser 275 280 285 Ser Pro Ser
Ala Val Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val 290 295 300 Ser
Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn 305 310
315 320 Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly Cys
Glu 325 330 335 Val Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala Leu
Val Gln Ile 340 345 350 Val Ser Ser Ser Ser Val Gly Tyr Ile Asn Pro
Ser Ala Val Asn Gln 355 360 365 Ile Thr Asn Val Val Ala Asn Ala Met
Ala Gln Val Met Gly 370 375 380 16270PRTEuprosthenops australis
16Gly Ser Gly Asn Ser Gly Gln Gly Gln Gly Gly Phe Ser Gln Gly Ala 1
5 10 15 Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala 20 25 30 Ala Ala Gln Gln Gly Gly Gln Gly Gly Phe Gly Gly Arg
Gly Gln Gly 35 40 45 Gly Phe Gly Pro Gly Ala Gly Ser Ser Ala Ala
Ala Ala Ala Ala Ala 50 55 60 Thr Ala Ala Ala Gly Gln Gly Gly Gln
Gly Arg Gly Gly Phe Gly Gln 65 70 75 80 Gly Ala Gly Ser Asn Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala 85 90 95 Ala Ala Ala Ala Gly
Gln Gly Gly Gln Gly Gln Gly Gly Phe Gly Gln 100 105 110 Gly Thr Val
Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 115 120 125 Ala
Ala Ala Gln Gln Gly Gly Gln Gly Gly Phe Gly Gly Gln Gly Gln 130 135
140 Arg Gly Phe Gly Gln Arg Ala Ala Ser Ala Val Ala Ser Ala Ala Ser
145 150 155 160 Ala Ala Asp Val Gly Asn Thr Val Ala Asn Thr Val Ser
Arg Leu Ser 165 170 175 Ser Pro Ser Ala Ala Ser Arg Val Ser Ser Ala
Val Ala Asn Leu Val 180 185 190 Ser Asn Gly Gln Leu Asn Met Ala Ala
Leu Pro Tyr Ile Ile Ser Asn 195 200 205 Ile Ser Ser Ser Val Ser Ala
Ser Val Pro Gly Ala Ser Gly Cys Glu 210 215 220 Val Ile Val Gln Ala
Leu Leu Glu Val Val Ala Ala Leu Cys Gln Ile 225 230 235 240 Val Ser
Ser Ser Asn Val Gly Tyr Ile Asn Pro Ser Ala Val Asn Asp 245 250 255
Ile Thr Asn Val Val Ala Asn Ala Met Ala Gln Val Met Gly 260 265 270
17513PRTEuprosthenops australis 17Met Lys Ala Ser His Thr Thr Pro
Trp Thr Asn Pro Gly Leu Ala Glu 1 5 10 15 Asn Phe Met Asn Ser Phe
Met Gln Gly Leu Ser Ser Met Pro Gly Phe 20 25 30 Thr Ala Ser Gln
Leu Asp Asp Met Ser Thr Ile Ala Gln Ser Met Val 35 40 45 Gln Ser
Ile Gln Ser Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn Lys 50 55 60
Leu Gln Ala Leu Asn Met Ala Phe Ala Ser Ser Met Ala Glu Ile Ala 65
70 75 80 Ala Ser Glu Glu Gly Gly Gly Ser Leu Ser Thr Lys Thr Ser
Ser Ile 85 90 95 Ala Ser Ala Met Ser Asn Ala Phe Leu Gln Thr Thr
Gly Val Val Asn 100 105 110 Gln Pro Phe Ile Asn Glu Ile Thr Gln Leu
Val Ser Met Phe Ala Gln 115 120 125 Ala Gly Met Asn Asp Val Ser Ala
Gly Tyr Gly Gln Gly Ala Gly Ser 130 135 140 Ser Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly 145 150 155 160 Arg Gly Gln
Gly Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala 165 170 175 Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Gln Gly Ser Gln Gly 180 185
190 Gly Gln Gly Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ser
195 200 205 Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ser Gly 210 215 220 Arg Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Gly
Asn Ala Ala Ala 225 230 235 240 Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Gly Gln Gly Gly Gln 245 250 255 Gly Gly Tyr Gly Gly Leu Gly
Gln Gly Gly Tyr Gly Gln Gly Ala Gly 260 265 270 Ser Ser Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly 275 280 285 Gln Gly Gly
Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser 290
295 300 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Gly 305 310 315 320 Arg Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly
Asn Ala Ala Ala 325 330 335 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Gly Gln Gly Gly 340 345 350 Gln Gly Gly Tyr Gly Arg Gln Ser
Gln Gly Ala Gly Ser Ala Ala Ala 355 360 365 Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Gly Ser Gly Gln Gly 370 375 380 Gly Tyr Gly Gly
Gln Gly Gln Gly Gly Tyr Gly Gln Ser Ser Ala Ser 385 390 395 400 Ala
Ser Ala Ala Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Val Ser 405 410
415 Arg Leu Ser Ser Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val Ser
420 425 430 Ser Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro
Asn Ile 435 440 445 Ile Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala
Pro Gly Ala Ser 450 455 460 Gly Cys Glu Val Ile Val Gln Ala Leu Leu
Glu Val Ile Thr Ala Leu 465 470 475 480 Val Gln Ile Val Ser Ser Ser
Ser Val Gly Tyr Ile Asn Pro Ser Ala 485 490 495 Val Asn Gln Ile Thr
Asn Val Val Ala Asn Ala Met Ala Gln Val Met 500 505 510 Gly
18640PRTEuprosthenops australis 18Ser His Thr Thr Pro Trp Thr Asn
Pro Gly Leu Ala Glu Asn Phe Met 1 5 10 15 Asn Ser Phe Met Gln Gly
Leu Ser Ser Met Pro Gly Phe Thr Ala Ser 20 25 30 Gln Leu Asp Asp
Met Ser Thr Ile Ala Gln Ser Met Val Gln Ser Ile 35 40 45 Gln Ser
Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn Lys Leu Gln Ala 50 55 60
Leu Asn Met Ala Phe Ala Ser Ser Met Ala Glu Ile Ala Ala Ser Glu 65
70 75 80 Glu Gly Gly Gly Ser Leu Ser Thr Lys Thr Ser Ser Ile Ala
Ser Ala 85 90 95 Met Ser Asn Ala Phe Leu Gln Thr Thr Gly Val Val
Asn Gln Pro Phe 100 105 110 Ile Asn Glu Ile Thr Gln Leu Val Ser Met
Phe Ala Gln Ala Gly Met 115 120 125 Asn Asp Gly Gly Gly Thr Pro Trp
Thr Asn Pro Gly Leu Ala Glu Asn 130 135 140 Phe Met Asn Ser Phe Met
Gln Gly Leu Ser Ser Met Pro Gly Phe Thr 145 150 155 160 Ala Ser Gln
Leu Asp Asp Met Ser Thr Ile Ala Gln Ser Met Val Gln 165 170 175 Ser
Ile Gln Ser Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn Lys Leu 180 185
190 Gln Ala Leu Asn Met Ala Phe Ala Ser Ser Met Ala Glu Ile Ala Ala
195 200 205 Ser Glu Glu Gly Gly Gly Ser Leu Ser Thr Lys Thr Ser Ser
Ile Ala 210 215 220 Ser Ala Met Ser Asn Ala Phe Leu Gln Thr Thr Gly
Val Val Asn Gln 225 230 235 240 Pro Phe Ile Asn Glu Ile Thr Gln Leu
Val Ser Met Phe Ala Gln Ala 245 250 255 Gly Met Asn Asp Val Ser Ala
Gly Tyr Gly Gln Gly Ala Gly Ser Ser 260 265 270 Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg 275 280 285 Gly Gln Gly
Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala 290 295 300 Ala
Ala Ala Ala Ala Ala Ala Ala Ser Gly Gln Gly Ser Gln Gly Gly 305 310
315 320 Gln Gly Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ser
Ser 325 330 335 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ser Gly Arg 340 345 350 Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Gly
Asn Ala Ala Ala Ala 355 360 365 Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala Gly Gln Gly Gly Gln Gly 370 375 380 Gly Tyr Gly Gly Leu Gly Gln
Gly Gly Tyr Gly Gln Gly Ala Gly Ser 385 390 395 400 Ser Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln 405 410 415 Gly Gly
Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala 420 425 430
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg 435
440 445 Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala
Ala 450 455 460 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln
Gly Gly Gln 465 470 475 480 Gly Gly Tyr Gly Arg Gln Ser Gln Gly Ala
Gly Ser Ala Ala Ala Ala 485 490 495 Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Gly Ser Gly Gln Gly Gly 500 505 510 Tyr Gly Gly Gln Gly Gln
Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala 515 520 525 Ser Ala Ala Ala
Ser Ala Ala Ser Thr Val Ala Asn Ser Val Ser Arg 530 535 540 Leu Ser
Ser Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val Ser Ser 545 550 555
560 Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile
565 570 575 Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala
Ser Gly 580 585 590 Cys Glu Val Ile Val Gln Ala Leu Leu Glu Val Ile
Thr Ala Leu Val 595 600 605 Gln Ile Val Ser Ser Ser Ser Val Gly Tyr
Ile Asn Pro Ser Ala Val 610 615 620 Asn Gln Ile Thr Asn Val Val Ala
Asn Ala Met Ala Gln Val Met Gly 625 630 635 640 19270PRTArtificial
SequenceEngineered 4RepCT variant 19Gly Pro Asn Ser Arg Gly Asp Ala
Gly Ala Ala Ser Gly Gln Gly Gly 1 5 10 15 Tyr Gly Gly Leu Gly Gln
Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser 20 25 30 Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gly 35 40 45 Gln Gly
Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala 50 55 60
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln 65
70 75 80 Gly Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala
Ala Ala 85 90 95 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly
Gly Gln Gly Gly 100 105 110 Tyr Gly Arg Gln Ser Gln Gly Ala Gly Ser
Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Ala Ala Ala Ala Ala Ala
Gly Ser Gly Gln Gly Gly Tyr Gly 130 135 140 Gly Gln Gly Gln Gly Gly
Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala 145 150 155 160 Ala Ala Ser
Ala Ala Ser Thr Val Ala Asn Ser Val Ser Arg Leu Ser 165 170 175 Ser
Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val 180 185
190 Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn
195 200 205 Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly
Cys Glu 210 215 220 Val Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala
Leu Val Gln Ile 225 230 235 240 Val Ser Ser Ser Ser Val Gly Tyr Ile
Asn Pro Ser Ala Val Asn Gln 245 250 255 Ile Thr Asn Val Val Ala Asn
Ala Met Ala Gln Val Met Gly 260 265 270 20270PRTArtificial
SequenceEngineered 4RepCT variant 20Gly Pro Asn Ser Arg Gly Glu Ala
Gly Ala Ala Ser Gly Gln Gly Gly 1 5 10 15 Tyr Gly Gly Leu Gly Gln
Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser 20 25 30 Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly Gly 35 40 45 Gln Gly
Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala 50 55 60
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln 65
70 75 80 Gly Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala
Ala Ala 85 90 95 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln Gly
Gly Gln Gly Gly 100 105 110 Tyr Gly Arg Gln Ser Gln Gly Ala Gly Ser
Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Ala Ala Ala Ala Ala Ala
Gly Ser Gly Gln Gly Gly Tyr Gly 130 135 140 Gly Gln Gly Gln Gly Gly
Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala 145 150 155 160 Ala Ala Ser
Ala Ala Ser Thr Val Ala Asn Ser Val Ser Arg Leu Ser 165 170 175 Ser
Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val 180 185
190 Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn
195 200 205 Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly
Cys Glu 210 215 220 Val Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala
Leu Val Gln Ile 225 230 235 240 Val Ser Ser Ser Ser Val Gly Tyr Ile
Asn Pro Ser Ala Val Asn Gln 245 250 255 Ile Thr Asn Val Val Ala Asn
Ala Met Ala Gln Val Met Gly 260 265 270 21272PRTArtificial
SequenceEngineered 4RepCT variant 21Gly Pro Asn Ser Ile Lys Val Ala
Val Ala Gly Ala Arg Ser Gly Gln 1 5 10 15 Gly Gly Tyr Gly Gly Leu
Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly 20 25 30 Ser Ser Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln 35 40 45 Gly Gly
Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala 50 55 60
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg 65
70 75 80 Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Asn Ala Ala
Ala Ala 85 90 95 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly
Gln Gly Gly Gln 100 105 110 Gly Gly Tyr Gly Arg Gln Ser Gln Gly Ala
Gly Ser Ala Ala Ala Ala 115 120 125 Ala Ala Ala Ala Ala Ala Ala Ala
Ala Ala Gly Ser Gly Gln Gly Gly 130 135 140 Tyr Gly Gly Gln Gly Gln
Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala 145 150 155 160 Ser Ala Ala
Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Val Ser Arg 165 170 175 Leu
Ser Ser Pro Ser Ala Val Ser Arg Val Ser Ser Ala Val Ser Ser 180 185
190 Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile
195 200 205 Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala
Ser Gly 210 215 220 Cys Glu Val Ile Val Gln Ala Leu Leu Glu Val Ile
Thr Ala Leu Val 225 230 235 240 Gln Ile Val Ser Ser Ser Ser Val Gly
Tyr Ile Asn Pro Ser Ala Val 245 250 255 Asn Gln Ile Thr Asn Val Val
Ala Asn Ala Met Ala Gln Val Met Gly 260 265 270 22267PRTArtificial
SequenceEngineered 4RepCT variant 22Gly Pro Asn Ser Tyr Ile Gly Ser
Arg Gly Gln Gly Gly Tyr Gly Gly 1 5 10 15 Leu Gly Gln Gly Gly Tyr
Gly Gln Gly Ala Gly Ser Ser Ala Ala Ala 20 25 30 Ala Ala Ala Ala
Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly Gln 35 40 45 Gly Gly
Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala Ala Ala Ala Ala 50 55 60
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr 65
70 75 80 Gly Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala
Ala Ala 85 90 95 Ala Ala Ala Ala Ala Ala Gly Gln Gly Gly Gln Gly
Gly Tyr Gly Arg 100 105 110 Gln Ser Gln Gly Ala Gly Ser Ala Ala Ala
Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Ala Ala Ala Gly Ser Gly
Gln Gly Gly Tyr Gly Gly Gln Gly 130 135 140 Gln Gly Gly Tyr Gly Gln
Ser Ser Ala Ser Ala Ser Ala Ala Ala Ser 145 150 155 160 Ala Ala Ser
Thr Val Ala Asn Ser Val Ser Arg Leu Ser Ser Pro Ser 165 170 175 Ala
Val Ser Arg Val Ser Ser Ala Val Ser Ser Leu Val Ser Asn Gly 180 185
190 Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn Ile Ser Ser
195 200 205 Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly Cys Glu Val
Ile Val 210 215 220 Gln Ala Leu Leu Glu Val Ile Thr Ala Leu Val Gln
Ile Val Ser Ser 225 230 235 240 Ser Ser Val Gly Tyr Ile Asn Pro Ser
Ala Val Asn Gln Ile Thr Asn 245 250 255 Val Val Ala Asn Ala Met Ala
Gln Val Met Gly 260 265 235PRTArtificial SequenceCell-binding motif
23Ile Lys Val Ala Val 1 5 245PRTArtificial SequenceCell-binding
motif 24Tyr Ile Gly Ser Arg 1 5 255PRTArtificial
SequenceCell-binding motif 25Glu Pro Asp Ile Met 1 5
265PRTArtificial SequenceCell-binding motif 26Asn Lys Asp Ile Leu 1
5
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