Growth of pancreatic islet-like cell clusters

A method for growing endocrine precursor cells in vitro, such as pancreatic islet precursors, by culturing such cells in the presence or absence of cell matrix proteins capable of promoting hemidesmosome formation, such as those produced by the rat bladder carcinoma cell line 804G.

BACKGROUND 
When organs of the body are formed, they develop as neatly organized arrays 
of cells. Often, cell groups of one kind are separated from cells of 
another kind by flat strips of connective tissue called basement 
membranes. In skin, for instance, the superficial layer of epidermal cells 
adheres to the underlying basement membrane. This skin basement membrane 
acts as a barrier between the epidermal cells on the outside, and the 
dermal cells underneath. A similar arrangement of cells occurs in the 
lining of the gut. 
Basement membranes have been implicated in the growth, attachment, 
migration, repair, and differentiation of their overlying cell 
populations. Three layers have been defined in basement membranes: a) the 
Lamina lucida, an electronmicroscopically clear region that resides in 
close approximation to the overlying cells; b) the lamina densa, an 
electron dense region of 20-300 nm in width; and c) the sublamina densa 
that contains anchoring fibrils, microfibrillar bundles and collagen 
fibers. 
Many different types of compounds have now been localized to the basement 
membrane. Some of these compounds are laminin, collagen IV and heparin 
sulfate proteoglycans (Verrando et al. Exp. Cell Res. (1987); 170: 
116-128). In addition, specific basement membranes include other 
biologically active components, such as nidogen and entactin. 
One major cell adhesion receptor that epidermal cells use to attach to the 
basement membrane is called .alpha.6 .beta.4. This transmembrane receptor 
is formed by a combination of two protein moieties .alpha.6 and .beta.4. 
The .alpha.6 and .beta.4 proteins are derived from different genes that 
have been found to be part of the integrin family. 
Integrins are versatile family cell adhesion receptors. So far, 
approximately twenty members have been discovered in the integrin family. 
These molecules are involved in many types of cell adhesion phenomena in 
the body. Integrins are signalling molecules that can translate 
environmental cues into cellular instructions. Further, integrins can also 
transmit signals in the reverse direction, from the cell interior to the 
exterior. This has been illustrated in non-adherent cells, such as 
lymphocytes. 
Stimulation of the T-cell antigen receptor, or of the CD3 complex, augments 
the affinity of certain integrins for their respective ligands. 
Unfortunately, in adherent cells, changes in the affinities of integrins 
have been more difficult to demonstrate. However, affinity modulation of 
one integrin in differentiating epidermal keratinocytes has been described 
by Adams et al. (Cell (1990); 63: 425-435). For this reason, modifications 
of cell status initiated by activation or differentiation of other 
receptors may influence integrin affinity, and ultimately, the adhesive 
behavior of cells. Further, as a consequence of adhering to a surface, an 
integrin may actively contribute to modifying cell shape or migration. 
Many epithelial cells interact with the underlying extracellular matrix via 
a junction called the hemidesmosome (Staehelin, 1974). Over the last few 
years there has been considerable progress in the biochemical 
characterization of this junction (Schwartz, et al., 1990). The 
hemidesmosome, with its associated structures such as intermediate 
filaments and anchoring fibrils, forms an adhesion complex. Disruptions of 
the epithelial-connective tissue interaction are often accompanied by 
disruption of the hemidesmosome complex. For example, in certain 
blistering skin diseases such as junctional epidermolysis bullosa where 
epithelial cell-connective tissue interaction is abnormal, it has been 
proposed that there is a biochemical modification in or loss of a basement 
membrane zone-associated component of the hemidesmosome. 
Two high molecular weight intracellular components of the hemidesmosome 
have been identified and characterized with the aid of antisera from 
patients suffering from bullous pemphigoid. This autoimmune disease 
results in a disruption of the interactions between epithelial cells and 
connective tissue simultaneously with loss of hemidesmosome integrity 
(Stanley, (1993) Adv. Immunol., 63: 291-325). Accordingly, it was 
discovered that bullous pemphigoid patients were producing antibodies 
against hemidesmosome components. Two hemidesmosome related bullous 
pemphigoid (BP) antigens have been previously described (Klatte, et al., 
1989). 
One BP antigen is a 230 kD polypeptide that may act as an anchor for 
cytoskeleton elements in the hemidesmosomal plaque (Jones and Green, 
1991). A second BP antigen is a type II membrane protein that possesses a 
collagen-like extracellular domain (Giudice, et al., 1991; Hopkinson, et 
al., 1992). In addition, it has been demonstrated that the interaction of 
the hemidesmosome with the underlying connective tissue involves the 
.alpha..sub.6 .beta..sub.4 integrin heterodimer (Stepp, et al., 1990; 
Jones, et al., 1991; Sonnenberg, et al., 1991; Kurpakus, et al., 1991). 
The .alpha..sub.6 .beta..sub.4 heterodimer has been localized to 
hemidesmosomes along the basal surfaces of the rat bladder carcinoma cell 
line 804G (Jones et al. Cell Regulation (1991); 2: 427-438). These results 
suggested that integrins (e.g. .alpha..sub.6 .beta..sub.4) may play an 
important role in the assembly and adhesive functions of hemidesmosomes. 
Various prior art efforts have focused on purifying adhesion-facilitating 
proteins found in basement membrane. For example, Burgeson, et al., Patent 
Cooperation Treaty Application No. WO92/17498, disclose a protein which 
they call kalinin. Kalinin is said to facilitate cell adhesion to 
substrates; however, this material is apparently inactive with respect to 
hemidesmosome formation. See also, Marinkovich, et al., J. Cell Biol. 
(1992); 119:695-703 (klaminin); laminin Rouselie, et al., J. Cell. Biol. 
(1991); 114:567-576 (kalinin); and Marinkovich, et al., J. Biol. Chem. 
(1992); 267:17900-17906 (kalinin). 
Similarly, a basement glycoprotein of about 600 kD made up of polypeptides 
in the range of 93.5 kD to 150 kD has been identified, and is known as GB3 
or nicein. See, e.g., Verrando, et al., Biochim. Biophys. Acta (1988); 
942:45-56; and Hsi, et al., Placenta (1987); 8:209-217. None of these 
materials have been effective in generating formation of hemidesmosomes, 
either in vitro or in vivo. 
When cultured on tissue culture plastic in vitro, most epithelial cells do 
not assemble bona fide hemidesmosomes despite the fact that they appear to 
express all of the hemidesmosomal plaque and transmembrane components 
mentioned above. Indeed, it is only recently that cell lines such as 804G 
were discovered to have the ability to readily assemble hemidesmosomes in 
vitro under regular culture conditions (Riddelle, et al., 1991; Hieda, et 
al., 1992). Such cells are at last allowing detailed cell and biochemical 
analysis of the dynamics of hemidesmosome assembly. 
For instance, it has been reported that substratum-associated staining by 
anti-hemidesmosome antibodies is greatly diminished in 804G cell cultures 
that enter in vitro wound sites (Riddelle et al., J. Cell Sci. (1992); 
103: 475-490). However, as closure of the wound became complete, 
anti-hemidesmosome staining along the substratum-attached surface was 
evident in the cells. 
There are, however, many epithelial cells that do not attach to tissue 
culture dishes in a normal fashion, even after treatment with various 
growth factors. These cells do not produce normal hemidesmosomes or grow 
to resemble their in vivo phenotype. It would provide a tremendous 
advantage to have a system that was capable of maintaining epithelial cell 
growth in vitro wherein the cells maintained their normal phenotype. 
Nearly two million Americans are afflicted with Type I (insulin-dependent) 
diabetes, in which the pancreas has lost its ability to secrete insulin 
due to an autoimmune disorder in which the insulin-secreting beta cells, 
found within the islet cells of the pancreas, are destroyed. Although 
insulin injections can compensate for beta cell destruction, blood sugar 
levels can still fluctuate dramatically. The impaired ability to take up 
glucose from the blood results in side reactions in which toxic products 
accumulate, leading to complications including blindness, kidney disease, 
nerve damage, and, ultimately, coma and death. 
Researchers have tried smaller, more frequent doses of insulin and 
mechanical pumps which mimic the action of the pancreas, but the results 
have been far from ideal. Another option, pancreatic transplant, requires 
major surgery and is accompanied by many complications. In addition, the 
limited number of donor pancreases leaves a significant number of 
diabetics without hope for transplantation. 
The most promising option thus far is islet cell transplantation using 
tissue derived from either cadavers or human fetuses. This procedure has 
had moderate success. Among the transplants from cadavers performed 
worldwide, the transplanted tissue survived for a full year in about 20% 
of recipients. Ten of these recipients are now insulin-independent, while 
others have a greatly reduced need for insulin. The main problems 
associated with islet cell transplantation include rejection by the immune 
system and the autoimmune disorder which caused the disease in the first 
place which, if left unchecked, will also destroy the transplanted islet 
cells. 
Islet-like cell clusters (ICCs) are composed of a heterogeneous cell 
population. In addition to epithelial cells which differentiate to form 
the endocrine, exocrine and ductal tissues, the clusters contain many 
stromal cells, primarily fibroblasts and endothelial cells. The presence 
of large numbers of stromal cells complicates the issue due to 
difficulties in quantitating important measures of differentiation such as 
insulin content per cell (Beattie et al., (1991) J. Clin. Endocrinol. 
Metab., 73: 93-98). Also, the effects of growth and differentiation 
factors on endocrine precursor cells from ICCs are complicated by the 
presence of stromal cells. 
Fetal pancreatic tissue has certain advantages over adult pancreas as a 
source of islet cells including its greater content of islets in 
proportion to its mass, its less mature endocrine cells and its greater 
capacity for proliferation (Voss et al., (1989) Transplantation Proc., 21: 
2751-2756). It is hoped that fetal islet cell transplants will 
dramatically reduce or eliminate diabetics' insulin dependence in a 
majority of patients in controlling blood sugar levels, thus minimizing 
the most severe diabetic complications. 
Earlier attempts at culturing pancreatic islet cells were complicated by 
fibroblast contamination (Leach et al., (1973) J. Endocrinol., 59: 65-79). 
Although partially digested fetal pancreas has been used to produce ICCs, 
the clinical use of these clusters is limited because only 100-200 can be 
obtained per pancreas (Sandler et al., (1985) Diabetes, 34: 1113-1119; 
Otonkoski et al., (1988) Acta. Endocrinol., 118: 68-76). Kover and Moore 
(Diabetes, 38: 917-924 (1989)) obtained 200-300 islets from a 17 week 
fetal pancreas, still not enough to be clinically useful. Finally, Simpson 
et al. (Diabetes, 40: 800-808 (1991)) were able to generate 
insulin-secreting, fibroblast-free monolayers of human fetal pancreas 
plated on bovine corneal matrix, although adequate numbers of cells for 
clinical transplantation were still not obtained. Although only a small 
number of cells within the clusters stain positively for the different 
pancreatic hormones, they differentiate efficiently into mature endocrine 
cells following transplantation into nude mice (Sandler et al., (1985) 
Diabetes, 34: 1113-1119). 
Expansion of the pool of available islet cells for transplantation is 
highly desirable because the current technology will not produce enough 
cells for routine transplantation. 
SUMMARY OF THE INVENTION 
One embodiment of the present invention is a method for enhancing the 
growth of endocrine precursor cells by culturing the cells in the presence 
of 804G matrix. Preferably, the endocrine precursor cells are pancreatic 
islet cell precursors. This embodiment further provides, prior to the 
culturing step, enzymatically digesting fetal pancreas and incubating the 
digested tissue in medium until islet-like cell aggregates are formed. 
Preferably, the pancreas is human and the 804G matrix proteins are 
attached to a substrate. Another aspect of the invention provides that the 
804G matrix is derived from 804G rat bladder carcinoma cells. 
Another embodiment of the invention is a method for the generation of 
hormone-producing cells by producing expanded endocrine precursor cells 
and transplanting these cells into a mammal. Preferably, these cells are 
pancreatic islet cells, the hormone is insulin and the transplantation 
site is either the kidney, lung or liver. 
The present invention also provides expanded endocrine precursor cells 
which are preferably fetal pancreatic islet precursor cells. 
Detailed Description of the Invention 
The present invention includes the discovery that certain cell lines 
produce an extracellular matrix that is capable of stimulating cellular 
adhesion and hemidesmosome assembly in other cells subsequently grown on 
the matrix. One such cell line is the bladder carcinoma cell line 804G. 
This cell line is described by Izumi, et al., Cancer Res. (1981); 
41:405-409, and is maintained in permanent collection in the laboratory of 
Jonathan C. R. Jones. This cell line is also available from Ryoichi Oyasu, 
Department of Pathology, Northwestern University Medical School, Chicago, 
Ill. The 804G cell line has also been deposited as a budapest Treaty 
Deposit with the American Type Culture Collection, Rockville, Md., on Feb. 
24, 1994, under Accession Number CRL 11555. The NBT II cell line referred 
to herein has been deposited as a Budapest Treaty Deposit with the 
American Type culture Collection, Rockville, Md., under Accession Number 
CRL 11556. 
Ultrastructural data have been developed demonstrating that the 804G matrix 
is capable of inducing a number of cells to develop mature hemidesmosomes 
and attach to their growth substrate. Further, it has been discovered that 
the 804G matrix contains novel laminin-like molecules that participate in 
hemidesmosome assembly (unlike laminins and related molecules that have 
been purified in the prior art). Three of these molecules have been cloned 
from a rat 804G cDNA library and encode proteins of 150, 140 and 135 kDa. 
Analysis of two cDNAs encoding the 140 kDa protein revealed sequence 
similarity with human laminin B2t (Kallunki et al., (1992) J. Cell Biol., 
119: 679-695). A novel matrix can now be prepared, produced by such cells 
as 804G cells, that can modulate the organization of hemidesmosomal 
antigens in unrelated cells maintained upon it. This effect appears 
specific to hemidesmosomal elements since adhesion plaque components do 
not obviously change their localization in cells maintained upon the 
matrix of the present invention. 
To demonstrate this new discovery, evidence is provided that the rat 804G 
matrix was capable of inducing assembly of "mature" hemidesmosomes in 
human epidermal carcinoma (SCC12) cells. It can be appreciated that it is 
uncommon to find compounds from rodent cells that have such a profound 
effect on human tissue. In these experiments, described in more detail 
below, an increased number of hemidesmosome-like structures were found in 
SCC12 cells maintained upon the 804G matrix as compared to control 
experiments wherein SCC12 cells were grown on rat tail collagen. Moreover, 
the majority of these hemidesmosome-like structures in the 804G matrix 
grown cells were in contact with the cell substrate and possessed basal 
dense plates. The latter structures are often used as indicators of mature 
or formed hemidesmosomes (Krawczyk and Wilgram, 1973). 
Although methods related to production and isolation of the 804G cell 
matrix are specifically disclosed, it can be appreciated that any cell 
matrix having the ability to support cell adhesion and hemidesmosome 
assembly is within the scope of the present invention. Matrices from other 
cell types, such as the rat bladder carcinoma cell line NBT II (ATCC CRL 
1655) also appear to be able to induce attachment and hemidesmosome 
assembly in vitro. It should be noted that the term "804G Matrix" is used 
to generically refer to any cell matrix with the ability to stimulate cell 
attachment and hemidesmosome formation. As defined herein, 804G matrix is 
comprised of one or more protein components secreted by 804G rat bladder 
carcinoma cells which facilitate hemidesmosome formation in epidermal 
cells and keratinocytes. 
One major use contemplated for the active components of the matrix of the 
present invention is in cell growth and attachment. A substrate upon which 
cells are to be grown is coated with the matrix or with purified 
hemidesmosome-promoting components thereof. The cells to be grown are then 
plated or applied to the substrate, and grown on the matrix. Such cells, 
including human cells in vitro and in vivo, will grow in an organized 
fashion on the substrate and will form hemidesmosomes. Hemidesmosome 
formation is a major advantage, because it greatly enhances the attachment 
of the cells to the substrate. Furthermore, it appears that the 
organization of cells growing on the matrix is significantly more 
advanced, more tissue-like, than cells grown without the matrix of the 
present invention. 
The substrate used herein may be any desired substrate. For laboratory use, 
the substrate may be as simple as glass or plastic. Alternatively, any 
suitable substrate may be used, including various shaped articles, 
fabrics, prosthetic implants, and the like. For use in vivo, the substrate 
may be any biologically compatible material on which cells can grow. 
Suitable substrate materials may include shaped articles made of or coated 
with such materials as collagen; regenerated collagen; polylactic acid; 
biocompatible metals such as stainless steel and titanium; ceramic 
materials including prosthetic materials such as hydroxylapatite; 
synthetic polymers, including polyesters and nylons; biological materials 
that are actually part of a patient, such as bones and teeth, and 
virtually any other material to which biological molecules can readily 
adhere. 
A specific use of the present invention is for generating skin for 
allograft use. Epidermal cells, for example, are seeded onto a substrate 
of the present invention. These cells are grown on the substrate using 
conventional skin growth conditions, including nutrients and growth 
factors. The improvement of the present invention, that is, the use of the 
hemidesmosome-promoting matrix on the substrate, improves such ex vivo 
growth of skin over prior art techniques that do not use that matrix. 
One particular use of the present invention is to increase epidermal cell 
adhesion to target surfaces. For instance, prostheses for dental 
implantation may be treated with the 804G matrix to stimulate periodontal 
cell attachment. Existing teeth may similarly be coated with the matrix as 
a treatment for gum (junctional epithelium) disease, such as gingivitis. 
Where a substrate is made of a natural or synthetic bioerodible material 
in the form of a sheet or fabric, such as woven or bonded collagen or 
polylactic acid, the matrix materials may be applied to the surface 
thereof or mixed in with the composition. Cells (such as epidermal cells) 
may then be grown on the matrix ex vivo to form transplantable or 
implantable materials; alternatively, the materials may be implanted and 
cells may be permitted to attach in vivo. 
Another preferred embodiment of the present invention is the growth of 
increased numbers of endocrine precursor cells. Particularly interesting 
are pancreatic islet cell precursors. For example, fetal pancreatic 
islet-like cell clusters may be grown in vitro in the presence of 804G 
matrix-type proteins for transplantation into diabetic patients. The 804G 
matrix will increase the yield of fetal ICCs for transplantation and will 
thus solve the established need for greater numbers of these cells. Since 
the matrix of the NBTII rat bladder carcinoma cell line is also able to 
promote increased epidermal cell growth, its use as a matrix for the 
growth of fetal pancreatic ICCs is advantageously envisioned, as is any 
such "804G" matrix protein, including all such proteins secreted by cell 
lines which are capable of promoting hemidesmosome formation in epidermal 
cells. In addition, the inclusion of growth factor in the ICC culture 
medium will further increase the yield of fetal pancreatic ICCs. 
The resulting cell clusters will differentiate into functional pancreatic 
endocrine cells after transplantation into mammals, preferably humans, and 
will reduce or eliminate the need for insulin injections. Interestingly, 
804G matrix has cross-species activity; even matrix derived from rat 
bladder carcinomas has the ability to promote growth and hemidesmosome 
formation in human tissue. 
The 804G matrix will also be of great use in studies concerning 
hemidesmosome morphogenesis, .alpha..sub.6 .beta..sub.4 integrin 
interactions with the extracellular matrix and for functional and 
structural analyses of new matrix components such as the laminin B2t-like 
rat molecule described below. Indeed, the 804G matrix may prove to be a 
tool that allows definition of hemidesmosome-mediated interactions between 
epithelial cells and the underlying connective tissues at the molecular 
level. 
The 804G matrix of the present invention comprises four 
concanavalin-binding glycosylated proteins, of approximately 135 kD, 140 
kD, 150 kD, and 400 kD, and a non-glycosylated, non-concanavalin binding 
protein of about 85 kD, all of which are recognized by polyclonal antibody 
raised against the 804G matrix. The methods of the present invention may 
be practiced with the complete, active matrix from 804G cells or a 
functionally equivalent "804G" matrix from other cells, and may also be 
practiced with any one of the individual protein components of the matrix 
which promotes hemidesmosome formation. Cell matrix and matrix proteins 
can be readily screened for the ability to facilitate hemidesmosome 
formation, using the techniques described herein. Only routine empirical 
testing is required. 
In addition to the active matrix and the active components thereof, the 
present invention also includes shaped articles coated with those 
materials. Preferably, those shaped articles are formed of materials other 
than glass, and include such forms as sheets, fabrics, prostheses, metal 
articles, bioerodible articles, and implantable articles. 
Furthermore, pharmaceutical preparations of the active matrix or its active 
components are contemplated. These preparations can be in any suitable 
form, and generally comprise the active ingredient in combination with any 
of the well known pharmaceutically acceptable carriers. The matrix 
material may be harvested (as by scraping, abrading, or treatment with low 
concentrations of SDS) from surfaces on which appropriate 
matrix-depositing cells have been grown. Alternatively, the matrix 
materials may be prepared synthetically or through recombinant DNA 
techniques, or through purification of deposited matrix material. Those 
carriers can include injectable carriers, topical carriers, transdermal 
carriers, and the like. The preparation may advantageously be in a form 
for topical administration, such as an ointment, gel, cream, spray, 
dispersion, suspension, or paste. The preparations may further 
advantageously include preservatives, antibacterials, antifungals, 
antioxidants, osmotic agents, and similar materials in composition and 
quantity as is conventional. For assistance in formulating the 
compositions of the present invention, one may refer to Remington's 
Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., Easton Pa. (1975), 
the relevant disclosure of which is hereby incorporated by reference. 
Finally, epithelial cells of various types may be grown on the substrates 
or with the compositions contemplated herein.

EXAMPLE 1 
Preparation of 804G Cell Matrix 
To begin biochemical characterization of the matrix secreted by the 804G 
cells, we followed the procedure of Gospodarowicz (1984). Briefly, rat 
bladder carcinoma 804G cells were maintained at 37.degree. C. in MEM with 
Earle's salts supplemented with 50 U/ml penicillin, 50 .mu.g/ml 
streptomycin and 10% FCS (GIBCO LABORATORIES, Grand Island, N.Y.). This 
medium contains approximately 1.9 mM Ca.sup.2+. 
The 804G cells were grown to confluency on either plastic Petri dishes or 
glass coverslips. The culture medium was then discarded and the cells 
washed in sterile PBS. The cells were removed from their matrix by 
treatment for 5 minutes in sterile 20 mM NH.sub.4 OH, followed by three 
rapid washes with sterile distilled water. 
The matrix was removed from the substrate by solubilization in 8M urea, 1% 
sodium dodecyl sulfate (SDS) in 10 mM Tris, pH 6.8. The 804G matrix 
polypeptide profile was analyzed by Sodium Dodecylsulfate Polyacrylamide 
Gel Electrophoresis (SDS-PAGE) using routine experimental methods known to 
those with skill in the art. 
A preparation having approximately 20 .mu.g of the solubilized 804G cell 
matrix was loaded onto an acrylamide gel and electrophoresed. As a 
control, an extract from the intact 804G cells, having approximately 
20.mu.g per lane, was also loaded onto the acrylamide gel. Following gel 
electrophoreses we noted that there were three major polypeptides in the 
matrix preparation ranging in molecular weight from 150-135 kD. A minor 
polypeptide of 85 kD was also present in the matrix preparation. After 
PAGE, the separated polypeptides were transferred to nitrocellulose by 
standard well known methods. Amido black stains of the dyed protein 
samples were transferred to the nitrocellulose indicating a successful 
completion of the Western Blotting procedure. 
EXAMPLE 2 
Concanavalin Binding to 804G Matrix 
A strip of the Western Blot nitrocellulose containing separated matrix 
proteins was incubated with Concanavalin A. Non-specific protein binding 
to the matrix molecules was blocked by first incubating the strip for 30 
minutes at room temperature with 2% (w/v) polyvinylpyrrolidone in PBS. 
Concanavalin A was added to the blocking buffer and the filter was then 
incubated with gentle shaking at room temperature. Horse radish peroxidase 
(HRP) was added to visualize Concanavalin A binding. 
Four matrix polypeptides of 135, 140, 150 and 400 kD were recognized by 
Concanavalin A. As is known in the art Concanavalin A binding indicates 
that these matrix components are glycosylated. To identify proteins on the 
Western Blot that were specific to the matrix, we raised polyclonal and 
monoclonal antibodies. 
EXAMPLE 3 
Production of Polyclonal Antibodies Against the 804G Matrix 
Antiserum was prepared by injecting urea/SDS solubilized 804G cell matrix, 
as described above, into a rabbit by standard methods. Briefly, 
solubilized 804G matrix was mixed with Freund's adjuvant and injected into 
a rabbit. Serum was collected at three weekly intervals following one 
booster injection as detailed by Harlow and Lane (1988). 
The isolated polyclonal antiserum (J18) had antibodies recognizing the four 
135-400 kD species that bound concanavalin A, as well as an 85 kD 
polypeptide. Therefore, there appears to be a non-glycosylated 85 kD 
species in the matrix along with four additional glycosylated 
polypeptides. 
Following our experiments with the polyclonal antibodies, we produced 
monoclonal antibodies specific for the 804G matrix by the following 
method. 
EXAMPLE 4 
Production of Monoclonal Antibodies Against the 804G Matrix 
A mouse monoclonal IgG (5C5) against the 804G cell matrix was prepared by 
injecting a solubilized 804G cell matrix sample into several mice. At two 
and three weeks after the initial injection the mice were boosted with 
further 804G matrix injections. Five days following the final boost their 
spleens were removed and isolated spleen cells were fused with the myeloma 
cell line Sp2 for the production of hybridomas using standard techniques 
(Galfre and Milstein, 1981). Hybridoma cells producing antibody against 
matrix elements were selected on the basis of their immunoblotting and 
immunofluorescence reactivities against matrix samples. Selected hybridoma 
cells were cloned twice by limited cell dilution as described in Harlow 
and Lane, 1988. 
Western Blots with one of the mouse monoclonal IgG antibodies (5C5) 
recognized only a 150 kD and a 135 kD polypeptide in the matrix 
preparation. Antibody 5C5 and the J18 serum were then used in 
immunoprecipitation studies to investigate potential protein-protein 
interactions in the matrix. 
EXAMPLE 5 
Immunoprecipitation Studies of the Matrix 
Immunoprecipitation of the 804G matrix was performed using conventional 
methodology. In brief, the 804G matrix was treated with RIPA buffer (0.1M 
Tris-HCl, pH 7.2, containing 0.15M NaCl, % Triton X100, 0.1% SDS, 1% Na 
deoxycholate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride), clarified 
by centrifugation, and incubated with either the rabbit serum J18 or 
monoclonal antibody 5C5. The resulting antibody-antigen complexes were 
immunoprecipitated with Staphylococcus aureus Protein A by methods known 
to those with skill in the art. 
The immunoprecipitated molecules were separated by SDS-PAGE and transferred 
to a Western blot by the methods described in more detail above. Lanes 1 
and 2 from the gel were immunoblotted with either goat anti-rabbit, or 
goat anti-mouse antibodies, conjugated to HRP for visualization. 
The polyclonal J18 antibodies recognized similar sets of polypeptides in 
both the matrix and 5C5 immunoprecipitate. Major protein bands were found 
in both samples at 150, 140 and 135 kD. This result indicated that the J18 
serum contained antibodies against all of the major proteins of the 
matrix. 
5C5 antibodies recognized primarily 150 kD and 135 kD polypeptides in both 
the 804G matrix and J18 immunoprecipitate. In contrast, the 5C5 antibodies 
apparently precipitated all of the molecular species in the matrix that 
are recognized by the J18 serum antibodies. As the 5C5 antibodies were 
able to precipitate most of the matrix proteins, yet only identified two 
proteins on a denaturing gel, we believe that the major proteins interact 
and are associated with one another in their normal state. 
To investigate the protein composition of the 804G matrix, we probed a 
Western Blot of solubilized matrix proteins with polyclonal serum against 
the 400 kD and 200 kD chains of Engelbreth-Holm-Swarm (EHS) laminin. 
Example 6 
Western Blot of Matrix Proteins Probed with Anti-laminin Antibodies 
Polyclonal antibodies against the 400 kD and 200 kD chains of EHS laminin 
were purchased from Collaborative Research Incorporated (Bedford, Mass.). 
A preparation of laminin (approximately 10.mu.g per lane) and a 
preparation of the solubilized 804G cell matrix (approximately 20.mu.g per 
lane) were denatured and run on a SDS gel, then subsequently transferred 
to nitrocellulose. We noted that the amido black stain used on the 
proteins run in lanes 1 and 2 was transferred to the nitrocellulose filter 
indicating that the blotting was successful. 
Incubation with the HRP conjugated anti-laminin polyclonal antibodies 
resulted in a strong reactivity in the laminin lanes, but there was very 
little detectable reactivity between the laminin polyclonal antibodies and 
the 804G cell matrix preparation. In a related experiment, the Western 
Blot was immunoblotted with labeled samples of either rabbit polyclonal 
anti-804G serum J18 or the monoclonal antibody 5C5, respectively. These 
antibodies failed to recognize any laminin polypeptides, although they did 
recognize polypeptides in the matrix preparation as expected from previous 
experiments described above. 
It appeared that there was little antibody cross-reactivity between laminin 
and the 804G matrix. For this reason, we attempted to isolate genes 
expressing polypeptides reactive with the J18 anti-804G antibodies. 
EXAMPLE 7 
Isolation of Clones Corresponding to Matrix Polypeptides 
A human keratinocyte lambda gt11 expression library was purchased from 
Clontech Labs., Inc., Palo Alto, Calif. and screened with the 804G matrix 
polyclonal serum J18 according to Huynh, et al., (1985). Antibodies 
absorbed by the fusion protein products of these three clones showed 
reactivity with both 140 kD and 85 kD molecular weight species in an 804G 
matrix preparation and a whole cell extract of SCC12 cells (Langhofer et 
al., (1993) J. Cell Sci., 105: 753-764). 
To further characterize positive clones, plaque lifts of 
nitrocellulose-bound fusion proteins were used to epitope select 
antibodies (Sambrook, et al., 1989). cDNA inserts were subcloned into M13 
vectors and sequenced by the Sanger dideoxychain termination method 
(Sanger, et al., 1977). Sequence analyses were made using the GCG sequence 
analysis software package (University of Wisconsin Biotechnology Center, 
Madison, Wis.). 
The nucleotide sequence of these clones revealed that they encode a region 
spanning amino acids 550-810 in domain I/II of a recently identified 
variant of the B2 chain of laminin that has been termed laminin B2t 
(Kallunki, et al., 1992). The B2t variant is not contained in EHS laminin, 
a and therefore represents a new subunit. This experiment illustrates the 
cross-reactivity of the matrix associated polypeptides with the laminin 
B2t variant. In addition, an 804G expression library (Invitrogen, San 
Diego, Calif.) was screened with both the J18 and 5C5 monoclonal antibody 
and clones were characterized by epitope selection. Clones containing the 
150, 140 and 135 kDa proteins of the matrix were isolated. Analysis of 
cDNAs encoding the 140 kDa protein revealed that their nucleotide 
sequences exhibited significant identity with regions of the human laminin 
B2t gene. The small stretches of sequence for the 150 and 135 kDa 
components have been obtained which do not exhibit similarity with any 
sequences in the database. 
Following this experiment we attempted to ascertain the location of 804G 
matrix polypeptides in intact tissue samples. 
EXAMPLE 8 
Immunofluorescence Localization of 804G Matrix Antigens in Intact Tissue 
804G cells were processed for immunofluorescence using the 5C5 monoclonal 
and J18 polyclonal antibodies. Initially, the 804G cells were fixed and 
extracted for 2-3 min in 20.degree. C. acetone prior to antibody 
incubation. Double labeling was carried out as detailed below. 
Cells on coverslips were first incubated in a mixture of primary antibodies 
for 1 hr at 37.degree. C. The coverslips were extensively washed in PBS 
and then overlaid with the appropriate mixture of rhodamine and 
fluorescein conjugated secondary antibodies by well known methods. 
Processed tissues were viewed on a Zeiss Photomicroscope III fitted with 
epifluorescence optics while cultured cells were viewed on a Zeiss laser 
scan microscope (LSM10) equipped with Argon and HeNe lasers for dual 
fluorescence confocal imaging (Carl Zeiss, Thornwood, N.Y.). As controls 
for the immunofluorescence analysis, cells were incubated in normal mouse, 
rat or rabbit IgG as well as secondary antibodies alone in order to assess 
staining due to non-specific antibody binding. Both J18 and 5C5 antibodies 
produced substrate staining in the 804G cell cultures. This staining is in 
a pattern and localizes with hemidesmosomal plaque staining in each 804G 
cell. 
Both the J18 serum, 5C5 antibodies and the antibodies selected from the J18 
serum using the laminin B2t fusion proteins were localized in 
cryo-sections of rat epithelial tissues by immunofluorescence microscopy. 
All of these antibody preparations show intense staining along the region 
of epithelial-connective tissue interaction. 
All of the above experiments have been related to the structure and 
function of the 804G matrix. Thus far, we have determined that the 804G 
matrix peptides immunologically related to the B2t laminin variant, and 
that antibodies directed against matrix proteins have been found at t2he 
epithelial-connective tissue juncture. 
One important aspect of the present invention is our discovery that the 
804G matrix, described in detail above, can unexpectedly provide a 
substrate capable of stimulating epithelial cell growth in vitro. We 
discovered that epithelial cells grown on the 804G matrix produced 
hemidesmosomes, as expected from normal cells exhibiting an in vivo 
morphology. To illustrate this aspect of the invention, we performed the 
following experiments. Initially, we grew the SCC12 human tumor cell line 
on the 804G matrix to determine its potential for normal growth in vitro. 
EXAMPLE 9 
Functional Analyses of Epithelial Cells Grown on the 804G Matrix 
Antibodies against a 230 kD plaque component of the hemidesmosome have been 
detailed before (Klatte et al., 1989). Monoclonal and polyclonal 
antibodies directed against the cytoplasmic domain (N-terminus) of a 180 
kD type II membrane element of the hemidesmosome have been described in 
Hopkinson et al., (1992) and Riddelle et al. (1992). An antibody against 
the .beta..sub.4 integrin subunit was purchased from Telios (San Diego, 
Calif.). 
SCC12 cells were maintained on the 804G cell matrix for 24 hrs to assess 
the impact of the matrix on hemidesmosome protein localization in a tumor 
cell line that, under normal circumstances, does not assemble bona fide 
hemidesmosomes in vitro. We chose to complete our studies in 24 hrs to 
minimize matrix degradation and/or modification by the added cells, a 
possibility that Carter, et al. (1990) have discussed. Each experiment was 
repeated at least four times involving the analysis of more than 500 
cells. As controls, the SCC12 were plated onto other matrices, such as 
glass and rat tail collagen. After 24 hrs the cells were processed for 
indirect immunofluorescence using antibodies directed against the 230 kD, 
180 kD and .alpha..sub.6 .beta..sub.4 integrin components of the 
hemidesmosome, double labelled with antibodies against the 804G cell 
matrix. 
Cells on coverslips were first incubated in a mixture of primary antibodies 
for one hour at 37.degree. C. The coverslips were extensively washed in 
PBS and then overlaid with the appropriate mixture of rhodamine and 
fluorescein conjugated secondary antibodies. Processed tissues were viewed 
on a Zeiss Photomicroscope III fitted with epifluorescence optics. As 
controls, cells were incubated with normal mouse, rat or rabbit IgG as 
well as secondary antibodies alone to assess staining due to non-specific 
background. 
In SCC12 cells maintained for 24 hrs on glass and rat tail collagen, the 
230 kD, 180 kD, .alpha..sub.6 .beta..sub.4 integrin subunits localized to 
the periphery of the cells along their substratum attached surfaces. The 
staining sometimes resembled a fuzzy band surrounding the cell periphery, 
or linear streaks near the cell edges (see also Hopkinson, et al., 1991). 
Anti-matrix antibodies in the J18 serum generated a diffuse staining along 
the region of cell-substrate interaction in cells maintained on rat tail 
collagen, with no obvious correlation to the staining generated by the 
hemidesmosomal antibody probes. The reactivity of J18 antibodies with the 
SCC12 cells by immunofluorescence is consistent with the positive 
immunoblotting reactivity using antibodies selected from the J18 serum by 
the human laminin B2t fusion proteins. Since antibodies in the J18 serum 
failed to recognize rat tail collagen alone, our results provide some 
indication concerning the matrix that the SCC12 cells themselves secrete. 
In SCC12 cells maintained on the 804G cell matrix, the 230 kD, 180 kD and 
.alpha..sub.6 .beta..sub.4 integrins show a dramatically different pattern 
of distribution compared with that observed in cells maintained on rat 
tail collagen or glass. The patterns that these hemidesmosomal antibodies 
generate are similar to that seen in 804G cells processed for 
immunofluorescence using the same antibodies, as described above. 
Furthermore, this staining, in most instances, appears coincident with 
those patterns generated by antibodies in the whole J18 serum. 
In addition, 5C5 antibodies or those J18 antibodies epitope selected from 
the laminin B2t fusion proteins were also localized in SCC12 cells 
maintained on the 804G matrix. The distribution of these antibodies 
compared with that of the 230 kD hemidesmosomal plaque component. It 
should be noted that the 230 kD antigen distribution in the SCC12 cells 
mirrors that of the staining generated by the 5C5 and epitope selected 
antibodies. 
Immunoblotting analyses were undertaken to examine whether there was a 
change in the amounts of both the 230 kD and 180 kD hemidesmosomal 
components in SCC12 cells maintained on 804G cell matrix for 24 hrs 
compared to SCC12 cells maintained for the same length of time on other 
matrices. There was no apparent difference in the quantity of both the 230 
kD and 180 kD polypeptides in SCC12 cells maintained on the various 
matrices as assessed by this procedure. 
In contrast to hemidesmosomal components, the .alpha..sub.5 .beta..sub.1 
integrin complex, a component of the microfilament associated-adhesion 
plaque (Burridge, et al. 1988), localize primarily at the peripheral 
cell-substratum associated surface of SCC12 cells regardless of whether it 
is maintained on rat tail collagen or the 804G cell matrix. 
Our studies of epithelial cell growth on the 804G matrix were not confined 
to SCC12 cells. Normal Human Keratinocytes (derived from human foreskins), 
HaCaT (immortalized cells), and SCC13 cells also exhibited almost 
identical responses when grown on the 804G matrix in comparison to the 
SCC12 cells discussed above. In each of these cell types, growth on the 
804G matrix led to a redistribution of integrins and mature hemidesmosome 
formation. 
In addition, experiments similar to those described above have been 
performed on the matrix produced by the NBTII cell line. The results from 
these experiments are virtually identical to those illustrated for the 
804G matrix. Cells grown on the NBTII matrix were stimulated to form 
mature hemidesmosomes and redistribute cell surface integrins. 
To further investigate the effect of growing epithelial cells on the 804G 
matrix, we examined SCC12 cells under the electron microscope. 
EXAMPLE 10 
Electron Microscopic Examination of the Impact of the 804G Cell Matrix on 
Hemidesmosome Assembly in SCC12 Cells 
SCC12 cells were fixed and processed for electron microscopy as described 
elsewhere (Riddelle, et al., 1991). Thin sections of cells were made 
perpendicular to their substrate, placed on 300 mesh electron microscope 
grids (Tousimis Corp., Rockville, Md.), stained and then viewed at 60 kV 
in a JEOL 100CX electron microscope. 
SCC12 cells maintained for 24 hrs on either rat tail collagen or the 804G 
matrix were examined by conventional electron microscopy. This procedure 
involved analyzing thin sections of the SCC12 cells cut perpendicularly to 
their substrate at intervals of 10 microns through a population of cells. 
By assessing sections at this distance apart we avoided the possibility of 
observing the same hemidesmosome more than once. 
In SCC12 cells maintained for 24 hrs on rat tail collagen, 
hemidesmosome-like structures were observed towards the cell periphery. In 
17 SCC12 cells incubated on rat tail collagen we observed 9 
hemidesmosome-like structures, none of which possessed a basal dense 
plate. This count was made over a distance of 306 microns (i.e. 1 
hemidesmosome-like structure/34 microns of the ventral surfaces of SCC12 
cells). The close apposition of three hemidesmosome-like structures was 
seen in one micrograph, however, this was highly unusual. In many basal 
profiles of SCC12 cells on rat tail collagen no hemidesmosomes were 
observed. 
In contrast, 103 hemidesmosome-like structures, of which 92 possessed basal 
dense plates, were observed in cross sectional profiles of SCC12 cells 
incubated on the 804G matrix. These observations were made over a distance 
of 504 microns (i.e., 1 hemidesmosome-like structure/4.9 microns of SCC12 
ventral surface). Unlike the "rudimentary" hemidesmosomes seen on cells 
incubated with rat tail collagen, these hemidesmosome-like structures were 
not confined to the periphery of the cell, but also were found underlying 
the nucleus. These SCC12 cells also appeared to possess tufts of 
intermediate filaments associated with their cytoplasmic face. 
In addition to electron microscopy of SCC12 cells, we looked for 
hemidesmosome assembly in Human Keratinocytes, HaCaT cells, and SCC13 
cells. As reported above in relation to immunofluorescence experiments, 
each of these other mammalian epithelial cells began redistributing 
integrins and forming mature hemidesmosomes. Our electron microscope 
studies revealed significant similarities in the effect of the 804G matrix 
on SCC12 cells, Human keratinocytes, HaCaT cells, and SCC13 cells. 
To demonstrate that the 804G cell matrix could retain its ability to induce 
changes in epithelial cells after solubilization, we coated glass 
coverslips with solubilized matrix elements. 
EXAMPLE 11 
Photolithography with 804G Matrix Elements 
To determine whether an isolated matrix sample could retain its ability to 
induce changes in hemidesmosomal and integrin localization 804G cells were 
grown and removed from their matrix as described above. A mild SDS buffer 
(RIPA) was used to solubilize and remove the matrix from its growth 
substrate. Following solubilization in RIPA buffer, the matrix elements 
were dialyzed extensively against phosphate buffered saline and then 
coated in a microscopic pattern onto glass coverslips using a 
photolithographic technique described by Hockberger et al. (Journal of 
Neuroscience (1988) 8 (11): 4098-4120). 
Briefly, a clean coverslip was first spin-coated with "photoresist". A mask 
was placed on top of the photoresist layer followed by illumination with 
UV light. At all of the points not covered by the mask the photoresist was 
UV cross-linked to the glass coverslip. Dialyzed 804G matrix elements were 
then added to the coverslip and bound along the entire surface of the 
coverslip. The photoresist and its bound matrix elements were removed from 
the non-UV linked areas of the coverslip by acetone treatment. A defined 
pattern of 804G matrix elements, configured as the inverse of the mask, 
was retained for further examination. 
In immunofluorescence studies using our matrix polyclonal antiserum, we 
demonstrated that SCC12 cells grown on these coverslips form 
hemidesmosomes in formations corresponding to the deposited pattern of 
804G elements. Remarkably, the location of .beta..sub.4 integrins on SCC12 
cells grown on these coverslips also followed the deposited matrix 
patterns. This indicated that the matrix maintained its functionality 
following mild SDS denaturation and deposit onto a solid substrate. By 
following this protocol, other solid substrates could be coated with the 
804G matrix to stimulate hemidesmosomal formation in epithelial cells. 
Thus, we have demonstrated that the 804G cell matrix is able to induce 
attachment and hemidesmosome assembly in many types of mammalian cells. 
EXAMPLE 12 
Expansion of fetal pancreas islet cells in vitro 
Human fetal pancreases are minced into 1 mm pieces in cold Hanks' balanced 
salt solution (HBSS) and digested with collagenase P by shaking vigorously 
for 15 min in a water bath at 37.degree. C. After several washes at 
4.degree. C. with HBSS, the digested tissue is washed with cold HBSS and 
placed into petri dishes in RPMI-1640 medium containing 10% pooled human 
serum and antibiotics for three days. Optionally, a growth factor is 
present during this procedure. 
Approximately 50 ICCs of uniform size (50-75 .mu.M diameter) and 
homogeneous translucent appearance are hand picked and plated on tissue 
culture dishes coated with either 804G matrix or bovine corneal matrix in 
RPMI-1640 containing 15% horse serum, 5% FCS, antibiotics and, optionally, 
a growth factor. ICCs attach overnight and monolayer formation is 
generally initiated by 24 hours. A significant increase is observed in the 
number of ICCs plated on 804G matrix compared to either no matrix or to 
bovine corneal matrix. 
To determine whether these fetal endocrine cells are capable of 
differentiating into insulin-producing cells in vivo, ICCs are 
transplanted as described below. 
EXAMPLE 13 
Transplantation of ICCs into nude mice 
ICCs from Example 12, cultured on 804G matrix, are transplanted under the 
kidney capsule of athymic nude mice (approximately 500 ICCs per mouse) and 
the grafts are analyzed after 3 months. An increased level of human 
C-peptide, released into the blood after processing of the insulin 
precursor molecule, is detected in the blood of grafted animals by 
radioimmunoassay after an intraperitoneal glucose challenge indicating 
that the grafted cells are able to produce insulin. In addition, 
immunocytochemistry of graft cells using an antibody to insulin indicates 
that the precursor cells differentiate into insulin-producing cells. 
Transplantation of ICCs into diabetic patients 
Human diabetes patients are administered a number of fetal ICCs to be 
optimized in clinical studies. Presumably, this number will be close to 
that used for adult-derived cells, approximately 2-8.times.10.sup.5, 
either by implantation under the kidney capsule or by direct injection 
into the liver. In addition, transplantation in other ectopic organ 
locations is also contemplated. C-peptide production and blood glucose 
levels are monitored over several months to determine whether transplanted 
endocrine precursor cells have differentiated into insulin-producing 
cells. The patients are still administered insulin during the monitoring 
period. 
It should be noted that the present invention is not limited to only those 
embodiments described in the Detailed Description. Any embodiment which 
retains the spirit of the present invention should be considered to be 
within its scope. However, the invention is only limited by the scope of 
the following claims.