U.S. patent application number 11/103924 was filed with the patent office on 2005-11-24 for process for obtaining mammalian insulin secreting cells in vitro and their uses.
This patent application is currently assigned to CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE LILLE. Invention is credited to Kerr-Conte, Julie, Pattou, Francois.
Application Number | 20050260750 11/103924 |
Document ID | / |
Family ID | 8854904 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050260750 |
Kind Code |
A1 |
Kerr-Conte, Julie ; et
al. |
November 24, 2005 |
Process for obtaining mammalian insulin secreting cells in vitro
and their uses
Abstract
The present invention relates to a process for obtaining
mammalian insulin secreting cells in vitro, characterized in that
it contains the following steps: a) preparation of the mammalian
pancreatic tissues by removal of a pancreas, b) dissociation of the
pancreatic tissues obtained in step (a) into isolated pancreatic
cells, c) possibly the elimination of the endocrine cells from the
pancreatic cells isolated in step (b), d) induction of
dedifferentiation of the cells isolated in step (b) into ductal
precursor cells, e) induction of redifferentiation of the ductal
precursor cells obtained in step (d) into insulin secreting cells.
It also concerns the use of the insulin secreting cells thus
obtained for the preparation of a pharmaceutical composition which
can be used for the treatment of pancreatic pathologies and
particularly diabetes.
Inventors: |
Kerr-Conte, Julie;
(Lambersart, FR) ; Pattou, Francois; (Lambersart,
FR) |
Correspondence
Address: |
HOFFMANN & BARON, LLP
6900 JERICHO TURNPIKE
SYOSSET
NY
11791
US
|
Assignee: |
CENTRE HOSPITALIER REGIONAL
UNIVERSITAIRE DE LILLE
|
Family ID: |
8854904 |
Appl. No.: |
11/103924 |
Filed: |
April 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11103924 |
Apr 12, 2005 |
|
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09960632 |
Sep 21, 2001 |
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6900051 |
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Current U.S.
Class: |
435/366 ;
435/455 |
Current CPC
Class: |
C12N 2506/22 20130101;
C12N 5/0676 20130101; C12N 2509/00 20130101; C12N 2500/25 20130101;
A61K 35/12 20130101; A61P 5/48 20180101 |
Class at
Publication: |
435/366 ;
435/455 |
International
Class: |
C12N 005/08; C12N
015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2000 |
FR |
00/12547 |
Claims
1-18. (canceled)
19. A cell preparation comprising mammalian insulin secreting cells
obtained by a process comprising: a) preparing mammalian pancreatic
tissues from a previously removed pancreas; b) dissociating the
pancreatic tissues into isolated pancreatic cells; c) eliminating
endocrine cells from the isolated pancreatic cells to obtain
exocrine cells; d) inducing dedifferentiation of the exocrine cells
into ductal precursor cells; and e) inducing redifferentiation of
the ductal precursor cells into insulin secreting cells, wherein
the elimination of endocrine cells in part (c) is carried out by
means of density gradient centrifugation, and wherein exocrine
cells devoid of endocrine cells are recovered in a pellet as a
result thereof.
20. A cell preparation according to claim 19, wherein the
dissociation of the pancreatic tissues is carried out by enzymatic
digestion.
21. A cell preparation according to claim 19, wherein the
elimination of the endocrine cells is carried out by withdrawal of
a fraction of the endocrine cells recovered in a density range
between 1.027 g/L to 1.104 g/L.
22. A cell preparation according to claim 19, wherein the
elimination of the endocrine cells is carried out by withdrawal of
a fraction of the endocrine cells recovered in a density range
between 1.045 g/L to 1.097 g/L.
23. A cell preparation according to claim 19, wherein the
dedifferentiation further comprises: i) culturing the isolated
pancreas cells obtained after the elimination of endocrine cells
for a duration of between 4 to 9 days, with a cell concentration
between 1.times.10.sup.6 and 10.times.10.sup.6 cells/mL, in a
culture medium containing glucose at a concentration between 1 and
10 g/l, and a mixture of insulin, transferrin, and selenium at a
concentration between 0.2 and 3%; and ii) recovering ductal
precursor cells.
24. A cell preparation according to claim 23, wherein the cells are
cultured with a cell concentration between 2.times.10.sup.6 and
6.times.10.sup.6 cells/ml.
25. A cell preparation according to claim 23, wherein the glucose
is at a concentration between 2 and 5 g/l.
26. A cell preparation according to claim 23, wherein the mixture
of insulin, transferrin, and selenium is used at a concentration
between 1.0 and 2.5%.
27. A cell preparation according to claim 23, wherein the cells are
cultured for a duration between 5 to 7 days.
28. A cell preparation according to claim 23, wherein the culture
medium further contains serum, wherein the serum is fetal calf
serum, bovine serum or human serum, and wherein the serum
concentration is greater than 8%.
29. A cell preparation according to claim 28, wherein the serum is
at a concentration between 10 and 15% final volume.
30. A cell preparation according to claim 23, wherein the culture
medium further contains factors preventing the growth of
fibroblasts, wherein the factors are present at a concentration
between 20 and 100 .mu.g/ml.
31. A cell preparation according to claim 30, wherein the factors
preventing the growth of fibroblasts are at a concentration between
30 and 60 .mu.g/ml.
32. A cell preparation according to claim 23, wherein the culture
medium further contains antibiotics and/or antifungal agents.
33. A cell preparation according to claim 19, wherein the induction
of redifferentiation further comprises: i) detaching the ductal
precursor cells to obtain separated ductal precursor cells; ii)
culturing the separated ductal precursor cells for a duration
between 12 and 36 hours, at cell concentration between
3.5.times.10.sup.5 cells/25 cm.sup.2 and 4.times.10.sup.6 cells/25
cm.sup.2, in a culture medium containing glucose at concentrations
between 1 and 10 g/L; iii) withdrawing said culture medium to
obtain non-adherent cells; iv) culturing the non-adherent cells for
a duration between 4 and 12 days, in a culture medium containing
glucose at a concentration between 1 and 10 g/L to obtain insulin
secreting endocrine cells; and v) recovering the insulin secreting
cells.
34. A cell preparation according to claim 33, wherein the separated
ductal precursor cells are cultured at a concentration between
7.times.10.sup.5 cells/25 cm.sup.2 to 3.times.10.sup.6 cells/25
cm.sup.2.
35. A cell preparation according to claim 33, wherein the culture
medium contains glucose at a concentration between 2 and 5 g/l.
36. A cell preparation according to claim 33, wherein the culture
medium contains serum, wherein the serum is fetal calf serum,
bovine serum or human serum at a concentration greater than 2.5% of
final volume.
37. A cell preparation according to claim 40, wherein the serum is
at a concentration between 5 and 15% final volume.
38. A cell preparation according to claim 33, wherein the culture
medium contains a mixture of insulin, transferrin, and selenium, at
a concentration between 0.2 and 5%.
39. A cell preparation according to claim 42, wherein the mixture
of insulin, transferrin, and selenium is at a concentration between
0.5 and 2%.
40. A cell preparation according to claim 33, wherein the culture
medium contains antibiotics and antifungal agents.
41. A cell preparation according to claim 33, wherein the ductal
precursor cells are cultured in the presence of a matrix.
42. A cell preparation according to claim 33, wherein the culture
medium contains growth factors.
43. A cell preparation according to claim 33, wherein the ductal
precursor cells are cultured for a duration between 5 and 10
days.
44. A cell preparation according to claim 33, wherein the
separation of the ductal precursor cells is done with trypsin at a
concentration between 0.01 and 0.1% and EDTA at a concentration
between 0.1 and 1 mM.
45. A cell preparation according to claim 33, wherein the trypsin
is at a concentration between 0.015 and 0.03% and the EDTA is at a
concentration between 0.25 and 0.75 mM.
46. A cell preparation according to claim 41, wherein the matrix is
collagen type IV, 804G, collagen type I, or Matrigel.
47. A cell preparation according to claim 19, wherein the
pancreatic tissues are obtained from a previous removal of a
fragment of a pancreas of a brain dead adult human.
48. A cell preparation according to claim 19, wherein the
pancreatic tissues are obtained from a previous removal of a
fragment of a pancreas of a living patient suffering from a
pancreatic pathology.
49. A cell preparation according to claim 19, wherein the
pancreatic tissues are obtained from a previous removal of a
fragment of a pancreas of a living patient suffering from
diabetes.
50. A method of pancreatic pathologies comprising administering to
a patient a cell preparation according to claim 19.
51. A method of treating diabetes comprising administering to a
patient a cell preparation according to claim 19.
Description
[0001] The present invention concerns a process for obtaining
insulin secreting cells in vitro from pancreatic tissue. It
particularly concerns obtaining insulin secreting cells from
pancreata of patients suffering from pancreatic pathologies and
particularly diabetes. It also concerns the use of these cells for
preparations intended for therapeutic treatment of diabetes.
[0002] Cellular therapy is currently offering important
perspectives in the treatment of diabetes (Shapiro A. M. et al.
Islet transplantation in seven patients with type 1 diabetes
mellitus using a glucocorticoid-free immunosuppressive regimen. N
Engl J Med 343:230-238,2000). The concept of treatment of diabetes
by ectopic transplantation of insulin secreting cells has already
been validated. In the course of a total pancreatectomy, isolation
and intraportal transplantation of the endocrine islets of the
pancreas make it possible to maintain endogenous, almost
physiological, secretion of insulin and to maintain glucide
homeostasis for more than ten years (Pyzdrowski, K. L., et al.
Preserved insulin secretion and insulin dependence in recipients of
islet autografts. New England J. Med. 1992, 327:220-226).
[0003] However, in spite of numerous technical advances, the
insufficient and often random yield from isolation of the islets
remains a major hindrance to the development of cellular therapy
for diabetes. Currently, the assembly of islets coming from several
donors is still most often necessary in order to transplant a
sufficient mass of endocrine tissue in a diabetic patient, and
given the limited number of available donors, the current use of
primary human islets of Langerhans nevertheless prohibits any hope
of large scale development of cellular therapy for this
disease.
[0004] Thus, new alternative processes for obtaining insulin
secreting cells which are less limiting and which can be used in
humans have been envisaged, such as genetic manipulation of somatic
cells in order to induce synthesis of insulin, immortalizing of
cell lines or use of animal cells. However, there are still
problems because on one hand the coordinated transcriptional
regulation of insulin secretion is complex, and on the other hand
clinical use of transformed cells of animal origin is very
controversial. Consequently, the design of alternative methods for
obtaining insulin secreting cells remains a major stake in the
context of cellular therapy for diabetes.
[0005] Another approach takes into account the recent experimental
studies which have revealed in the adult pancreas the presence of
pancreatic stem cells which are capable of proliferation and
differentiation.
[0006] It is known that the pancreas forms from the endoderm during
embryonic development (Le Douarin, N. M. On the origin of
pancreatic endocrine cells. Cell, 1988, 53:169-171) and that the
three existing pancreatic cell types are derived from the
proliferation of the pancreatic epithelium and its secondary
differentiation into ductal, endocrine or acinar tissue.
[0007] Although the mechanisms of differentiation between the
different pancreatic cell types have not been completely
elucidated, certain specific phenotypic markers of each of them are
known. Thus in rodents as in humans the phenotype of the pancreatic
stem cells is of the ductal type as demonstrated by the expression
by them of cytokeratins 20 and 19 (Bouwens L. et al. Cytokeratins
as markers of ductal cell differentiation and islet neogenesis in
the neonatal rat pancreas. Diabetes. 1994, 43:1279-1283; Bouwens L.
et al. Proliferation and differentiation in the human fetal
endocrine pancreas. Diabetologia, 1997, 40:398-404).
[0008] Also known is the phenomenon called nesidioblastosis, whose
mechanism remains unknown, which reproduces in the adult period the
mode of embryonic formation of the endocrine cells of the pancreas.
Nesidioblastosis is not a species specific phenomenon; moreover, it
is also observed in humans in certain pathological circumstances.
Nesidioblastosis is also frequent in the adjacent parenchyma of
endocrine tumors of the pancreas, whether they are sporadic or
caused by mutation of a tumor suppressing gene, in cases of
endocrine neoplasia. In certain exceptional cases, diffuse
neosidioblastosis [sic; nesidioblastosis] of the whole pancreas can
even be observed.
[0009] These observations therefore suggest the persistence in the
mature human pancreas of quiescent stem cells with a ductal
phenotype which are capable of endocrine differentiation under
certain conditions.
[0010] Processes for in vitro culturing of adult pancreas cells
which make use of these observations and reproduce this phenomenon
of nesidioblastosis in vitro have already been described. For
example, the European patent applications EP 758376 and EP 871455
describe a process for obtaining insulin secreting cells from
pancreatic preparations of adult tissue enriched with stem
cells.
[0011] However, the process for obtaining insulin secreting cells
described in these documents has several problems. On one hand, it
includes a first culture step intended for stem cell enrichment of
the pancreatic cell population isolated during the first step of
the process. This step, spread out over several weeks, provides for
the culturing of the pancreatic cells in a serum-poor medium so as
to eliminate 99% of the pancreatic cells, the majority of which are
differentiated exocrine cells. The aim is to maintain in culture
only a cell population which is enriched with islet precursor stem
cells. After having carried out this selection, the small
population of isolated stem cells undergoes an expansion step for
several weeks. The third step of the process involves the
differentiation of the stem cells into insulin secreting cells.
Another major problem inherent to this process lies in the small
number of stem cells which can be obtained after having eliminated
almost all (99%) of the differentiated pancreatic cells. This
process for obtaining endocrine islets in vitro in adult mammals
reproduces the route of pancreatic embryogenesis as it occurs in
vivo.
[0012] In the context of the present invention, the inventors
succeeded for the first time in vitro in inducing a
transdifferentiation of differentiated pancreatic exocrine cells to
another differentiated phenotype. This transdifferentiation is not
based on the physiological route followed by pancreatic
embryogenesis in vivo and thus opens up an alternative way to
obtain differentiated pancreatic cells.
[0013] In a completely surprising manner, the inventors succeeded
in inducing in vitro a dedifferentiation of the exocrine cells of
the adult pancreas, constituting more than 95% of the pancreatic
parenchyma, under certain culture conditions, in order to obtain
dedifferentiated cells, hereafter called ductal precursor
cells.
[0014] These ductal precursor cells are in turn grown in a suitable
medium in which redifferentiation is induced, which transforms them
into insulin secreting endocrine cells.
[0015] In the context of the present invention, the following
meanings are understood:
[0016] Differentiation: for a cell, the act of acquiring a
specialized function. This is the process which leads to the
expression of characteristic phenotypic properties of a
functionally mature cell in vivo.
[0017] Redifferentiation: for a cell, the act of reacquiring a
specialized function which it previously lost following
dedifferentiation.
[0018] Dedifferentiation or retrodifferentiation: for a specialized
cell, the act of regressing to a less specialized embryonic form.
Dedifferentiation entails the loss, temporary or definitive, of the
differentiated genotypic and/or phenotypic characteristics which
said specialized cell was able to acquire during its development.
Dedifferentiation is either an adaptive process implying that the
differentiated phenotype can be attained by administering the
suitable inducers, or a selective process, in that case implying
that the precursor cells were chosen because of their high
proliferation potential.
[0019] Transdifferentiation: This is a biological process of
reprogramming of the genetic expression from one cell phenotype to
another. Transdifferentiation includes a first step of
dedifferentiation and a second step of redifferentiation.
[0020] Stem cell: Cell which has unlimited self-replication
capacities, which is capable of producing at least a highly
differentiated lineage of
unipotential/bipotential/multipotential/pluripotential/totipotential
cells.
[0021] Dedifferentiated cell: any cell which does not express the
phenotype of the original cell or that of the subsequent
differentiated cells.
[0022] Epithelial precursor cell: Cell which is capable of
differentiating, but only in order to become a cell belonging to
its own epithelial tissue type and not to another. The ductal
precursor cells belong to this category.
[0023] Beta-cell: cell of the islets of Langerhans of the pancreas
which secretes the hormone insulin in response to glucose and other
secretagogues.
[0024] The inventors carried out extensive research studies in
order to prove from the molecular standpoint as well as from the
protein standpoint that, under suitable culture conditions, the
pancreas exocrine cells dedifferentiate into precursor cells
bearing a characteristic phenotype of the ductal epithelial cells.
Later redifferentiation of these exocrine precursor cells under
suitable culture conditions makes possible the preparation of
insulin secreting cells in large quantity, with said ductal
epithelial cells reexpressing the factor IPF-1, a specific marker
of the insulin secreting beta-cells.
[0025] Manifestations in vivo of this cellular dedifferentiation
had already been observed in vitro, but the inventors reproduced it
in vitro for the first time and used it for the purpose of
obtaining an abundant source of endocrine precursor pancreatic
cells. The process which they used opens the way to great clinical
innovations in the domain of therapeutic treatment of pancreatic
pathologies, particularly diabetes. The new process for obtaining
insulin secreting cells by transdifferentiation used in the context
of the invention provides an abundant source of precursors of
beta-cells. This makes it possible to easily envisage on one hand
an allogenic therapy for the pancreatic pathologies, particularly
of diabetes, and on the other hand an autologous cellular therapy.
In effect, a partial pancreatectomy by the process of the invention
will allow one to produce from a small fragment of the pancreas of
the patient autologous insulin secreting cells in sufficient
quantity to restore the pancreatic functions.
[0026] The present invention uses a process for obtaining insulin
secreting cells in vitro starting from a preparation of pancreatic
cells of an adult pancreas, which is possibly devoid of endocrine
cells.
[0027] The process for obtaining insulin secreting cells of the
invention is remarkable because it is carried out precisely using
the exocrine cell population which counts for more than 95% of the
cells present in the pancreatic tissue, and not using isolated stem
cells only.
[0028] It is thus possible by this route to obtain up to 1.5
billion ductal precursor cells from a single human pancreas, or
100,000 times more than from the pancreatic ducts themselves.
[0029] Thus, the invention relates to a process for obtaining
mammalian insulin secreting cells in vitro, characterized in that
it contains the following steps:
[0030] a) preparation of the mammalian pancreatic tissues by
removal of a pancreas,
[0031] b) dissociation of the pancreatic tissues obtained in step
(a) into isolated pancreatic cells,
[0032] c) possibly, elimination of endocrine cells from the
isolated pancreatic cells obtained in step (b),
[0033] d) induction of dedifferentiation of the cells isolated in
step (c) into ductal precursor cells,
[0034] e) induction of redifferentiation of the ductal precursor
cells obtained in step (d) into insulin secreting cells.
[0035] According to a preferred embodiment of the process of the
invention, the dissociation of the pancreatic tissues in step (b)
is carried out by means of enzymatic digestion.
[0036] Advantageously, the pancreatic cells isolated in step (b)
are devoid of endocrine cells in step (c) before step (d) of
induction of differentiation [sic; dedifferentiation].
[0037] Advantageously, the elimination of the endocrine cells from
the pancreatic cells of step (c) is carried out by means of density
gradient centrifugation.
[0038] Preferably, the elimination of the endocrine cells from the
pancreatic cells in step (c) is carried out by withdrawal of the
fraction of endocrine cells recovered in the density range between
1.027 g/L to 1.104 g/L, preferably between 1.045 g/L to 1.097
g/L.
[0039] Quite preferably, the pancreatic cells devoid of endocrine
cells obtained in step (c) are exocrine cells recovered in the
density gradient residue.
[0040] According to another embodiment of the process of the
invention, the elimination of the endocrine cells in step (c) is
carried out by means of a cell separator.
[0041] Advantageously, the dedifferentiation of step (d) includes
the following substeps:
[0042] i) culturing of the cells obtained in step (c) with a cell
concentration between 1.times.10.sup.6 and 10.times.10.sup.6
cells/mL, preferably between 2.times.10.sup.6 and 6.times.10.sup.6
cells/mL, in a culture medium containing:
[0043] glucose at a concentration between 1 and 10 g/L, preferably
between 2 and 5 g/L.
[0044] possibly serum, chosen from fetal calf serum, bovine serum
or human serum, at concentrations greater than 8%, preferably
between 10 and 15% final volume.
[0045] a mixture of insulin, transferrin, selenium used at a
concentration between 0.2 and 3%, preferably between 1.0 and
2.5%,
[0046] possibly factors stopping the growth of fibroblasts at a
concentration between 20 and 100 .mu.g/mL, preferably between 30
and 60 .mu.g/mL,
[0047] possibly antibiotics, antifungal agents,
[0048] for a duration between 4 to 9 days, preferably 5 to 7
days,
[0049] ii) recovery of the ductal precursor cells obtained in step
(i).
[0050] Advantageously, the induction of the redifferentiation of
step (e) includes the following substeps:
[0051] i) possibly the separation of the ductal precursor cells
obtained in step (d)
[0052] ii) culturing of the ductal precursor cells obtained in step
(i) at cell concentrations between 3.5.times.10.sup.5 cells
25/cm.sup.2 and 4.times.10.sup.6 cells/25 cm.sup.2, preferably
7.times.10.sup.5 cells/25 cm.sup.2 to 3.times.10.sup.6 cells/25
cm.sup.2, in a culture medium containing:
[0053] glucose at concentrations between 1 and 10 g/L, preferably
between 2 and 5 g/L.
[0054] possibly serum, chosen from fetal calf serum, bovine serum
or human serum, at concentrations greater than 2.5%, preferably
between 5 and 15% final volume.
[0055] possibly a mixture of insulin, transferrin, selenium at a
concentration between 0.2 and 5%, preferably between 0.5 and
2%,
[0056] possibly antibiotics and antifungal agents,
[0057] possibly in the presence of a matrix,
[0058] for a duration between 12 and 36 h,
[0059] iii) withdrawal of said culture medium and of the
non-adherent cells possibly present,
[0060] iv) culturing of the cells obtained in step (iii) in a
culture medium such as that used in step (i), possibly containing
growth factors,
[0061] for a duration between 4 and 12 days, preferably between 5
and 10 days,
[0062] in order to obtain insulin secreting endocrine cells,
and
[0063] v) recovery of the insulin secreting cells obtained in step
(iv).
[0064] According to a preferred implementation of the process of
the invention, the separation of the cells in substep (i) of step
(e) is done with trypsin/EDTA at concentrations between 0.01 and
0.1% of trypsin, preferably 0.015-0.03, and EDTA between 0.1 and 1
mM, preferably 0.25-0.75 mM.
[0065] According to a preferred embodiment of the process of the
invention, the matrix used for the culturing of the cells in
substep (ii) of step (e) is chosen from collagen type IV, 804G,
collagen type I, Matrigel or its equivalents which are known to the
expert in the field.
[0066] Advantageously, the pancreatic tissues dissected in step (a)
are obtained from the pancreas of a brain dead adult human.
[0067] Preferably, the pancreatic tissues dissected in step (a) are
obtained from a fragment of a pancreas of a living patient
suffering from a pancreatic pathology and quite preferably from a
fragment of a pancreas of a living patient suffering from
diabetes.
[0068] The invention also concerns insulin secreting cells prepared
by the process of the invention.
[0069] The invention also concerns the use of the insulin secreting
cells prepared by the process of the invention for the
manufacturing of a pharmaceutical composition intended for the
treatment of human pancreatic pathologies, and more particularly
intended for the treatment of diabetes.
[0070] The subject of the invention is also a method of
administration of the insulin secreting cells prepared according to
the process of the invention by means of a percutaneous intraportal
catheter.
[0071] The subject of the invention is also a bioartificial
pancreas made up of insulin secreting cells prepared according to
the process of the invention grown after microencapsulation
according to processes which are known in themselves to the expert
in the field.
[0072] The inventors carried out extensive studies aiming to prove
that a population of non-beta 1 exocrine pancreatic cells can be
effectively obtained in vitro from exocrine pancreatic tissue.
[0073] In the first place, the proof of the cellular
dedifferentiation in vitro of the pancreatic exocrine cells in a
suitable culture medium was supplied by verification of the almost
complete loss of amylase expression and an increase of the
expression of ductal markers (cytokeratin 19, cytokeratin 7,
carbohydrate antigen 19-9).
[0074] For the first time, the studies carried out by the inventors
demonstrated a reexpression of the insulin promotor factor-1
(IPF-1) or of its equivalents: the pancreatic duodenal homeotic
sequence (PDX-1), the islet duodenal homeotic sequence 1 (IDX-1),
the somatostatin transactivation factor 1 (STF-1) by the pancreatic
cell cultures in the protein and mRNA.
[0075] The factor IPF-1 is a homeodomain protein essentially
present in the differentiated beta-cells of the adult pancreas
(Ohlsson H. et al. IPF-1, a homeodomain-containing transactivator
of the insulin gene.; The EMBO journal, 12:4251-4259, 1993),
functioning as principal regulator of phenotype b.
[0076] The expression of IPF-1/PDX-1 is preserved in human
beta-cells which have lost their capacity to express insulin after
a 30,000 fold expansion.
[0077] During the pancreatic ontology, the expression of the factor
IPF-1 in the primitive ducts appears to be essential for the
formation of endocrine and exocrine cells in mice (Johnson J. et
al. Insulin-promoter-factor 1 is required for pancreas development
in mice. Nature 371:606609, 1994); and in humans (Stoffers D. A. et
al. Pancreatic agenesis attributable to a single nucleotide,
deletion in the human IPF1 gene coding sequence. Nat. Genet.
15:106-110, 1997), its absence leads to pancreatic agenesis. The
factor IPF-1 is also reexpressed in a significant manner in the
ductal cells in the course of proliferation during pancreatic
regeneration in adult rodents. The authors recently observed the
expression of IPF-1 in adult human pancreatic ducts of patients
with nesidioblastosis. Consequently, the factor IPF-1 proves to be
a marker of the ductal cells which recover their pluripotentiality
in order to redifferentiate later into any pancreatic cell type
(Sharma A. et al. The homeodomain protein IDX-1 increases after an
early burst of proliferation during pancreatic regeneration.
Diabetes 48:507-513, 1999).
[0078] Given that the expression of the factor IPF-1 in adult
ductal cells seems to be a precondition for their redifferentiation
into beta-cells in the animal models, the expression of the factor
IPF-1 in the human ductal cells in culture provides evidence of
their potential redifferentiation and proves that these cells are
endocrine precursor candidates.
[0079] The later redifferentiation of these endocrine precursor
cells under suitable culture conditions will make possible the
preparation of the insulin secreting cells in large quantity.
[0080] Other advantages and characteristics of the invention will
appear upon reading of the examples and figures which follow,
reporting the research work which made it possible to verify that,
under the culture conditions used, dedifferentiation in vitro of
the exocrine pancreatic cells into ductal precursor cells is
induced. These ductal precursor cells are then redifferentiated
into insulin secreting endocrine cells.
[0081] FIG. 1 represents a diagram of the pancreatic embryogenesis
with identification of the origin of the different pancreatic
tissues and the markers which identify the cellular phenotypic
changes used in the context of the invention.
[0082] FIG. 2 is a diagram of the process for preparation of
insulin secreting cells used in the context of the invention.
[0083] FIG. 3 illustrates the phenotypic transition of the cultures
which is determined by a slot blot technique.
[0084] FIG. 4 shows the expression of the mRNA determined in the
exocrine culture preparations.
[0085] FIG. 5 illustrates the immunohistochemical analysis by
Western Blot, and the PCR-RT results of the exocrine preparations
with specific immunolabeling of the ductal antigens CK19, CK7.
[0086] Human Ductal Precursor Cells.
[0087] Human cells with a phenotype of ductal precursor cells are
obtained in culture from pancreatic preparations. Human pancreata
were removed from brain dead adult human donors. The pancreata were
possibly weakened with 80 mL of a cold solution of collagenase (0.5
mg/mL, Liberase.RTM. or Collagenase of type P, Roche Diagnostics,
Meylan, France), diluted in Hanks medium.
[0088] The pancreata are dissociated according to the automated
method of Ricordi (Ricordi, C. Automated method for isolation of
human pancreatic islets. Diabetes 37:413-420, 1988), with some
modifications (Kerr-Conte, J. et al. Simple dithizone-stained
multilayer test for selection of density gradient before human
islet mass purification. Transplant Proc. 26:4013-4015, 1994).
After the selection of the densities leading to an optimal
separation, the islets are isolated by purification using
discontinuous gradient of EuroFicoll.RTM. or Histopaque.RTM. with a
COBE 2991 cell separator.
[0089] The exocrine fraction is recovered in the pellet, washed
three times in Hanks solution and cultured in a proportion of
2.times.10.sup.6 to 6.times.10.sup.6 cells per 75 cm.sup.2 culture
dish in minimum essential Dulbecco culture medium (DMEM, with 3 g/L
of glucose), containing 10% fetal calf serum (FCS, Laboratoires
Eurobio, Les Ulis, France), 1% insulin, transferrin, selenium
(ITS), and 50 .mu.g/mL of Geneticine.RTM. (G418) in order to limit
the growth of fibroblasts.
[0090] After 12 h of attachment and every two/three days the
culture medium is changed; the monolayer cultures are maintained
for 2 weeks.
[0091] Cellular Proliferation
[0092] In order to verify cell proliferation estimated in the
exocrine preparations, one .mu.Ci/mL of tritiated thymidine is
added to the culture medium on days 1, 1.5, 2, 3, 4, 6 and 10. The
cells thus treated were washed, precipitated with 5%
trichloroacetic acid and solubilized in sodium hydroxide (0.5M) and
counted in a beta-counter after addition of scintillation liquid to
them. The number of counts per minute (cpm) was expressed with
respect to the DNA, measured with the PicoGreen.RTM. reagent.
[0093] RNA
[0094] The expression of IPF-1, insulin, and beta-actin were
assessed by an RT-PCR reaction using the exocrine cell
preparations.
[0095] The total RNA was isolated with RNAzol.RTM. B and quantified
by spectrophotometry (260 nm). The cDNA was synthesized from 2
.mu.g total RNA with oligo(DT)12-18 primers and a reverse
transcriptase (M-MLV). The PCR reaction was carried out on an
aliquot of one pL of the product of the RT reaction in the presence
of 200 mM dNTP, 1.5 mM MgCl.sub.2, the primers: 25 pM (IPF-1) or 5
pM (beta-actin) and 5 U AmpliTaq DNA polymerase. The sets of
primers include primers for the amplification of IPF-1:
[0096] 5'CCATGGATGAAGTCTACC-3', 5'-GTCCTCCTCCTTTTTCCAC-3'
[0097] primers for the insulin:
[0098] 5'-TGTGAACCAACACCTGTG-3', 5'-CCTCTAGTTGCAGTAGT-3'
[0099] and primers for the beta-actin:
[0100] 5'- ATCATGTTTGAGACCTCCAA-3', 5'-CATCTCTTGCTCGAAGTCCA-3'
[0101] The PCR reaction is carried out in a programmable PCR
apparatus with 35 cycles for IPF-1 (94.degree. C.: one
minute/52.degree. C.: one minute/72.degree. C.: one minute) and
with 27 cycles for the insulin (94.degree. C.: 30 sec/53.degree.
C.: 1 min/72.degree. C.: 30 sec). All the PCR products are
subjected to electrophoresis using 2% agarose gel. After
digitalization with a digital camera with integration (CDD) (COHU
4912), the intensities of the bands, expressed in arbitrary units,
are quantified by means of the GelAnalysts.RTM. software, version
3.01 FR (GreyStone-Iconix). The expression of each specific product
is standardized according to the levels of the internal control
consisting of the expression of beta-actin.
[0102] Protein
[0103] A kinetics of expression of amylase, cytokeratin 19, and
IPF-1 was done on the protein extracts of the cultures.
[0104] For the execution of the slot and Western Blot techniques,
the exocrine cells which were grown are trypsinized (0.025%
trypsin-5 mM EDTA) in a buffer of Hank's free of Ca++/Mg++ ions
(Sigma-Aldrich) and washed in the culture medium. The cells are
homogenized in ice in a phosphate-buffered saline (PBS) buffer
supplemented with 0.25M glucose and lysed by ultrasound treatment.
The protein concentrations were measured with the bicinchinic [sic;
bicinchoninic] acid reagent. For the slot blots, the total proteins
(25 .mu.g) were deposited on nitrocellulose membranes using the
Slot Blot Filtration manifolds filtration apparatus (Amersham Life
Science). The membranes were saturated with 5% milk in PBS,
incubated with antibodies directed against amylase, chromogranin A,
factor IPF-1, cytokeratin CK19, in a diluted (1:10) saturation
buffer for two hours. The membranes were then washed twice with PBS
and incubated with a solution containing a secondary antibody
marked with horseradish peroxidase diluted 1/2000 in the diluted
(1/10) saturation solution for one hour. After washing in PBS, the
binding of the antibodies is visualized with the reagent for
augmentation of the luminescence (ECL.RTM. Kit, Amersham). The
intensities of the spots were quantified with the Image quant 5.0
apparatus (Molecular Dynamics) and expressed in arbitrary units
(Phosphoimager). For the Western Blot, a total quantity of 50 .mu.g
of protein was separated by electrophoresis in polyacrylamide gel
containing 10% sodium dodecyl sulfate and transferred onto a
polyvinylidene fluoride membrane (PVDF, Amersham). The saturation
of the membranes and the immunochemoluminescence reaction were
carried out as described in the preceding.
[0105] Immunohistochemistry
[0106] The immunohistochemistry was analyzed on cells fixed in 80%
cold ethanol (-20.degree. C., 10 min), with cytocentrifuges, fixed
in 1% paraformaldehyde (PFA) or on paraffin sections of pancreatic
tissue fixed immediately after collection in 10% formalin or
PFA.
[0107] The antibodies (IPF-1, cytokeratin 19 and 7, insulin,
chromogranin A) are revealed with the Envision.RTM. system (Dako),
using various chromogenic substrates, 3,3'-diaminobenzidine (DAB),
3-amino-9-ethylcarbazole (AEC), or PhThaloBlue (HistoMark
BLUE.RTM.). The nuclei were counterstained with Carazzi's
hematoxylin.
[0108] Apoptosis
[0109] The specific cellular apoptosis of the acini is evaluated
after immunolabeling with antiamylase antibodies visualized with a
biotinylated goat antibody directed against the rabbit antibodies
(KPL, Gaithersburg, Md., USA) and streptavidin conjugated with
fluorescein isothiocyanate (streptavidin-FITC) (Sigma-Aldrich). The
apoptotic nuclear alterations are visualized with Hoechst 33258 (5
.mu.g/mL 10 min, 37.degree. C., Sigma-Aldrich).
[0110] Selective Adherence of the Cells
[0111] In order to exclude nonacinar cells from preferentially
adhering, the amylase positive cells were counted in dishes with
respect to the total number of cells (nuclei) before and after 12 h
of culture.
[0112] The number of acinar cells in the growing fraction is
determined by double labeling of cytocentrifuged cells after 12 h
and 2 days of culturing with amylase and Ki-67, which labels the
cells in all phases of their growth cycle with the exception of the
cells in GO phase.
[0113] Results
[0114] The human exocrine cell clusters adhere after 12 h of
culture and gradually spread out to form monolayer cultures. The
cell proliferation, determined by incorporation of tritiated.
(.sup.3H) thymidine, increases rapidly, with a peak after three
days and only after a slow decrease of expansion. The incorporation
of (.sup.3H) thymidine expressed in cpm/.mu.g
DNA.times.10.sup.3.+-.[sic; font conversion error] SEM (n=3) is:
40.5.+-.8.7 (day 1), 79.3.times.24.2 (day 1.5), 83.7.+-.12.8 (day
2), 95.4.+-.5.4 (day 3), 82.1.+-.5.0 (day 4), 42.9.+-.21.5 (day 6)
and 68.5.+-.5.1 (day 10).
[0115] The DNA and protein levels correlate with the levels of
proliferation with tritiated thymidine. FIG. 3 illustrates the
phenotypic transition of the cultures determined by slot blot using
25 .mu.g of total proteins coming from the cultures. The proteins
are expressed in arbitrary units of integration. This FIG. 3
illustrates the effective loading by proteins confirmed in the
preparations (n=3) by measuring the levels of beta-actin between
the wells; there are no statistical differences all along the
culture. This figure represents the analyses by Slot Blots on 25
.mu.g of total protein. The levels of beta-actin were measured
using 3 preparations as internal standard, with control of the
quantity of protein loaded per well.
[0116] FIG. 3A, in which the exocrine phenotype is revealed with
antiamylase (.box-solid.), the ductal phenotype with anti-CK19
(.quadrature.), the endocrine phenotype with antichromogranin A
(.largecircle.), illustrates the high levels of amylase protein
expressed by the exocrine preparations after their isolation, while
the levels of ductal (CK19) and endocrine (chromogranin A) proteins
are lower. An extensive reduction of the amylase protein is
observed after a day of culturing (92.+-.3.3, p<0.05 versus day
1). FIG. 3B, with the anti-IPF-1 (mean.+-.SEM, p<0.05 versus day
0), in which the intensity of the spots is expressed in arbitrary
units after digital integration, illustrates slot blots which prove
that the IPF-1 protein is present in minute levels in the exocrine
preparations after isolation increase during their culture, and
remain high. FIG. 3C shows the Western Blots representative of the
five human pancreata, reveals that the 46 kd band characteristic of
the IPF-1 protein is weak or undetectable immediately after
isolation of the exocrine cells (day 0) and intensifies as the
culture progresses. The Western Blot results are confirmed with two
different anti-IPF-1 antibodies directed against the C-terminal and
N-terminal domains. FIG. 3D illustrates the kinetic study obtained
by using a more sensitive visualization with the ECL kit and a
Phosphoimager.RTM. image analyzer, underlining the fact that this
increase in the IPF-1 protein (3.2 fold) rapidly appears in the
first two days of culture.
[0117] FIG. 4 shows the expression of the mRNA determined in the
exocrine culture preparations (n=5), standardized for the
expression of beta-actin.
[0118] FIG. 4A illustrates the PCR products with the specific bands
of IPF-1 (262 bp) and of beta-actin (314 bp).
[0119] FIG. 4B illustrates that the average expression of
IPF-1/beta-actin is low before culturing and that it increases
rapidly 10.5 fold after 3 days (n=5, p<0.001) with respect to
day 1 and remains high, with levels of expression eight times
higher after one week, and seven times higher after two weeks in
comparison with day 1 (9 [sic; p]=0.08 versus day 1; p<0.001
versus day 0).
[0120] FIG. 4C, in which (.largecircle.) represents the expression
of insulin standardized with respect to beta-actin (n=5; p>0.05)
compared with day 0) and (.box-solid.) represents the expression of
insulin from purified endocrine preparations (n=5), illustrates the
determination of the mRNA of the insulin in the exocrine
preparations in order to control the contamination with endocrine
cell populations during culturing (islets (n=4, 71.+-.6% pure) are
used as positive control). The levels of mRNA in the exocrine
cultures remain lower (for example, between 7% (day 0) and 2.5%
(days 3, 7)) than those of the control islets. No significant
differences between the levels of days 0, 3 or 7 were observed.
[0121] FIG. 5 illustrates the immunohistochemical analysis,
complementary to the Slot blot, the Western Blot and the PCR-RT
results, showing that the culturing of the exocrine preparations
for one week leads to the loss of the specific immunolabeling of
the amylase (not shown), and to an increase of the labeling of the
ductal antigens CK19, CK7.
[0122] FIG. 5A illustrates that, after 7 days of culture, the cells
show a ductal phenotype revealed with a dominant labeling for the
expression of CK19. The labeling of the insulin is always negative
in the exocrine preparations which were grown; consequently, the
neuroendocrine marker chromogranin A was used for evaluating the
contamination of the exocrine preparations with islets. FIG. 5B
(arrow) illustrates that the contamination with endocrine cells is
limited and remains lower than 5%, both in the initial preparations
as well as at the end of culture. Double immunolabeling was done in
order to establish that the majority of cells in the cultures
derived from the exocrine preparations are ductal cells (CK19/CK7
positive cells) and IPF-1 positive cells either in the cytoplasmic
compartment or in the nuclear compartment (CK7/IPF-1, illustrated
in FIG. 5C). The rare IPF-1 positive and CK7 negative cells
(arrowhead) probably correspond to contaminating beta-cells.
[0123] In order to exclude selective adherence of the exocrine
tissue, the inventors compared the preparations immunolabeled with
the amylase/Hoechst before day 0 and 12 h after the beginning of
culture.
[0124] Approximately 60% (n=2, 59%.+-.1 in triplicate) of all of
the cells have [sic; typo] an amylase stain on day 0 (therefore 41%
non-acinar) and this percentage remains the same after 12 h of
culturing.
[0125] The apoptosis is monitored by immunolabeling with an
antiamylase antibody/Hoechst 33258 (n=2).
[0126] The nuclear signs of apoptosis were virtually absent from
the exocrine cultures, in particular during the principal
phenotypic change (day 3). On day 5, a small number of cells in
culture have nuclei in the shape of a half moon, indicators of a
process of apoptosis; however, they remain negative for the
staining with annexin V, an early marker of apoptosis.
[0127] Inversely, double immunolabeling of the preparations after
12 h and after two days (n=2) for amylase and Ki-67, a nuclear
antigen expressed during all the phases of the cell cycle with the
exception of G0, shows that the majority of acinar cells constitute
part of the growing fraction. After approximately 12 h of culture,
40% of all of the cells are acinar cells in the cell cycle
(Amy+/Ki67+), 15% are acinar cells which are not in the cycle
(Amy+/Ki-67-), 43% of the cells are nonacinar cells in the cycle
(Amy-/Ki-67+); thus only 17% of the cells are in G0 phase of the
cell cycle and are consequently Ki-67 negative. After two days of
culture, when the expression of the amylase is still visible by
immunohistochemical techniques and the levels of proliferation
(tritiated thymidine) are close to the peaks, 51.5% of the cells
are acinar cells (Amy+/Ki-67-), 42% of the cells are nonacinar
cells in the cycle (Amy-/Ki-67+).
[0128] The role of the IPF-1 transcription factor in the neogenesis
of the islets is supported by its increased expression in the
pancreatic ducts, the site of the endocrine cell precursors, in
several models of pancreatic regeneration. The expression of the
factor IPF-1 in recently divided ductal cells is based on the
hypothesis that all the adult ductal cells can recover their
pluripotentiality (for example, their stem cell capacity)
(Bonner-Weir S. et al. "Partial pancreatectomy as a model of
pancreatic regeneration" in Pancreatic Growth and Regeneration.
Sarvetnick N. Ed. Paris, Karger Landes Systems, 1997, pp.
138-153).
[0129] The decisive role of the factor IPF-1/PDX-1 in the endocrine
cell differentiation of digestive endodermal cells was recently
confirmed in the liver (Ferber S. et al. Pancreatic duodenal
homeobox gene 1 induces expression of insulin genes in liver and
ameliorates streptozotocin induced hyperglycemia. Nature Med
6:568-572, 2000).
[0130] The transition of the exocrine cell preparations in culture
to a ductal phenotype has been well characterized in various
species, but the exact mechanism involved in it remains
controversial. Ligation of the rat duct is followed by an apoptotic
deletion of the acinar cells simultaneously with a proliferation of
ductal cells. Logsdon et al., working on acinar cells of mice, also
showed a substantial loss of exocrine tissue (DNA, proteins)
preceding the cell proliferation.
[0131] Inversely, in experiments conducted by the inventors in the
context of the present invention, the rapid increases of cell
proliferation, of the levels of DNA and of protein observed
immediately after cell attachment (12 h) correlate with the initial
increase of the ductal cell markers and with the decrease of the
level of amylase.
[0132] A selective attachment of the ductal cells and death and/or
apoptosis of the acinar cells cannot explain these facts in the
human model, because the proportion of amylase positive cells
initially present in the preparations remains the same after 12 h
of culture when the plates are cleaned of the unattached cells.
Alternatively, this rapid loss of amylase may be due to a reduction
of the levels of amylase in the acinar cells.
[0133] None of the methods used to evaluate the apoptosis,
including early and later markers, detects increasing levels of
apoptosis during the phenotypic transition (day 3).
[0134] In contrast, the early presence of Ki-67/amylase positive
cells in the cultures invokes their potential proliferation and
confirms that the phenotypic transition happens with the cell
proliferation as demonstrated using other models.
[0135] The appearance of a small number of nuclei with a half moon
shape is observed only after 5 days of culture, after the principal
phenotypic transition. The absence of significant apoptosis in
these cultures and the presence of high levels of proliferation
proves that the acinar cells have dedifferentiated into cells with
a ductal phenotype, simultaneously with the rapid growth of the
preexisting CK19 positive cells.
[0136] These ductal cells derived from exocrine populations express
low or nonexistent levels of protein IPF-1 as well as low levels of
IPF-1 mRNA which increase rapidly during culture. The 46 kd band
characteristic of IPF-1 is confirmed by two anti-IPF antibodies; in
order to exclude detection by these sensitive techniques of IPF-1
derived from minimal fractions of contaminating endocrine cells,
simultaneous quantifications of insulin mRNA were made. The levels
of this insulin mRNA are initially detectable in the preparations
when they are compared with the levels found for the islets, and
remain practically constant during the whole culture period.
[0137] The immunohistochemistry with two anti-IPF-1 antibodies
locates the expression of IPF-1 on the ductal cells (CK-7
positive). Only a few endocrine cells are present (FIG. 5). Using
pan-neuroendocrine markers including chromogranin A and
synaptophysin, the total number of endocrine cells in the initial
preparation is estimated to be 4.7.+-.1.8% of the initial
preparation and 3.5.+-.0.8% of the cells after 7 days of culture
(results not shown).
[0138] The disagreement between this low (<5%) endocrine
contamination of the exocrine cultures and the intensity of the
IPF-1 and CK19 (CK7) immunolabeling contributes towards confirming
that the factor IPF-1 does not come essentially from the CK19
negative contaminating beta-cells. The studies of double labeling
with IPF-1 and synaptophysin (not shown) confirm these data.
[0139] The Western Blots done with extracts of total proteins
isolated from purified human islets show the two forms of proteins
with the visualization of two bands at 31 kd and 46 kd (data not
shown), revealed with the antibody directed against the N-terminal
domain of PDX-1. The protein extracts from preparations of human
exocrine cell cultures show a principal 46 kd band and a weak or
undetectable 31 kd cytoplasmic band.
[0140] Unlike the IPF-1 positive but CK19 negative cells shown by
Beattie et al., which are of endocrine origin, the majority of
cells in the cultures derived from the exocrine cells show a ductal
phenotype (positive for CK19, CK7 and carbohydrate 19-9 (results
not shown) and are simultaneously IPF-1 positive. The initial
cultures shown by Beattie et al. were insulin and IPF-1 positive,
quite unlike the cells described in the experiments conducted in
the context of the present invention, implying that the IPF-1
positive cells of their study are dedifferentiated beta-cells.
[0141] Thus, the inventors show that the rapid
dedifferentiation/transdiff- erentiation of exocrine cells in vitro
is associated simultaneously with an increase of the ductal markers
and with transcription of the factor IPF-1 at the mRNA level as
well as at the protein level.
Sequence CWU 1
1
6 1 18 DNA artificial sequence Artificial sequence are primer
designed to amplify sequences 1 ccatggatga agtctacc 18 2 19 DNA
artificial sequence Artificial sequence are primer designed to
amplify sequences 2 gtcctcctcc tttttccac 19 3 18 DNA artificial
sequence Artificial sequence are primer designed to amplify
sequences 3 tgtgaaccaa cacctgtg 18 4 17 DNA artificial sequence
Artificial sequence are primer designed to amplify sequences 4
cgtctagttg cagtagt 17 5 19 DNA artificial sequence Artificial
sequence are primer designed to amplify sequences 5 atcatgtttg
agacctcca 19 6 20 DNA artificial sequence Artificial sequence are
primer designed to amplify sequences 6 catctcttgc tcgaagtcca 20
* * * * *