U.S. patent application number 12/937610 was filed with the patent office on 2011-02-10 for method for obtaining ngn3-expressing cells and insulin producing-beta cells.
Invention is credited to Cecile Haumaitre-Sarron, Olivia Lenoir, Raphael Scharfmann.
Application Number | 20110033930 12/937610 |
Document ID | / |
Family ID | 41707669 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110033930 |
Kind Code |
A1 |
Scharfmann; Raphael ; et
al. |
February 10, 2011 |
METHOD FOR OBTAINING NGN3-EXPRESSING CELLS AND INSULIN
PRODUCING-BETA CELLS
Abstract
The present invention relates to a method for obtaining
Ngn3-expressing cells and insulin producing-beta cells by
contacting a Pdx1-expressing pancreas explant with an amount of at
least one histone deacetylase inhibitor (HDACi). The inventive
methods have the advantage of being simple and quick, and of
providing large amounts of Ngn3-expressing cells and insulin
producing-beta cells, that are useful therapeutic tools. The
invention also relates to a pharmaceutical composition for the
treatment of diabetes which comprises an amount of at least one
HDACi.
Inventors: |
Scharfmann; Raphael; (Paris,
FR) ; Haumaitre-Sarron; Cecile; (Paris, FR) ;
Lenoir; Olivia; (Paris, FR) |
Correspondence
Address: |
WHITHAM, CURTIS & CHRISTOFFERSON & COOK, P.C.
11491 SUNSET HILLS ROAD, SUITE 340
RESTON
VA
20190
US
|
Family ID: |
41707669 |
Appl. No.: |
12/937610 |
Filed: |
August 21, 2009 |
PCT Filed: |
August 21, 2009 |
PCT NO: |
PCT/US09/54588 |
371 Date: |
October 13, 2010 |
Current U.S.
Class: |
435/375 ;
564/163 |
Current CPC
Class: |
F02C 1/05 20130101; Y02E
20/14 20130101; F01K 25/08 20130101 |
Class at
Publication: |
435/375 ;
564/163 |
International
Class: |
C12N 5/071 20100101
C12N005/071; C07C 237/20 20060101 C07C237/20 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 2008 |
US |
12197014 |
Claims
1. A method for obtaining a population of insulin producing-beta
cells which comprises a step of contacting a Pdx1-expressing
pancreatic explant with an amount at least one histone deacetylase
inhibitor (HDACi).
2. The method according to claim 1, wherein the Pdx1-expressing
pancreatic explant is a non-human embryonic pancreatic explant or a
human foetal pancreatic explant.
3. A method for obtaining a population of insulin producing-beta
cells which comprises a step of contacting Ngn3-expressing cells
with an amount at least one HDACi.
4. The method according to claim 1, wherein the histone deacetylase
inhibitor is selected from the group consisting of selective class
II HDACi and classical HDACi.
5. The method according to claim 4, wherein the HDACi is
trichostatin A (TSA).
6. A method for obtaining a population of Ngn3-expressing cells
which comprises a step of contacting a Pdx1-expressing pancreatic
explant with an amount at least one HDACi.
7. The method according to claim 6, wherein the HDACi is selected
from the group consisting of selective class I HDACi, selective
class II HDACi and classical HDACi.
8. The method according to claim 7, wherein the HDACi is valproic
acid (VPA) or TSA.
9. A pharmaceutical composition for the treatment of diabetes which
comprises an amount of at least one HDACi.
10. The pharmaceutical composition according to claim 9, wherein
the HDACi is selected from the group consisting of selective class
_11 HDACi and classical HDAC1.
11. The pharmaceutical composition according to claim 10, wherein
the HDACi is TSA.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a method for obtaining a population
of Ngn3-expressing cells or a population of insulin producing-beta
cells by contacting a Pdx1-expressing pancreatic explant with an
amount of at least one histone deacetylase inhibitor (HDACi). The
invention also relates to a pharmaceutical composition for the
treatment of diabetes which comprises an amount of at least one
HDACi.
BACKGROUND OF THE INVENTION
[0002] Nowadays, more than 150 million people suffer from diabetes
in the world. This disease is rising heavily and it is estimated
than in the next 20 years, 300 million people could be affected.
Insulinotherapy is the large-scale diabetes mellitus treatment. It
consists in recurrent injections of insulin every day. The hope is
to replace this heavy treatment, which is associated with side
effects, by a definitive cure. In this goal, islets transplantation
was tested according to the Edmonton protocol (66). However, 5 to
10 organ donors are required to transplant a single diabetic
patient. Thus, one of the major problems limiting islet
transplantation therapy is the lack of organ donors. An alternative
source of beta cells (insulin producing-beta cells) is therefore
required. Different approaches are being considered: xenografts,
transdifferentiation of bone marrow, liver or intestine cells, as
well as differentiation of embryonic or adult stem cells. Yet,
despite of the intensive efforts which have been devoted to define
an effective method for obtaining beta cells, none of these methods
really are satisfying. Indeed, even if some of these strategies may
be useful, each of these approaches has shown limitations (e.g.,
raised problems of rejection and potential transfer of viruses form
animal to human with xenograft; important risk of carcinogenesis
and difficulties to control in vitro some steps of development with
embryonic stem cells; early stage of approaches based on adult stem
cells, liver and intestine cells, and poor reproducibility
encountered with bone marrow.)
[0003] Thus, there still remains, in the art, an ongoing need for
an effective method for obtaining islet progenitors and more
importantly insulin producing-beta cells useful in therapy, and
more particularly in cell replacement in diabetes. With regard to
this serious public health problem, well understanding the
pancreatic development and processes controlling it is an essential
and critical challenge. However, the mechanisms regulating cell
fate choices during pancreatic development are still unclear.
[0004] Currently, there is known that the mature pancreas contains
exocrine tissue composed of acinar cells that secrete digestive
enzymes via a branched network of ductal cells into the intestine
and endocrine islets that produce hormones such as insulin (.beta.
cells), glucagon (.alpha. cells), somatostatin (.delta. cells) and
pancreatic polypeptide (PP cells). The pancreas originates from the
dorsal and ventral regions of the foregut endoderm directly behind
the stomach. Signals derived from adjacent mesodermal structures,
notochord and dorsal aorta, (32, 36) and mesenchyme, which
condenses around the underlying committed endoderm (4, 52) are
involved in the control of pancreas development.
[0005] It has also been shown during pancreas development,
transcription factors play critical roles in exocrine and endocrine
differentiation. Indeed, studies in genetically engineered mice
have identified a hierarchy of transcription factors regulating
pancreatic specification, growth and differentiation. Thus, recent
work has identified transcription factors that regulate pancreatic
cell lineages (10, 29). The pancreas-committed endodermal region
expresses the homeodomain factor Pdx1 (28, 49). Next, the basic
helix-loop-helix factor Neurogenin3 (Ngn3) initiates the endocrine
differentiation program in epithelial pancreatic progenitor cells.
Indeed, Ngn3-deficient mice fail to generate any endocrine cells
(18) and lineage tracing experiments have also provided direct
evidence that Ngn3-expressing cells are islet progenitors (20).
Subsequently, additional transcription factors determine the
specific endocrine cell fate. Gain- and loss-of-function
experiments are consistent with antagonistic roles for Pax4 and Arx
in specifying endocrine sub-types (for .beta./.delta. or .alpha./PP
cells, respectively). Whereas Pax4-deficient mice display a
selective loss of .beta.- and .delta.-cells with a proportional
increase in .alpha.-cells, Arx-deficient mice present the opposite
phenotype (11, 54). Furtheimore, Arx and Pax4 display mutual
transcriptional inhibition (8).
[0006] In other respects, transcriptional regulation in eukaryotes
occurs within chromatin and is influenced by post-translational
histone modifications (e.g. acetylation) involving histone
deacetylases. Histone modifications play crucial roles in the
transcriptional regulation of most eukaryotic genes and have been
linked to cell differentiation control (in muscle and cardiac
cells, for example (3, 43)). Acetylation or deacetylation of
histone terminal domains can regulate gene expression. Thus,
acetylation generally contributes to the formation of a
transcriptionally competent environment by relaxing the chromatin
structure, allowing transcription factors to access the target DNA.
In contrast, histone deacetylation compacts chromatin and leads to
transcriptional repression (19, 34). More precisely, histone
acetyltransferases (HATS) and histone deacetylases (HDACs),
respectively loosen or compact chromatin structures, regulate cell
proliferation/differentiation in various tissues (6, 7, 35, 43, 47,
57, 59). Recent invalidation studies in mice revealed that the
various HDACs are not functionally redundant. Thus, HDAC1 and -2
were shown to regulate cardiac development (57). HDAC5 and HDAC9
are involved in the heart's response to stress signals (6), HDAC4
in chondrocyte hypertrophy (59) and HDAC7 in maintenance of
vascular integrity (7).
[0007] Small-molecule, called histone deacetylasc inhibitors
(HDACi) are major tools for studying the connection between overall
chromatin effects and cell lineage specification. Pharmacological
inhibition of HDACs enables experimental manipulation and
systematic analysis of chromatin remodelling (41). The effects of
HDACi are selective (39, 58) and are thus often used to
specifically inhibit HDACs (41, 45, 60). For example, valproic acid
(VPA) preferentially targets class I HDACs (17), whereas
trichostatin A (TSA) inhibits both class I and class II HDACs
(Yoshida et al., 1990). HDACi were successfully used to demonstrate
the roles of HDACs in intestine (56), oligodendrocyte (40, 53),
neuron (25), adipocyte (63), osteoblast (37) and T cell (55)
differentiation programs and are now being clinically evaluated as
cancer drugs (45). However, to date, pancreatic phenotypes have not
been reported in specific HDAC-deficient mice.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method for obtaining a
population of insulin producing-beta cells which comprises a step
of contacting a Pdx1-expressing pancreatic explant with an amount
at least one histone deacetylase inhibitor (HDACi). In such a
method, the HDACi is selected from the group consisting of
selective class II HDACi and classical HDACi. In preferred
embodiments, the HDACi is trichostatin A (TSA).
[0009] In another aspect, the invention also provides a method for
obtaining a population of insulin producing-beta cells which
comprises a step of contacting Ngn3-expressing cells with an amount
at least one HDACi.
[0010] In another aspect, the invention also provides a method for
obtaining a population of Ngn3-expressing cells which comprises a
step of contacting a Pdx1-expressing pancreatic explant with an
amount at least one HDACi. In such a method, the HDACi is selected
from the group consisting of selective class I HDACi, selective
class II HDACi and classical HDACi. In preferred embodiments, the
HDACi is valproic acid (VPA) or TSA.
[0011] In yet another aspect, the invention provides a
pharmaceutical composition for the treatment of diabetes which
comprises an amount of at least one HDACi. The suitable HDACi is
selected from the group consisting of selective class II HDACi and
classical HDACi. In a preferred embodiment, the HDACi is TSA.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention encompasses the recognition by the
inventors that the maintenance of acetylation (via histone
deacetylase inhibition) has a specific, dominant function in
pancreatic lineage development. The inventors have indeed
highlighted the ability of histone deacetylase inhibitors to
modulate pancreatic cell determination and amplify specific
cellular sub-types. Such approach may thus be very useful for
developing novel cell replacement therapies in diabetes.
[0013] Indeed, the present inventors have now demonstrated that
HDACi treatment promoted the Ngn3 pro-endocrine lineage leading to
an increased pool of endocrine progenitors and insulin
producing-beta cells with potential applications in cell
replacement therapies notably in diabetes.
[0014] Definitions:
[0015] As used herein, the term "pancreatic explant" generally
refers to a tissue harvested after its isolation from a piece of
pancreas and placed in a culture dish containing an appropriate
culture medium.
[0016] As used herein, the term "Pdx1-expressing pancreatic
explants" means that pancreatic explants which are used in the
context of the invention express the marker Pdx1 which is the
transcription factor pancreatic duodenal homeobox-1. Therefore,
Pdx1-expressing pancreatic explants include embryonic pancreas
explants such as the established rat embryonic pancreas explants as
previously described (2, 21). Furthermore, Pdx1-expressing
pancreatic explants do not produce insulin (at least no detectable
amount of insulin). Pdx1-expressing pancreatic explants do not
contain insulin producing-beta cells. Thus, the unique type of cell
expressing Pdx1 present in such pancreatic explants is pancreatic
progenitors.
[0017] As used herein, the term "marker" refers to a protein,
glycoprotein, or other molecule expressed on the surface of a cell
or into a cell, and which can be used to help identify the cell. A
marker can generally be detected by conventional methods. Specific,
non-limiting examples of methods that can be used for the detection
of a cell surface marker are immunohistochemistry, fluorescence
activated cell sorting (FACS), and enzymatic analysis.
[0018] As used herein, the term "providing" refers to a process in
which tissue or organ are isolated and provided in a state suitable
for in vitro culture.
[0019] As used herein, the term "isolated" refers to a tissue or
organ which has been separated from at least some components of its
natural environment. This term includes gross physical separation
of the tissue or organ from natural environment (e.g., removal from
the donor).
[0020] As intended herein, the term "contacting" refers to a
process in which pancreatic explants are put in contact in an
appropriate culture medium with an amount of at least of HDACi of
interest.
[0021] As used herein, the term "an appropriate culture medium"
refers to a culture medium that contains nutrients necessary to
support the growth and/or survival of the pancreatic explants and
the Ngn3-expressing cells and insulin producing beta-cells. Said
appropriate culture medium may or may not further comprise growth
factors. By way of example, growth factors of interest may be
serum, fibroblast growth factors and any combination of these or
other growth factors.
[0022] The term "progenitor cells" refers to cells arc partially
specialized. These cells divide and give rise to differentiated
cells. Progenitor cells belong to a transitory amplifying
population of cells derived from stem cells. Compared to stem
cells, they have a limited capacity for self-renewal and
differentiation. Moreover, since progenitor cells are committed to
a particular differentiation process, progenitor cells also express
specific markers.
[0023] The terms "Ngn3-expressing cells", "Ngn3+ cells",
"pancreatic endocrine progenitor cells", insulin producing-beta
cell progenitor" or "islet progenitors" are used herein
interchangeably. They refer to progenitor cells that can divide and
give rise to the different pancreatic endocrine cells (i.e. .alpha.
cells, .beta. cells, .delta. cells and PP cells). Such progenitor
cells express the specific marker Ngn3 which is a transcription
factor which is a marker of choice for detecting the onset of
pancreatic endocrine cell differentiation. Ngn3 thus represents the
earliest pancreatic endocrine-cell-specific transcription factor in
embryonic development and is a well-established marker for
embryonic insulin producing-beta cells progenitors. Moreover, such
Ngn3-expressing cells express other specific markers such as Pdx1,
Pax4, Isl1, Nkx2.2, Nkx6.1, Isl1, NeuroD1 and Pax6 such as
described by Chiang et al. 2003 (68).
[0024] The terms "beta cells", ".beta. cells" or "insulin
producing-beta cells" are used herein interchangeably. They refer
to a type of cell present in the pancreas in areas called the
islets of Langerhans. They make up 65-80% of the cells in the
islets. Beta cells make and release insulin, a hormone that
controls the level of glucose in the blood.
[0025] The terms "histone deacetylases" or "HDACs" are used herein
interchangeably. They refer to enzymes that remove acetyl groups
from histones.
[0026] As previously described, mammalian HDACs are grouped into
the classical class I, II and IV HDAC family and the structurally
unrelated Sirtuin family (class III HDACs). Thus, class I HDACs
includes HDAC1-3 and 8, class II HDACs includes HDAC4-7, 9 and 10
and class IV includes HDAC11.
[0027] The term "histone deacetylase inhibitor" or "HDACi" are used
herein interchangeably. They refer to a compound natural or not
which inhibits the histone deacetylase activity. There exist
different classes of HDACi in function of their selectivity for
their substrates. The classes of HDACi useful in the present
invention are selective class I HDACi, selective class II HDACi and
classical HDACi.
[0028] A "classical HDACi" refers thus to a compound natural or not
which has the capability to inhibit the histone deacetylase
activity independently of the class of HDACs. Therefore a classical
HDACi is a non selective HDACi. By "non selective" it is meant that
said compound inhibits the activity of classical HDACs (i.e. class
I, II and IV) with a similar efficiency independently of the class
of HDAC. Examples of classical HDACi include, but are not limited
to, trichostatin A (TSA), SAHA, scriptaid, oxamflatin, NVP-LAQ824,
PDX101, LBH-589, ITF2357 and PCI-24781; cyclic peptides such as
trapoxine and apicidin; sodium butyrate; benzamides and
2'-amino-anilides such as acetyldinaline and histacin.
[0029] In the context of the invention, "selective class I HDACi"
is selective for class I HDACs (i.e. HDAC1-3 and 8) as compared
with class II HDACs (i.e. HDAC4-7, 9 and 10). By "selective" it is
meant that selective class I HDACi inhibits class I HDACs at least
5-fold, preferably 10-fold, more preferably 25-fold, still
preferably 100-fold higher than class II HDACs. Selectivity of
HDACi for class I or class II HDACs may be determined according to
previously described method (Kahn et al. 2008). Examples of
selective class I HDACi include, but are not limited to, valproic
acid (VPA), sodium valproate, the cyclic peptide FK-228 and
2'-amino-anilides such as MS275, CI994 and MGCD0103.
[0030] In the same way, "selective class II HDACi" is selective for
class II HDACs (i.e. HDAC4-7, 9 and 10) as compared with class I
HDACs (i.e. HDAC1-3 and 8). By "selective" it is meant that
selective class II HDACi inhibits class II HDACs at least 5-fold,
preferably 10-fold, more preferably 25-fold, still preferably
100-fold higher than class I HDACs. Examples of selective class II
HDACi include, but are not limited to, tubacin and
(aryloxopropenyl)pyrrolyl hydroxamates.
[0031] In the context of the invention, the term "treating" or
"treatment", as used herein, refers to a method that is aimed at
delaying or preventing the onset of a pathology, at reversing,
alleviating, inhibiting, slowing down or stopping the progression,
aggravation or deterioration of the symptoms of the pathology, at
bringing about ameliorations of the symptoms of the pathology,
and/or at curing the pathology.
[0032] As used herein, the term "subject" refers to a mammal,
preferably a human being, that can suffer from a condition
associated with tissue or organ damage, but may or may not have the
pathology. The term "subject" does not denote a particular age, and
thus encompasses adults, children, and newborns.
[0033] As used herein, the term "amount" refers to any amount of at
least HDACi (or a pharmaceutical composition thereof) that is
sufficient to achieve the intended purpose.
[0034] As used herein, the term "pathologies" refers to any disease
or condition associated with tissue or organ damage. The term
"pathology associated with tissue or organ damage" refers to any
disease or clinical condition characterized by tissue or organ
damage, injury, dysfunction, defect, or abnormality. Thus, the term
encompasses, for example, injuries, degenerative diseases and
genetic diseases. Such pathologies may affect any tissues or
organs.
Methods for Obtaining Ngn3-Expressing Cells and Insulin
Producing-Beta Cells
[0035] In a first aspect, the invention relates to a method for
obtaining a population of Ngn3-expressing cells which comprises a
step of contacting Pdx1-expressing pancreatic explants with at
least one histone deacetylase inhibitor (HDACi).
[0036] In a particular embodiment, the method comprises the
following steps: [0037] a) providing a Pdx1-expressing pancreatic
explant, and [0038] b) contacting said explant with an amount of at
least one HDACi.
[0039] In such method, the HDACi is selected from the group
consisting of selective class I HDACi, selective class II HDACi and
classical HDACi.
[0040] In an embodiment, the HDACi is valproic acid (VPA).
[0041] In another embodiment, the HDACi is trichostatin A
(TSA).
[0042] In an attempt to overcome the problems of scarcity of
insulin producing-beta cells described in the state of the art, the
present inventors have developed a simple method for obtaining
insulin producing-beta cells.
[0043] Thus, in a second aspect the invention relates to a method
for obtaining a population of insulin producing-beta cells which
comprises a step of contacting a Pdx1-expressing pancreatic explant
with an amount at least one HDACi.
[0044] In a particular embodiment, the method comprises the
following steps: [0045] a) providing a Pdx1-expressing pancreas
explant, and [0046] b) contacting said pancreas explant with an
amount of at least one HDACi.
[0047] In such method, the HDACi is selected from the group
consisting of selective class II HDACi and classical HDACi.
[0048] In a particular embodiment, the HDACi is TSA.
[0049] In a third aspect, the invention relates to a method for
obtaining a population of insulin producing-beta cells which
comprises a step of contacting Ngn3-expressing cells with an amount
at least one HDACi.
[0050] In a particular embodiment, the method comprises the
following steps: [0051] a) providing a population of
Ngn3-expressing cells, and [0052] b) culturing said population with
an amount of at least one HDACi.
[0053] In such method, the HDACi is selected from the group
consisting of class II HDACi and classical HDACi.
[0054] In a particular embodiment, the HDACi is TSA.
[0055] The inventive method thus provides a valuable alternative to
obtain a high number of insulin producing-beta cells that can be
used as pharmacological and/or therapeutic tools.
[0056] Thus, a first step consists in providing a pancreas explant.
As already mentioned above, pancreatic explants useful in a method
according to the present invention express the transcription factor
Pdx1.
[0057] In one embodiment, the Pdx1-expressing pancreatic explant is
a non-human embryonic pancreatic explant.
[0058] In another embodiment, the Pdx1-expressing pancreatic
explant is a human foetal pancreatic explant.
[0059] Indeed, within the context of the invention, Pdx1-expressing
pancreas explants may be from any appropriate mammal origin (e.g.,
mouse, rat, rabbit, pig, dog or human origin).
[0060] The Pdx1-expressing pancreatic explants of the present
invention include, but are not limited to, embryonic rat pancreas
as previously described (2, 21) or foetal human pancreas as
previously described (67). As used herein, the term "foetal" refers
to a human developing organism from the eighth week after
fertilization.
[0061] Embryonic rat pancreas explant may be obtained by dissecting
pancreatic epithelia after harvesting embryos at E13.5. The
stomach, the pancreas, and a small portion of the intestine were
dissected together; then the pancreatic primordium was dissected.
It must be noted that at E13.5, the pancreas is composed of an
epithelium surrounded by mesenchymal tissue and that in the
presence of mesenchyme, the epithelium grew rapidly, spread into
the mesenchyme, and developed lobules. It must be further noted
that at this stage E13.5, the pancreatic epithelium is mainly
composed of early Pdx1 expressing-progenitors, with few glucagon
expressing-endocrine cells.
[0062] It must also be noted that Pdx1-expressing pancreatic
explants may also be characterized by other additional markers such
as Pft1a and NKx6.1 (29).
[0063] In another embodiment, pancreas explants may be explants
harvested from human foetuses (67) from the eighth week after
fertilization.
[0064] Methods of harvesting samples from tissues and organs are
known in the art and can be used in the practice of the present
invention. Preferably, methods of harvesting are not excessively
destructive for the tissue being harvested. Isolation of samples of
interest from a tissue sample preferably occurs in an aseptic
environment. For example, the tissue sample may be washed with a
buffer solution (e.g., buffered saline) optionally comprising
antimytotic and/or antibiotic agents.
[0065] A second step consists in contacting a Pdx1-expressing
pancreas explant or a population of Ngn3-expressing cells with an
amount of at least HDACi in an appropriate culture medium.
[0066] An appropriate culture medium according to the invention may
consist in a minimal medium in which cells can grow, such as for
example an RPMI medium complemented with serum. Such medium may
also include others factors of interest such antibiotics or amino
acids. A defined medium consisting of RPMI 1640 supplemented with
penicillin (100 units/ml), streptomycin (100 .mu.g/ml), HEPES (10
mmol/l), L-glutamine (2 mmol/l), nonessential amino acids (1x), and
10% heat-inactivated calf serum may be typically used as
appropriate culture medium.
[0067] In certain embodiments, the culture medium is changed every
day.
[0068] Cultures are often grown in a suitable vessel in a sterile
environment at 37.degree. C. in an incubator containing a
humidified 95% air--5% CO2 atmosphere. Vessels may contain stirred
or stationary cultures. Cell culture techniques are well known in
the art and established protocols are available for the culture of
diverse cell types.
[0069] Pdx1-expressing pancreatic explants may be put in contact
with at least appropriate HDACi for any efficient amount of time,
i.e., any amount of time that is necessary to allow the formation
of pancreatic endocrine progenitor cells or insulin producing-beta
cells. One skilled in the art will know how to determine such an
amount of time.
[0070] In embodiments destined for obtaining a population of
Ngn3-expressing cells, Pdx1-expressing pancreatic explants are put
in contact with at least one HDACi for at least about 3 days,
preferably between 3 days and more preferably for 7 days in an
appropriate culture medium. In embodiments destined for obtaining
insulin producing-beta cells, Pdx1-expressing pancreatic explants
are put in contact for at least about 7 days and preferably for at
least about 15 days in an appropriate culture medium with at least
one HDACi.
[0071] Moreover, Pdx1-expressing pancreatic explants or
Ngn3-expressing cells may be put in contact with at least one
appropriate HDACi in any efficient amount, i.e., any amount that is
necessary to allow the formation of Ngn3-expressing cells or
insulin producing-beta cells. One skilled in the art will know how
to determine such an amount. It must be underlined that useful
HDACi concentration depends on kind of used pancreatic explant
(e.g. higher for human explant than rat explant).
[0072] Thus, in a method according to the present invention, the
concentration of VPA added in the culture medium is preferably
comprised between about 1 mM and about 3 mM. In another method
according to the present invention, the concentration of TSA added
in the culture medium is preferably comprised between about 70 nM
and about 700 nM.
[0073] In a preferred embodiment involving embryonic rat pancreas
explant, the optimal concentration of VPA added in the culture
medium is about 1 mM. In another preferred embodiment, the optimal
concentration of TSA added in the culture medium is about 100
nM.
[0074] A large number of known HDACi can be used in the practice of
the invention.
[0075] The selective class I HDACi of the present invention
include, but are not limited to, valproic acid (VPA), sodium
valproate; the cyclic peptide FK-228 and 2'-amino-anilides such as
MS275, CI994 and MGCD0103.
[0076] The selective class II HDACi of the present invention
include, but are not limited to, tubacin and
(aryloxopropenyl)pyrrolyl hydroxamates.
[0077] The classical HDACi of the present invention include, but
are not limited to, hydroxamates such as trichostatin A (TSA),
SAHA, scriptaid, oxamflatin, NVP-LAQ824, PDX101, LBH-589, ITF2357
and PCI-24781; cyclic peptides such as trapoxine and apicidin;
sodium butyrate (NaB); benzamides and 2'-amino-anilides such as
acetyldinaline and histacin.
[0078] Cells of interest obtained with a method according to the
present invention may also be expanded by culturing in an
appropriate culture medium comprising at least one factor that
stimulates the proliferation of the cells.
[0079] Thus, a method according to the present invention
constitutes a quick and easy way to obtain a high number of
Ngn3-expressing cells and insulin producing-beta cells that can be
used in therapeutic applications.
[0080] It must further be noted that the population of
Ngn3-expressing cells or the population of insulin producing-beta
cells that are obtained by any method disclosed herein may be
incorporated in a pharmaceutical composition, optionally with a
pharmaceutically acceptable carrier or excipient. Thus, a
population of pancreatic endocrine progenitor cells or a population
of insulin producing-beta cells obtained by a method of the present
invention, or a pharmaceutical composition thereof, may be used in
the treatment of pathologies, in particular pathologies associated
with pancreas damage, injury, dysfunction, degeneration or
abnormality such as diabetes, and for reconstruction or
regeneration of the pancreatic endocrine tissue.
Pharmaceutical Composition for the Treatment of Diabetes which
Comprises an Amount of at least one HDACi.
[0081] A further aspect of the invention relates to pharmaceutical
composition for the treatment of diabetes which comprises an amount
of at least one HDACi.
[0082] Such pharmaceutical composition may be useful for inducing
in a subject the differentiation of endogenous progenitors in
insulin producing-beta cells.
[0083] In such pharmaceutical composition, the HDACi is selected
from the group consisting of class II HDACi and classical
HDACi.
[0084] In a particular embodiment, the HDACi is TSA.
[0085] In certain embodiments, a pharmaceutical composition may
further comprise at least another biologically active substance or
bioactive factor.
[0086] As used herein, the term "pharmaceutically acceptable
carrier or excipient" refers to a carrier medium which does not
interfere with the effectiveness of the biological activity of the
HDACi, and which is not excessively toxic to the host at the
concentrations at which it is administered. Examples of suitable
pharmaceutically acceptable carriers or excipients include, but are
not limited to, water, salt solution (e.g., Ringer's solution),
alcohols, oils, gelatins, carbohydrates (e.g., lactose, amylase or
starch), fatty acid esters, hydroxymethylcellulose, and polyvinyl
pyroline. Pharmaceutical compositions may be formulated as liquids,
semi-liquids (e.g., gels) or solids (e.g., matrix, lattices,
scaffolds, and the like). If desired, the pharmaceutical
composition may be sterilized.
[0087] As used herein the term "biologically active substance or
bioactive factor" refers to any molecule or compound whose presence
in a pharmaceutical composition of the invention is beneficial to
the subject receiving the composition. As will be acknowledged by
one skilled in the art, biologically active substances or bioactive
factors suitable for use in the practice of the present invention
may be found in a wide variety of families of bioactive molecules
and compounds. For example, a biologically active substance or
bioactive factor useful in the context of the present invention may
be selected from anti-inflammatory agents, anti-apoptotic agents,
immunosuppressive or immunomodulatory agents, antioxidants, growth
factors, and drugs.
[0088] A related aspect of the invention relates to a method for
treating a subject suffering from a pathology associated with
tissue or organ damage, said method comprising a step of
administering to the subject an efficient amount of HDACi as
described herein, or a pharmaceutical composition thereof.
[0089] Another aspect of the invention relates to a method for
treating a subject suffering from a pathology associated with
tissue or organ damage, said method comprising a step of
administering to the subject a population of insulin producing-beta
cells as obtained by a method according to the present invention,
or a phaimaceutical composition thereof.
[0090] In a particular embodiment, the method comprises the
following steps: [0091] a) providing a population of insulin
producing-beta cells, and [0092] b) administrating said population
to a subject in need thereof.
[0093] Still another aspect of the invention relates to a method
for treating a subject suffering from a pathology associated with
tissue or organ damage, said method comprising a step of
administering to the subject a population of Ngn3-expressing cells
as obtained by a method according to the present invention, or a
pharmaceutical composition thereof.
[0094] In a particular embodiment, the method comprises the
following steps: [0095] a) providing a population of
Ngn3-expressing cells, and [0096] b) administrating said population
to a subject in need thereof.
[0097] Yet, in preferred embodiments, pathology of interest is
diabetes mellitus. Diabetes mellitus often simply diabetes is a
syndrome characterized by disordered metabolism and inappropriately
high blood sugar (hyperglycaemia) resulting from either low levels
of the hormone insulin or from abnormal resistance to insulin's
effects coupled with inadequate levels of insulin secretion to
compensate. The World Health Organization recognizes three main
forms of diabetes mellitus: type 1, type 2, and gestational
diabetes (occurring during pregnancy), which have different causes
and population distributions. While, ultimately, all forms are due
to the beta cells of the pancreas being unable to produce
sufficient insulin to prevent hyperglycemia, the causes are
different. Type 1 diabetes is usually due to autoimmune destruction
of the pancreatic beta cells. Type 2 diabetes is characterized by
insulin resistance in target tissues. This causes a need for
abnormally high amounts of insulin and diabetes develops when the
beta cells cannot meet this demand. Gestational diabetes is similar
to type 2 diabetes in that it involves insulin resistance; the
hormones of pregnancy can cause insulin resistance in women
genetically predisposed to developing this condition.
[0098] Effective dosages and administration regimens can be readily
determined by good medical practice based on the nature of the
pathology of the subject, and will depend on a number of factors
including, but not limited to, the extent of the symptoms of the
pathology and extent of damage or degeneration of the tissue or
organ of interest, and characteristics of the subject (e.g., age,
body weight, gender, general health, and the like).
[0099] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0100] FIG. 1 is a schematic representation of pancreatic
differentiation.
[0101] During pancreatic development, precursor cells expressing
the PDX1-homeodomain transcription factor give rise to both
exocrine and endocrine pancreatic cell lineages. Mature exocrine
acinar cells express digestive enzymes such as amylase. The
endocrine cell sub-types including insulin-expressing .beta.-cells
as well as .alpha., .delta. and PP cells, arise from endocrine
progenitors expressing the NGN3 transcription factor.
[0102] The morphology of a E13.5 rat pancreatic explant cultured 1
day is shown, with the epithelium circled. Immunohistological
analysis shows PDX1 staining in epithelial cells (in green). Ngn3
transcripts (in blue) are revealed by in situ hybridization in the
pancreatic explant cultured 5 days. Exocrine acinar cells are
detected by amylase staining (in green), and endocrine
insulin-expressing cells are detected by insulin staining (in red),
with immunohistological analysis of pancreatic explants cultured 7
days.
[0103] FIG. 2 is a schematic representation of pancreatic cell
lineage differentiation. Pdx1 expressing-progenitor cells give rise
to both exocrine and endocrine cells. Among others, the
transcription factor P48 is involved in the commitment of the
acinar exocrine cells. Endocrine progenitors express the
transcription factor NGN3. Downstream of Ngn3, the transcription
factors Arx and Pax4 among others, are involved in the
determination of the different endocrine sub-types.
[0104] FIG. 3 shows that HDACi treatment enhances the pool of Ngn3
endocrine progenitor cells.
[0105] FIG. 5A is a graph representing real-time PCR quantification
of Ngn3 mRNA after 0, 1, 3, 5, 7, 9, 11 and 14 days of culture,
with and without VPA or TSA treatment. Values are meansSEM of at
least three independent experiments; **p<0.005;
***p<0.001.
[0106] FIG. 5B is a set of pictures showing at top the detection of
Ngn3 transcripts by in situ hybridization in pancreases cultured
for 5 days, with and without VPA or TSA treatment; and at bottom
the detection of Ngn3 protein by immunohistochemistry in pancreases
cultured 7 days, with and without VPA or TSA treatment. Scale
bar=100 .mu.m.
[0107] FIG. 4 shows the opposite effects of VPA and TSA treatment
on the endocrine .beta./.delta. lineage differentiation.
[0108] FIG. 4A is a set of pictures representing pancreases
cultured 9, 11 and 14 days with and without VPA or TSA treatment.
Note that with TSA treatment, at day 11 and 14, translucent buds
can be seen (black arrows).
[0109] FIG. 4B is a set of graph representing real-time PCR
quantification of insulin mRNA after 7, 9, 11 and 14 days of
culture, with and without VPA or TSA treatment.
[0110] FIG. 4C is a set of pictures illustrating the
mmunohistological analyses of pancreases after 14 days of culture,
with and without VPA or TSA treatment. .beta.-cell development was
evaluated using anti-insulin staining, in red. Nuclei were stained
in blue with Hoechst. Note that in TSA-treated explants, insulin
staining (white arrow) corresponds to the translucent bud seen in A
(black arrow). Quantification of the absolute surface areas
occupied by insulin+ cells that developed after 14 days of culture,
without or with VPA or TSA treatment. Values are meansSEM of at
least three independent experiments. NS: no significant difference;
*p<0.05; **p<0.005; ***p<0.001. Scale bar=100 .mu.m.
[0111] FIG. 5 is a schematic representation of class I HDAC
inhibitors effects on pancreas differentiation.
[0112] VPA and MS275 (class I HDAC inhibitors) repress acinar
lineage to the benefit of ductal cell differentiation. These HDACi
promote endocrine NGN3 progenitor cells. The differentiation of
.alpha. and PP cells is enhanced while the .beta./.delta. lineage
is abolished. More glucagon- and PP-expressing endocrine cells are
thus induced by VPA and MS275.
[0113] FIG. 6 is a schematic representation of class I and class II
HDAC inhibitors effects on pancreas differentiation.
[0114] TSA and NaB (both class I and class II HDAC inhibitors)
repress acinar lineage to the benefit of ductal cell
differentiation. These HDACi promote endocrine NGN3 progenitors
cells that differentiate into the .alpha./PP and .beta./.delta.
lineages. More endocrine cells, especially insulin-expressing
.delta. cells, are thus generated by TSA and NaB.
[0115] FIG. 7 represents a model for the role of HDACs on pancreas
differentiation.
[0116] Simple and double lines delineate the proposed involvement
of class I and class II HDACs, respectively, in the regulation of
pancreatic lineages. Based on the effects of HDACi, we propose that
class I HDACs regulate the exocrine lineage commitment in acinar
and ductal cells. We propose that class I HDACs have a major effect
on controlling the NGN3 pro-endocrine lineage. We propose that
class I and class II HDACs have distinct role in the regulation of
endocrine sub-types. Class I and class II HDACs might regulate the
balance between Pax4 and Arx expression, two transcription factors
involved in the .beta./.delta. and .alpha./PP lineages
respectively. According to our model, when class I HDACs are
inhibited (by VPA or MS275) Arx is promoted, favoring Pax4
inhibition and repression of the .beta./.delta. lineage. When all
HDACs are repressed (by TSA or NaB), Arx and Pax4 are enhanced and
both .beta./.delta. and .alpha./PP lineages are induced.
EXAMPLE 1
Material & Methods
[0117] Animals and dissection of dorsal pancreatic rudiments:
Pregnant Wistar rats were purchased from the CERJ (Le Genest,
France). The first day post-coitum was taken as embryonic day 0.5
(E0.5). Pregnant female rats at 13.5 days of gestation were killed
by CO2asphyxiation, according to the French Animal Care Committee's
guidelines. Dorsal pancreatic buds from E13.5 rat embryos were
dissected as described previously (2).
[0118] Organ culture, HDACi treatments, BrdU incorporation:
Pancreases were laid on 0.45 .mu.m filters (Millipore) at the
air-medium interface in Petri dishes, containing RPMI 1640
[0119] (Invitrogen) supplemented with penicillin (100 U/mL),
streptomycin (100 .mu.g/mL), HEPES (10 mmol/L), L-glutamine
(2mmol/L), non-essential amino acids (1x, Invitrogen) and 10%
heat-inactivated calf serum (HyClone). Cultures were maintained at
37.degree. C. in humidified 95% air/5% CO2. Medium was changed
every other day. Explants were cultured in the presence of VPA or
TSA (Sigma). We determined the optimal concentration of both
inhibitors by testing increasing doses of VPA (from 0.75 mM to 3
mM) and TSA (from 50 nM to 200 nM). Because VPA concentrations
above 1.5 mM and TSA concentrations above 125 nM result in
increased cell death (data not shown), we used concentrations that
lead to phenotypic effects without toxicity: 1 mM VPA and 100 nM
TSA. MS275 and NaB (Sigma) were used at 1 .mu.M and 125 .mu.M,
respectively. For cell proliferation assay, 10 .mu.M BrdU (Sigma)
was added to the medium during the last hour of culture.
[0120] Immunohistochemistry and quantification: Tissues were fixed
in 10% formalin, pre-embedded in low gelling agarose and embedded
in paraffin. All sections (4 .mu.m thick) of each pancreatic
explant were collected and processed for immunohistochemistry, as
described previously (14, 46). Antibodies were used at the
following dilutions: mouse anti-insulin (Sigma, 1/2000), guinea pig
anti-insulin (DAKO, 1/200), mouse anti-glucagon (Sigma, 1/2000),
rabbit anti-amylase (Sigma, 1/300), goat anti-osteopontin/SPP1
(R&D systems, 1/200), rabbit anti-PDXI ((14), 1/1000), mouse
anti-BrdU (Amersham, 1/2), rabbit anti-Ngn3 ((21), 1/1000). The
fluorescent secondary antibodies were: fluorescein anti-rabbit and
anti-goat antibodies (Jackson Immunoresearch, 1/200), Texas-red
anti-mouse antibody (Jackson Immunoresearch, 1/200) Alexa fluor 488
anti-rabbit antibody (Biogenex, 1/400) and AMCA anti-guinea pig
antibody (Jackson Immunoresearch, 1/200). Nuclei were stained blue
with Hoechst 33342 (0.3 .mu.g/ml, Invitrogen). Ngn3 detection was
perfointed as previously described (21) using the Vectastain elite
ABC kit (Vector Laboratories). Photographs were taken using a
fluorescence microscope (Leitz DMRB, Leica) and digitized using
cooled 3CCD cameras (C5810 or C7780, Hamamatsu). The surface area
of each staining was quantified with IPLab (Scanalytics). The
surface areas per section were summed to obtain the total surface
area per explant in mm2. At least three explants were analyzed per
condition and results are expressed as means.+-.SEM. Statistical
significance was determined using Student's t test.
[0121] TUNEL labelling: Terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
(TUNEL) experiments were performed using an in situ cell death
detection kit (Roche) and followed by insulin and amylase
immunostaining.
[0122] In situ hybridization: In situ hybridization was performed
as previously described (15) and colorimetric development was
performed with 5-bromo-4-chloro-3-indolyl phosphate (Promega) and
nitroblue tetrazolium (Roche). No signal was obtained when a sense
riboprobe was used.
[0123] RNA extraction and real-time PCR: Total RNA was extracted
from pools of at least three pancreases using RNeasy Microkit
(Qiagen) and reverse transcribed using Superscript reagents
(Invitrogen). Real-time PCR was performed with the 7300 Fast
real-time PCR System; each reaction consisted of a mix of
Taqman.RTM. universal PCR master mix with a specific labelled probe
(Applied Biosystem) (21). The comparative method of relative
quantification (2.sup.-.DELTA..DELTA.ct) (38) was used to calculate
expression levels of each target gene, normalized to peptidylpropyl
isomerase A (ppia/cyclophilin A). The data are presented as fold
changes in gene expression. At least three pools of explants were
analyzed per condition and results are expressed as means.+-.SEM.
Statistical significance was determined using Student's t test. PCR
primer sequences are available upon request.
[0124] Protein extracts and Western-Blot analysis: Tissue lysates
were prepared from pools of at least five pancreases using a
Complete Lysis-M kit (Roche). Equal amounts of proteins were loaded
on SDS-PAGE gels for separation and transferred onto 0.45 .mu.m
nitrocellulose membranes. After blocking with milk, the membranes
were probed with different antibodies: rabbit anti-HDAC1, rabbit
anti-HDAC2, rabbit anti-HDAC3, rabbit anti-HDAC4, rabbit
anti-HDAC6, rabbit anti-HDAC7 (Abeam) and rabbit anti-HDAC5
(Sigma), rabbit anti-acetyl-histone H3 (Lys9), rabbit
anti-acetyl-histone H4 (Lys12) (Upstate) and mouse anti-actin
antibody (Sigma). Results were visualized with horseradish
peroxidase-conjugated secondary antibodies (Cell Signaling) and
enhanced chemiluminescence (LumiGLO, Cell Signaling).
[0125] HDAC enzyme activity assay: Total cellular HDAC enzyme
activity was measured using an HDAC assay kit (Millipore). Briefly,
2 .mu.g of control or VPA- or TSA-treated cell extracts (prepared
as described above) were incubated in a 96-well plate with a
fluorometric substrate in HDAC assay buffer for 45 min at
37.degree. C. An activator solution was then added to release the
fluorophore from the deacetylated substrates, and fluorescence was
measured in a plate-reading fluorimeter (excitation=390 nm,
emission detection=460 nm).
EXAMPLE 2
[0126] In vivo HDACi injections in rat: Experiments were submitted
for ethical evaluation to the "Comite Regional d'Ethique pour
l'Experimentation Animale Ile-de-France-Paris Descartes" and
approved (register number P2.CHS.081.09). Pregnant rat wistar
females are injected intraperitoneally daily with HDACi between
E13.5 and E20.5. The NaB treated group received 1000 mg/kg whereas
the VPA treated group received 300 mg/kg, in Phosphate Buffer
Saline solution (PBS). After 7 days of treatment, the animals are
killed by CO2 asphyxiation, according to the French Animal Care
Committee's guidelines. Embryos are dissected and each pancreas is
fixed or frozen for subsequent analysis by immunohistochemistry or
western-blot respectively.
EXAMPLE 3
[0127] In vitro HDACi treatment of human pancreas: Human pancreases
are extracted from foetal tissue fragments obtained immediately
after voluntary abortions performed around 9 weeks of development,
in compliance with the current French legislation. Human pancreases
are cultured in the same conditions described above used for rat
pancreases, and treated until 14 days with 800nM TSA.
Results
[0128] HDACs are down-regulated during pancreas development: The
inventors first analyzed the expression of different HDACs during
rat pancreas development. Using Western blotting, we found that
both class I (HDAC1-3) and class II (HDAC4-7) HDACs were expressed
at E13.5, E17.5 and in the adult pancreas. The expression levels of
most HDACs (with the exception of HDAC3) decreased during
development. The inventors next measured total HDAC activity and
observed a 86.1% .+-.6.5% decrease at E17.5 compared with E13.5. To
determine whether decreased HDAC activity correlated with increased
histone acetylation, they performed Western blot analysis with
antibodies directed against histone acetyl-H3 and acetyl-H4
residues. The inventors found that the overall degree of histone
acetylation increased from E13.5 to the adult. These results show
that HDACs are expressed and then developmentally regulated in the
pancreas. Decreased HDAC expression is associated with decreased
HDAC activity and increased histone acetylation throughout pancreas
development.
[0129] To study the role of HDACs in pancreas development, the
inventors treated rat pancreatic explants with VPA and TSA. They
cultured E13.5 rat pancreases on floating filters at the air-medium
interface for 14 days. Under such conditions, acinar and endocrine
cells develop in a way that replicates pancreas development in vivo
(2, 21). The inventors found that the in vitro HDAC expression
pattern replicates the one found in vivo, with decreased expression
of most HDACs. They next measured total HDAC activity from day 0 to
day 14 and found a 65.4%.+-.1.6% decrease between the two stages.
They verified that VPA and TSA were acting as HDACi, and found
average decreases of 93.2%.+-.2.3% and 99.2%.+-.0.9% with VPA and
TSA, respectively. The decreased HDAC activity was correlated with
an increase in histone acetylation by Western Blot analysis with
histone acetyl-H3 and -H4 antibodies and found that both HDACi
induced histone hyper-acetylation. This correlated with
93.2%.+-.2.3% and 99.2%.+-.0.9% decreases in HDAC activity with VPA
and TSA, respectively. Moreover, they found that both HDACi induced
histone hyperacetylation.
[0130] Altogether, these results show that HDAC expression and
activity are regulated during pancreas development in vivo and in
vitro. Thus, data validate the use of VPA and TSA for potently
modulating HDAC activity in pancreatic explants.
[0131] HDACi treatment suppresses acinar differentiation and
promotes ductal differentiation: The inventors first compared the
morphology of pancreases cultured in the presence or absence of
HDACi. At E13.5, the pancreas is composed of an epithelium
surrounded by mesenchymal tissue. In control conditions, the
epithelium grew and spread into the mesenchyme. With VPA or TSA
treatment, epithelial growth occurred, branching increased and,
after 7 days of culture, the tips of the branched epithelium formed
cystic structures. Haematoxylin/eosin staining revealed cystic
structures and less-developed acinar structures with VPA and TSA,
compared with controls. They found similar results with MS275 and
NaB.
[0132] To precisely define the molecular events resulting from HDAC
inhibition, the inventors analyzed markers for the different
pancreatic lineages. They found no effect of HDACi on the ratio of
epithelial to mesenchymal tissue (by analyzing E-cadherin and
vimentin expression, respectively; data not shown). They next
analyzed the effects of HDACi on cell differentiation, focusing
first on the exocrine lineage. They used real-time PCR to monitor
the expression of the P48/Ptfla (33) and Mist1 (51) transcription
factors involved in acinar cell development, and amylase, a marker
of differentiated acinar cells. In control conditions, the
expression of P48 and Mist1 increased over the first 5 days of
culture, followed by increased amylase expression. The increase in
levels of P48, Mist1 and amylase was strongly reduced by VPA or
TSA. Quantification of the surface area occupied by amylase+ cells
indicated that acinar cell development was much lower in pancreases
cultured with VPA or TSA. For ductal cell differentiation, the
inventors used real-time PCR to evidence a major increase in SPP1
(31) expression, especially after 5 and 7 days of culture with VPA
or TSA. Immunohistological analysis showed that SPP1 staining was
greater in HDACi-treated pancreases and was mainly found around
cystic structures. Quantification of the surface area occupied by
SPP1+ cells showed a 4-fold increase on HDACi treatment, indicating
enhanced ductal cell development. Similar results with exocrine
differentiation were found with MS275 and NaB.
[0133] Overall, these results show that HDACi treatment leads to a
dramatic decrease in acinar lineage differentiation, whereas ductal
lineage differentiation is strongly enhanced.
[0134] HDACi treatment strongly promotes Ngn3 pro-endocrine
lineage: To define whether HDAC activity was involved in the
regulation of endocrine lineage, the inventors focused on
expression of Ngn3, a specific pancreatic endocrine progenitor
marker (18). In control pancreatic explants, Ngn3 mRNA levels
measured by real-time PCR increased after 1 day of culture, peaked
at day3 and decreased thereafter. With VPA or TSA, the inventors
observed a dramatic increase in Ngn3 expression from day1 and a
peak at day7 (14-fold higher than in controls). Hence, the Ngn3
expression profile was longer and amplified with HDACi treatment.
They found similar results with MS275 and NaB.
[0135] At day5, the inventors visualized Ngn3 mRNA expression by in
situ hybridization and observed enhanced Ngn3 expression, with
positive cells throughout the epithelium in VPA- and TSA-treated
explants. Finally, immunohistochemical analysis at day7 showed that
NGN3+ cells were extremely scarce in control pancreases, while many
were present in VPA- and TSA-treated explants.
[0136] Thus, these results show that the expression profile of the
pro-endocrine transcription factor Ngn3 was strongly enhanced and
maintained with HDACi treatment, leading to an increased pool of
endocrine progenitor cells.
[0137] HDACi treatment enhances the endocrine .alpha./PP lineage:
The inventors next analyzed the effect of HDACi on each of the
pancreatic endocrine cell types. They first focused on expression
of Arx, known to support an .alpha. and PP cell fate (8, 9, 11).
They observed a major increase in Arx expression with VPA or TSA
treatment at all stages of culture. At day 7, Arx expression was
enhanced around 3-fold with VPA and 11-fold with TSA. They next
used real-time PCR to monitor the expression of glucagon and PP
synthesized by .alpha. and PP cells, respectively. Both VPA and TSA
treatment produced a major increase in glucagon and PP expression
at all stages of culture. At day7, we observed a 5-fold and a
25-fold increase in glucagon expression and a 4-fold and a 6-fold
increase in PP expression with VPA and TSA, respectively.
Immunohistological analysis revealed a 4-fold increase in the
number of glucagon-expressing cells upon VPA or TSA treatment.
Similar results were found with MS275 and NaB.
[0138] Together, these results show that HDACi treatment promotes a
and PP lineage differentiation.
[0139] Opposing effects of VPA and TSA on the endocrine
.beta./.delta. lineage: The inventors used real-time PCR to monitor
expression of Pax4, known to support the .beta./.delta. cell fate
(54) over 7 days of culture. VPA produced a dramatic decrease in
Pax4 expression as early as day1, associated with a dramatic
decrease in insulin expression and almost full abolition of
somatostatin expression. Moreover, the expression of NeuroD1,
another Ngn3 target subsequently expressed in .delta. cells (26,
48) was significantly lower in VPA-treated pancreases. These
results were confirmed immunohistologically, with a major decrease
in the number of insulin+ cells at days 5 and 7. Even in tissues
cultured for 14 days with VPA, insulin expression remained
dramatically low. Few insulin+ cells were observed
immunohistochemically.
[0140] In contrast, TSA strongly activated Pax4 and NeuroD1
expression. However, no differences in insulin and somatostatin
expression were found after 7 days in culture either by real-time
PCR or by immunohistochemistry. Hence, the inventors extended the
culture period. After 11 days, TSA-treated pancreases developed
translucent buds at the periphery of the pancreas corresponding to
an outer mass of insulin+ cells. Quantification of insulin staining
showed that the .delta. cell mass had increased almost 2-fold under
such conditions. Such results were confirmed using real-time PCR:
insulin expression was increased 2-fold at day9, 3-fold at day11
and 5-fold at day14 compared with controls. Somatostatin expression
was also increased after 14 days in culture (data not shown),
demonstrating that the .beta./.delta. lineage was promoted by TSA.
Interestingly, MS275 showed results similar to those with VPA,
whereas NaB showed results similar to those with TSA.
[0141] Altogether, these results indicate that VPA suppresses the
.beta./.delta. lineage, whereas TSA enhances it.
[0142] The effects of HDACi treatment on pancreatic differentiation
are independent of proliferation and apoptosis: The observed
effects of HDACi on pancreatic development could be attributed to a
direct effect of HDAC inhibition on the differentiation program
itself or to an indirect effect on proliferation/apoptosis. First,
the inventors asked whether the massive increase in the number of
NGN3+ cells was accompanied by an increased proliferation of early
PDX1+ pancreatic precursor cells that would give rise to more NGN3+
endocrine progenitor cells (20). Thus, they cultured pancreases for
1 day with or without VPA and TSA and added BrdU to the medium
during the last hour of culture. No difference in BrdU
incorporation was seen. The percentage of PDX1+/BrdU+ was
30.4.+-.1.4% in control conditions, 29.3.+-.2.3% with VPA and
31.9.+-.1.4% in TSA-treated pancreases. They next determined
whether the greater number of NGN3+ cells was associated with
increased proliferation of such cells. After 3 days of culture,
very few NGN3/BrdU+ cells could be found in control conditions and
neither VPA nor TSA increased the proliferation of NGN3+ cells.
Given the observed increase in glucagon-expressing cells with HDACi
treatment, the inventors also tested for increased proliferation of
such cells. As for NGN3+ cells, they observed a low proliferative
rate for glucagon+ cells with no difference between controls and
5-day VPA or TSA. Finally using the TUNEL method, they found that
neither VPA nor TSA treatments modified the number of apoptotic
cells expressing amylase or insulin, indicating that the lower
number of amylase and insulin cells observed with VPA treatment was
not due to apoptosis.
[0143] These results show that HDACi treatment does not modify the
proliferation/apoptosis balance and further suggest that the
effects of HDACi treatment on pancreas differentiation are
direct.
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