U.S. patent application number 17/196063 was filed with the patent office on 2021-06-24 for maturation of mammalian hepatocytes.
This patent application is currently assigned to Takara Bio Europe AB. The applicant listed for this patent is Takara Bio Europe AB. Invention is credited to Barbara KUPPERS-MUNTHER.
Application Number | 20210189332 17/196063 |
Document ID | / |
Family ID | 1000005444148 |
Filed Date | 2021-06-24 |
United States Patent
Application |
20210189332 |
Kind Code |
A1 |
KUPPERS-MUNTHER; Barbara |
June 24, 2021 |
MATURATION OF MAMMALIAN HEPATOCYTES
Abstract
Directed differentiation and maturation of mammalian
hepatocytes, such as human hepatocytes. The hepatocyte obtained
show a phenotype which is more similar to that of primary
hepatocytes than previously shown. In particular, exposure of
mammalian hepatocytes, such as human hepatocytes, to at least one
maturation factor selected from the group consisting of Src kinase
inhibitors, vitamin D including precursors, metabolites and analogs
thereof, hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), PKC
inhibitors, and combinations thereof.
Inventors: |
KUPPERS-MUNTHER; Barbara;
(Goteborg, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Takara Bio Europe AB |
Goteborg |
|
SE |
|
|
Assignee: |
Takara Bio Europe AB
Goteborg
SE
|
Family ID: |
1000005444148 |
Appl. No.: |
17/196063 |
Filed: |
March 9, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
17066546 |
Oct 9, 2020 |
|
|
|
17196063 |
|
|
|
|
15578899 |
Dec 1, 2017 |
10913932 |
|
|
PCT/EP2016/062670 |
Jun 3, 2016 |
|
|
|
17066546 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2500/36 20130101;
C12N 2500/30 20130101; C12N 2501/02 20130101; C12N 2501/39
20130101; C12N 2501/237 20130101; C12N 2501/727 20130101; C12N
2501/06 20130101; C12N 2506/45 20130101; C12N 2501/11 20130101;
C12N 2501/12 20130101; C12N 2501/415 20130101; C12N 2501/405
20130101; C12N 2533/52 20130101; C12N 2501/385 20130101; C12N
2506/02 20130101; C12N 2501/999 20130101; C12N 2533/54 20130101;
C12N 2500/25 20130101; C12N 2500/38 20130101; C12N 2501/16
20130101; C12N 5/067 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 2015 |
DK |
PA201570345 |
Claims
1. A composition comprising: at least two maturation factors
selected from the group consisting of Src kinase inhibitors,
vitamin D including precursors, metabolites and analog thereof,
hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), PKC
inhibitors, and combinations thereof.
2. The composition according to claim 1, wherein said composition
comprises at least one Src kinase inhibitor and at least one
further maturation factor selected from the group consisting of,
vitamin D including precursors, metabolites and analog thereof,
hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), and PKC
inhibitors.
3. The composition according to claim 2, wherein said composition
comprises at least one Src kinase inhibitor selected from the group
consisting of PP1, PP2, 1-NA PP1, 1-NM-PP1, Src Inhibitor-1
(Src-11), Src Kinase Inhibitor I, Src Kinase Inhibitor II,
A-419529, A-770041, AZM 475271, bosutinib, CGP77675, Damnacanthal,
dasatinib, dasatinib monohydrate, ER 27319 maleate, Fingolimod
(FTY720), Geldanamycin, Herbimycin A, KB SRC 4, KX2-391, KX1-004,
Lavendustin A, Lavendustin C, LCK inhibitor 2, Lyn peptide
inhibitor, MLR-1023, MNS, N-Acetyl-O-phosphono-Tyr-Glu
Dipentylamide, N-Acetyl-O-phosphono-Tyr-Glu-Glu-Ile-Glu,
NVP-BHG712, PD 166285, PD173952, PD 180970, Piceatannol, pp60
c-src, quercetin, radicicol from Diheterospora chlamydosporia
solid, saracatinib, SU 6656, TC-S 7003, TG 100572, WH-4-023, ZM
306416, and combinations thereof.
4. The composition according to claim 2, wherein said at least one
Src kinase inhibitor is PP1.
5. The composition according to claim 2, comprises at least one
vitamin D, vitamin D precursor, vitamin D metabolite or vitamin D
analog.
6. The composition according to claim 5, wherein said vitamin D is
a vitamin D3, selected from the group consisting of
cholecalciferol, calcifediol, calcitriol, and combinations
thereof.
7. The composition according to claim 2, wherein said composition
comprises at least one hypoxia inducing compound selected from the
group consisting of RAR-related orphan receptor alpha (ROR-alpha)
ligand, selected from the group consisting of CGP52608, CGP52608
analogs, melatonin, melatonin analogs, cholesterol, cholesterol
derivatives, and combinations thereof, CoCl.sub.2, and
NaN.sub.3.
8. The composition according to claim 2, wherein said composition
comprises a sphingosine or sphingosine derivative.
9. The composition according to claim 8, wherein said sphingosine
is D-erythro-sphingosine.
10. The composition according to claim 2, wherein said composition
comprises at least one activator of peroxisome
proliferator-activated receptors (PPARs), preferably selected from
the group consisting of thiazolidinediones, free fatty acids
(FFAs), eicosanoids including eicosanoid precursors and eicosanoid
analog, and combinations thereof.
11. The composition according to claim 10, wherein said free fatty
acids (FFAs) are selected from the group consisting of dodecanoic
acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid,
hexadecanoic acid, heptadecanoic acid, eicosanoic acid,
heneicosanoic acid, docosanoic acid, tricosanoic acid,
tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, and
combinations thereof.
12. The composition according to claim 10, wherein said eicosanoid,
eicosanoid precursor or eicosanoid analog are selected from the
group consisting of Diacylglycerol, Eicosapentaenoic acid,
Dihomo-gamma-linolenic acid, Arachidonic acid, ETYA
(5,8,11,14-eicosatetraynoic acid), members of the
hydroxyeicosatetraenoic acid (HETE) family, including 5-HETE and
15-HETE, members of the hydroxyoctadecadieonic acid (HODE) family,
including 9-HODE and 13-HODE, classic eicosanoids, and non-classic
eicosanoids.
13. The composition according to claim 2, wherein said composition
comprises at least one platelet-activating factor (PAF).
14. The composition according to claim 2, wherein said composition
comprises at least one PKC inhibitor, preferably selected from the
group consisting of Bisindolylmaleimide I, Bisindolylmaleimide II,
Bisindolylmaleimide III, Bisindolylmaleimide V, Bisindolylmaleimide
VI, Bisindolylmaleimide VII, Bisindolylmaleimide VIII,
Bisindolylmaleimide X, HBDDE, Rottlerin, Palmitoyl-DL-carnitine,
R-Stearoyl Carnitine Chloride, Piceatannol, H-9, H-8,
1-(5-lsoquinolinesulfonyl)-3-methylpiperazine, HA-100
dihydrochloride, HA-1004, HA-1077, 5-lodotubericidin, Ro-32-0432,
Ro-31-7549, Enzastaurin (LY317615), Sotrastaurin, Dequalinium
Chloride, Go 6976, Go 6983, Go 7874, Myricitrin,
4-Hydroxy-Tamoxifen, N-Desmethyltamoxifen HCl, Safingol, Phloretin,
UCN-01, 7-Oxostaurosporine, K-252a, K-252b, K-252c, Melittin,
Hispidin, Calphostin C, Ellagic acid, PKC Inhibitor Peptide 19-31,
PKC Inhibitor Peptide 19-36, PKC epsilon Translocation Inhibitor
II, EGF-R Fragment 651-658, PKC beta inhibitor (CAS 257879-35-9),
PKC 20-28, PKCpII/EGFR Inhibitor (CAS 145915-60-2), PKC6
Pseudosubstrate Inhibitor, PKC6/5 Inhibitor, [Ala107]-MBP
(104-118), [Ala113]-MBP (104-118), ZIP, C-1, Bryostatin 1, LY
333531 hydrochloride, CGP 53353, Chelerythrine Chloride, TCS 21311,
CID 755673, Gossypol, ET-18-OCH3,
1-O-Hexadecyl-2-O-methyl-rac-glycerol, NPC-15437 dihydrochloride,
NGIC-I, MDL-27,032, DAPH-7, 7-Aminoindole, 5-Amino-2-methylindole,
rac-2-Methoxy-3-hexadecanamido-1-propylphosphocholine,
Copperbis-3,5-diisopropylsalicylate,D,L-3,4-Dihydroxymandelic Acid,
rac-3-Octadecanamido-2-Methoxypropan-1-ol Phosphocholine, KRIBB3,
Ilmofosine, rac-2-Methoxy-3-hexadecanamido-1-propylphosphocholine,
and combinations thereof.
15. A culture medium comprising the composition according to claim
2.
16. A kit comprising the composition according to claim 2,
optionally further comprising at least one extracellular matrix
(ECM) component or ECM component mixture.
17. A kit comprising the culture medium according to claim 14,
optionally further comprising at least one extracellular matrix
(ECM) component or ECM component mixture.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 17/066,546, filed on Oct. 9, 2020, which is a
continuation of U.S. application Ser. No. 15/578,899, filed on Dec.
1, 2017, which is a U.S. National Stage of International
Application No. PCT/EP2016/062670, filed on Jun. 3, 2016, which
claims the benefit of Danish Application No. PA 2015-70345, filed
on Jun. 3, 2015. The entire contents of each of U.S. application
Ser. No. 17/066,546, U.S. application Ser. No. 15/578,899,
International Application No. PCT/EP2016/062670, and Danish
Application No. PA 2015-70345 are hereby incorporated herein by
reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to directed differentiation
and maturation of mammalian hepatocytes, such as human hepatocytes.
The hepatocyte obtained in accordance with the present invention
show a phenotype which is more similar to that of primary
hepatocytes than previously shown for stem cell derived
hepatocytes. In particular, the present invention relates to
exposure of mammalian hepatocytes, such as human hepatocytes, to at
least one maturation factor selected from the group consisting of
Src kinase inhibitors, vitamin D including precursors, metabolites
and analogs thereof, hypoxia inducing compounds, sphingosine and
sphingosine derivatives, activators of peroxisome
proliferator-activated receptors (PPARs), platelet-activating
factor (PAF), PKC inhibitors, and combinations thereof. The
inventors have, as disclosed herein, found that exposing mammalian
hepatocytes, such as human hepatocytes, to at least one maturation
factor as disclosed herein leads to the development of more mature
and functional features for the hepatocytes, compared to currently
available state of the art methods.
BACKGROUND OF THE INVENTION
[0003] The development of novel pharmaceuticals faces a number of
challenges, not least the problem of overcoming adverse
toxicological effects. Indeed, adverse liver reactions remain the
most prominent side effect. Metabolism and ultimate clearance of
the majority of small molecule drugs occurs in the liver, and thus
one of the main areas of focus in drug development concerns whether
such compounds or their metabolites possess any hepatotoxic effect.
Moreover, it is also of paramount importance to discover whether
the secondary metabolites of such compounds also display any
cytotoxic effects before the drug can begin clinical trial
programmes.
[0004] Accordingly there is an urgent need for a hepatic model
system that mimics mammalian liver cells, and especially human
liver cells, and that is able to predict effects of candidate
molecules in the development of new drugs or chemicals.
Traditionally, researchers have been forced to rely on primary
liver-derived hepatocytes for such screening but these have a
number of serious drawbacks including difficulty of maintaining the
cells in long term culture and difficulty of obtaining consistent,
homogeneous cell populations. A solution to this has been offered
in the form of hepatocytes derived from human pluripotent stem
cells.
[0005] Human pluripotent stem cells (hPS) have already begun to
revolutionise the ways in which relevant human cell types can be
obtained. The possibility to indefinitely propagate pluripotent
human embryonic-derived stem (hES) cells and human induced
pluripotent stem (hiPS) cells and subsequently differentiate them
into the desired target cell types is now providing a stable and
virtually unlimited supply of cells for a range of applications in
vivo and in vitro.
[0006] Unfortunately, currently available hepatocyte cell types do
not always accurately model the hepatic environment, due to
differences in morphology and function. For example, one often used
alternative to primary cells are hepatic cell lines which often
contain very low levels of (or totally lack) metabolising enzymes
and have expression of other important proteins substantially
different from the native hepatocyte in vivo. This is of particular
relevance in relation to drug metabolism since one of the major
deficiencies in hepatic cell lines is the absence or abnormally
high expression of drug transporter proteins which are essential
for drug screening purposes. Other available hepatic cell lines
suffer from having a morphology and physiology which is more
reminiscent of fetal or juvenile hepatocytes than the more
clinically relevant adult hepatocytes. For these reasons there is a
strong need to develop hepatocyte cell lines which are not only
easy to culture and propagate but which also possess a more mature
phenotype and which behave in a manner more akin to adult primary
hepatocytes.
[0007] Derivation of hepatocytes from pluripotent stem cells is
well established in the art. For in vitro purposes, several groups
have developed protocols for deriving hepatocytes from hES cells
(Hay et al., 2007; Hay et al., 2008; Brolen et al. 2010; Funakoshi
et al. 2011) as well from hiPS cells (U.S. Pat. No. 8,148,151B;
Song et al. 2009; Sullivan et al. 2010; Si-Tayeb et al. 2010; Chen
et al. 2012). However, common to all of these is a specific low
mRNA and protein expression of genes typical for mature
hepatocytes, like phase I and II genes (e.g. CYP1A2, 2B6, 2C9, 2D6,
3A4), nuclear receptors (e.g. CAR and PXR), and other adult hepatic
markers (e.g. Albumin). In addition, these hESC- and hiPSC-derived
hepatocytes have high expression of fetal hepatic genes like
.alpha.-fetoprotein (AFP) and CYP3A7, with the result that the cell
types described therein have a fetal and not adult phenotype (for
overview see e.g. Baxter et al. 2010). Furthermore, in most of the
published studies on hESC- and hiPSC-derived hepatocytes,
expression and functionality of drug transporters has not been
investigated at all.
[0008] It has recently been shown that exposure of hepatocytes to
an activator of a retinoic acid responsive receptor leads to the
development of more mature and functional features for the
hepatocytes as well as to more pure and homogenous populations of
hepatocytes (WO2014/083132).
[0009] However, there remains the need for further improving the
maturation of developing hepatocyte.
SUMMARY OF THE INVENTION
[0010] The above objective has been addressed by the present
inventors in that maturation factors have been identified which
improve the levels of mature hepatic markers such as CYP1A, CYP3A4,
CYP2C9, CYP2C19, CYP2B6 and CYP2D6.
[0011] The present invention thus provides inter alia improved
methods, compositions and kits by which mammalian hepatocytes, such
as human hepatocytes, derived from e.g. pluripotent stem (PS)
cells, may be further matured into hepatocytes possessing a
phenotype more closely resembling that of ex vivo primary liver
hepatocytes. More specifically, the present invention may be
summarized by the following items: [0012] 1. A method for promoting
the maturation of mammalian hepatocytes, such as human hepatocytes,
the method comprising: [0013] Exposing said hepatocytes to at least
one maturation factor selected from the group consisting of Src
kinase inhibitors, vitamin D including precursors, metabolites and
analogs thereof, hypoxia inducing compounds, sphingosine and
sphingosine derivatives, activators of peroxisome
proliferator-activated receptors (PPARs), platelet-activating
factor (PAF), PKC inhibitors, and combinations thereof. [0014] 2.
The method according to item 1, comprising culturing mammalian
hepatic progenitor cells under differentiation conditions to obtain
said hepatocytes. [0015] 3. A method for producing mammalian
hepatocytes, the method comprising: [0016] Culturing mammalian
hepatic progenitor cells under differentiation conditions to obtain
hepatocytes, and [0017] Exposing said hepatocytes to at least one
maturation factor selected from the group consisting of Src kinase
inhibitors, vitamin D including precursors, metabolites and analogs
thereof, hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), PKC
inhibitors, and combinations thereof. [0018] 4. The method
according to item 2 or 3, further comprising initially culturing
cells of the definitive endoderm (DE) under differentiation
conditions to obtain said hepatic progenitor cells. [0019] 5. The
method according to item 2 or 3, further comprising initially
culturing mammalian pluripotent stem (PS) cells under
differentiation conditions to obtain said hepatic progenitor cells.
[0020] 6. The method according to item 5, wherein the initial
culturing of PS cells includes culturing the mammalian PS cells
under differentiation conditions to obtain cells of the definitive
endoderm (DE cells) and further culturing the obtained cells under
differentiation conditions to obtain said hepatic progenitor cells.
[0021] 7. The method according to item 5 or 6, wherein said
mammalian pluripotent stem cells are embryonic stem (ES) cells.
[0022] 8. The method according to item 5 or 6, wherein said
mammalian pluripotent stem cells are artificial pluripotent stem
cells. [0023] 9. The method according to item 8, wherein said
artificial pluripotent stem cells are induced pluripotent stem
(iPS) cells. [0024] 10. The method according to any one of items 1
to 9, wherein said mammalian cells are human cells. [0025] 11. The
method according to any one of items 1 to 10, wherein said
mammalian hepatocytes are exposed to at least one Src kinase
inhibitor. [0026] 12. The method according to item 11, wherein said
mammalian hepatocytes are exposed to at least one Src kinase
inhibitor selected from the group consisting of PP1, PP2, 1-NA PP1,
1-NM-PP1, Src Inhibitor-1 (Src-11), Src Kinase Inhibitor I, Src
Kinase Inhibitor II, A-419529, A-770041, AZM 475271, bosutinib,
CGP77675, Damnacanthal, dasatinib, dasatinib monohydrate, ER 27319
maleate, Fingolimod (FTY720), Geldanamycin, Herbimycin A, KB SRC 4,
KX2-391, KX1-004, Lavendustin A, Lavendustin C, LCK inhibitor 2,
Lyn peptide inhibitor, MLR-1023, MNS, N-Acetyl-O-phosphono-Tyr-Glu
Dipentylamide, N-Acetyl-O-phosphono-Tyr-Glu-Glu-Ile-Glu,
NVP-BHG712, PD 166285, PD173952, PD 180970, Piceatannol, pp60
c-src, quercetin, radicicol from Diheterospora chlamydosporia
solid, saracatinib, SU 6656, TC-S 7003, TG 100572, WH-4-023, ZM
306416, and combinations thereof. [0027] 13. The method according
to item 11 or 12, wherein said mammalian hepatocytes are exposed to
PP1 or PP2. [0028] 14. The method according to any one of items 11
to 13, wherein said mammalian hepatocytes are exposed to said Src
kinase inhibitor at a concentration in the range of 0.05 to 50
.mu.M. [0029] 15. The method according to any one of items 1 to 14,
wherein said mammalian hepatocytes are exposed to at least one
vitamin D, vitamin D precursor, vitamin D metabolite or vitamin D
analog. [0030] 16. The method according to item 15, wherein said
mammalian hepatocytes are exposed to at least one vitamin D3,
vitamin D3 precursor, vitamin D3 metabolite or vitamin D3 analog.
[0031] 17. The method according to item 15 or 16, wherein said
mammalian hepatocytes are exposed to at least one vitamin D3
selected from the group consisting of cholecalciferol, calcifediol,
calcitriol, and combinations thereof. [0032] 18. The method
according to any one of items 15 to 17, wherein said mammalian
hepatocytes are exposed to cholecalciferol, calcitriol or a
combination thereof. [0033] 19. The method according to any one of
items 15 to 18, wherein said mammalian hepatocytes are exposed to
said at least one vitamin D, vitamin D precursor, vitamin D
metabolite or vitamin D analog at a concentration of 0.05 to 15
.mu.M. [0034] 20. The method according to any one of items 1 to 19,
wherein said mammalian hepatocytes are exposed to at least one
hypoxia inducing compound. [0035] 21. The method according to any
one of items 1 to 20, wherein said mammalian hepatocytes are
exposed to at least one hypoxia inducing compound selected from the
group consisting of retinoic acid receptor (RAR)-related orphan
receptor alpha (ROR-alpha) ligands, CoCl.sub.2, and NaN.sub.3.
[0036] 22. The method according to item 21, wherein said mammalian
hepatocytes are exposed to at least one RAR-related orphan receptor
alpha (ROR-alpha) ligand. [0037] 23. The method according to item
21 or 22, wherein said mammalian hepatocytes are exposed to at
least one RAR-related orphan receptor alpha (ROR-alpha) ligand
selected from the group consisting of CGP52608, a CGP52608 analog,
melatonin, melatonin analogs, cholesterol, cholesterol derivatives,
and combinations thereof. [0038] 24. The method according to item
22 or 23, wherein said mammalian hepatocytes are exposed to
CGP52608 or a CGP52608 analog. [0039] 25. The method according to
any one of items 22 to 24, wherein said mammalian hepatocytes are
exposed to said RAR-related orphan receptor alpha (ROR-alpha)
ligand at a concentration in the range of 0.05 to 50 .mu.M. [0040]
26. The method according to any one of items 1 to 25, wherein said
mammalian hepatocytes are exposed to at least one sphingosine or
sphingosine derivative. [0041] 27. The method according to item 26,
wherein said sphingosine is D-erythro-sphingosine. [0042] 28. The
method according to item 26, wherein said sphingosine derivative is
sphingosine-1-phosphate. [0043] 29. The method according to item
26, wherein said sphingosine derivative is a sphingolipid. [0044]
30. The method according to item 29, wherein said sphingolipid is a
ceramide or a ceramide analog. 31. The method according to any one
of items 26 to 30, wherein said mammalian hepatocytes are exposed
to a D-erythro-ceramide or an analog thereof. [0045] 32. The method
according to item 31, wherein said D-erythro-ceramide is
N-palmitoyl-D-erythro-sphingosine. [0046] 33. The method according
to item 30, wherein said ceramide analogue is L-erythro MAPP or
D-erythro MAPP. [0047] 34. The method according to any one of items
26 to 33, wherein said mammalian hepatocytes are exposed to said
sphingosine or sphingosine derivative at a concentration of 0.05 to
15 .mu.M. [0048] 35. The method according to any one of items 1 to
34, wherein said mammalian hepatocytes are exposed to at least one
activator of peroxisome proliferator-activated receptors (PPARs).
[0049] 36. The method according to item 35, wherein said mammalian
hepatocytes are exposed to at least one activator of peroxisome
proliferator-activated receptors (PPARs) selected from the group
consisting of thiazolidinediones, free fatty acids (FFAs),
eicosanoids including eicosanoid precursors and eicosanoid analog,
and combinations thereof. [0050] 37. The method according to item
35 or 36, wherein said mammalian hepatocytes are exposed to at
least one thiazolidinedione. [0051] 38. The method according to
item 37, wherein said mammalian hepatocytes are exposed to at least
one thiazolidinedione selected from the group consisting of
CGP52608, CGP52608 analogs, ciglitazone, rosiglitazone,
pioglitazone, lobeglitazone, troglitazone, TS5444, and combinations
thereof. [0052] 39. The method according to any one of items 35 to
38, wherein said mammalian hepatocytes are exposed to at least one
free fatty acid. [0053] 40. The method according to item 39,
wherein said at least one free fatty acid is a saturated fatty acid
selected from the group consisting of dodecanoic acid, tridecanoic
acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid,
heptadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic
acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid,
hexacosanoic acid, and combinations thereof. [0054] 41. The method
according to item 39 or 40, wherein said mammalian hepatocytes are
exposed to tetradecanoic acid. [0055] 42. The method according to
item 39, wherein said at least one free fatty acid is an
unsaturated fatty acid selected from the group consisting of
10Z-heptadecenoic acid, arachidonic acid (AA),
9(Z),11(E)-Conjugated Linoleic Acid, eicosadienoic acid,
eicosatrienoic acid (ETE), eicosapentaenoic acid (EPA),
docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), linoleic
acid, gamma-linolenic acid, dihomo-gamma-linolenic acid,
docosadiennoic acid, adrenic acid, mead acid, ricinoleic acid,
docosatrienoic acid, and combinations thereof. [0056] 43. The
method according to any one of items 39 to 42, wherein said
mammalian hepatocytes are exposed to 10Z-heptadecenoic acid. [0057]
44. The method according to any one of items 39 to 43, wherein said
mammalian hepatocytes are exposed to arachidonic acid (AA). [0058]
45. The method according to any one of items 39 to 44, wherein said
mammalian hepatocytes are exposed to Docosahexaenoic acid (DHA).
[0059] 46. The method according to any one of items 1 to 45,
wherein said mammalian hepatocytes are exposed to at least one
eicosanoid, eicosanoid precursor or eicosanoid analog. [0060] 47.
The method according to item 46, wherein said mammalian hepatocytes
are exposed to at least one eicosanoid, eicosanoid precursor or
eicosanoid analog selected from the group consisting of
Diacylglycerol, Eicosapentaenoic acid, Dihomo-gamma-linolenic acid,
Arachidonic acid, ETYA (5,8,11,14-eicosatetraynoic acid), members
of the hydroxyeicosatetraenoic acid (HETE) family, including 5-HETE
and 15-HETE, members of the hydroxyoctadecadieonic acid (HODE)
family, incuding 9-HODE and 13-HODE, classic eicosanoids, and
non-classic eicosanoids. [0061] 48. The method according to item 46
or 47, wherein said mammalian hepatocytes are exposed to at least
one classic eicosanoid selected from the group consisting of
prostaglandins, prostacyclines, leukotriens, eoxins, thromboxanes,
and analogs thereof. [0062] 49. The method according to item 48,
wherein said prostaglandins are selected from the group consisting
of pgd.sub.2, pgd.sub.3, pge.sub.1, pge.sub.2, pge.sub.3,
pgf.sub.1c, pgf.sub.2a, pgf.sub.3, and pgj.sub.2. [0063] 50. The
method according to item 48, wherein said prostacyclins are
selected from the group consisting of pgi.sub.2 and pgi.sub.3.
[0064] 51. The method according to item 48, wherein said
leukotriens are selected from the group consisting of Lta.sub.4,
Lta.sub.5, Ltb.sub.4, Ltb.sub.5, Ltc.sub.4, Ltc.sub.5, Ltd.sub.4,
Ltd.sub.5, Lte.sub.4, and Lte.sub.5. [0065] 52. The method
according to item 48, wherein said eoxins are selected from the
group consisting of 14,15-leukotriene A4, 14,15-leukotriene C4,
14,15-leukotriene D4, and 14,15-leukotriene E4. [0066] 53. The
method according to item 48, wherein said thromboxanes are selected
from the group consisting of Txa.sub.1, Txa.sub.2, and Txa.sub.3.
[0067] 54. The method according to any one of items 46 to 53,
wherein said mammalian hepatocytes are exposed to at least one
nonclassic eicosanoid selected from the group consisting of
endocannabinoids, hepoxilins, resolvins, isofurans, isoprastanes,
lipoxins, epi-lipoxins, epoxyeicosatrieonic acids (EETs). [0068]
55. The method according to item 54, wherein said endocannabionoids
are selected from the group consisting of anandamides, WIN55,
212-2, palmitylethanolamide, mead ethanolamid, R-mathandamide,
BML-190, N-arachidonylglycine, and arachidonamide. [0069] 56. The
method according to any one of items 35 to 55, wherein said
mammalian hepatocytes are exposed to said activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration of 0.05
to 15 .mu.M. [0070] 57. The method according to any one of items 1
to 56, wherein said mammalian hepatocytes are exposed to at least
one platelet-activating factor (PAF). [0071] 58. The method
according to item 57, wherein said mammalian hepatocytes are
exposed to said platelet-activating factor at a concentration of
0.05 to 50 .mu.M. [0072] 59. The method according to any one of
items 1 to 58, wherein said mammalian hepatocytes are exposed to at
least one protein kinase C (PKC) inhibitor. [0073] 60. The method
according to item 60, wherein said mammalian hepatocytes are
exposed to said PKC inhibitor at a concentration of 0.05 to 50
.mu.M. [0074] 61. The method according to any one of items 1 to 60,
comprising the exposure of said mammalian hepatocytes to a matrix
overlay. [0075] 62. The method according to item 61, wherein said
matrix overlay comprises fibronectin and collagen 1. [0076] 63. The
method according to item 62, wherein the concentration of
fibronectin is from 2 to 10 .mu.g/cm.sup.2 and the concentration of
collagen I is from 30 to 150 .mu.g/cm.sup.2. [0077] 64. The method
according to any one of items 2 to 63, wherein said mammalian
hepatic progenitor cells are exposed to said at least one
maturation factor and/or matrix overlay. [0078] 65. The method
according to any one of items 1 to 64, wherein the mammalian
hepatocytes obtained are for therapeutic use. [0079] 66. Use of at
least one maturation factor selected from the group maturation
factor selected from the group consisting of Src kinase inhibitors,
vitamin D including precursors, metabolites and analog thereof,
hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), PKC
inhibitors, and combinations thereof, for maturing mammalian
hepatocytes. [0080] 67. A composition comprising at least one
maturation factor selected from the group Src kinase inhibitors,
vitamin D including precursors, metabolites and analog thereof,
hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), PKC
inhibitors, and combinations thereof.
[0081] 68. The composition according to item 67, wherein said
composition comprises at least one Src kinase inhibitor. [0082] 69.
The composition according to item 68, wherein said at least one Src
kinase inhibitor is selected from the group consisting of PP1, PP2,
1-NA PP1, 1-NM-PP1, Src Inhibitor-1 (Src-11), Src Kinase Inhibitor
I, Src Kinase Inhibitor II, A-419529, A-770041, AZM 475271,
bosutinib, CGP77675, Damnacanthal, dasatinib, dasatinib
monohydrate, ER 27319 maleate, Fingolimod (FTY720), Geldanamycin,
Herbimycin A, KB SRC 4, KX2-391, KX1-004, Lavendustin A,
Lavendustin C, LCK inhibitor 2, Lyn peptide inhibitor, MLR-1023,
MNS, N-Acetyl-O-phosphono-Tyr-Glu Dipentylamide,
N-Acetyl-O-phosphono-Tyr-Glu-Glu-Ile-Glu, NVP-BHG712, PD 166285,
PD173952, PD 180970, Piceatannol, pp60 c-src, quercetin, radicicol
from Diheterospora chlamydosporia solid, saracatinib, SU 6656, TC-S
7003, TG 100572, WH-4-023, ZM 306416, and combinations thereof.
[0083] 70. The composition according to item 68 or 69, wherein said
at least one Src kinase inhibitor is PP1. [0084] 71. The
composition according to any one of items 68 to 70, wherein the
concentration of said at least one Src kinase inhibitor Is in the
range of 0.05 to 50 .mu.M. [0085] 72. The composition according to
item 71, wherein the concentration of said at least one Src kinase
inhibitor Is in the range of 0.1 to 10 .mu.M, such as 2.5 to 7.5
.mu.M. [0086] 73. The composition according to any one of items 67
to 72, wherein said composition comprises at least one vitamin D,
vitamin D precursor, vitamin D metabolite or vitamin D analog.
[0087] 74. The composition according to item 73, wherein said
vitamin D is a vitamin D3. [0088] 75. The composition according to
item 74, wherein said vitamin D3 is selected from cholecalciferol,
calcifediol, calcitriol, and combinations thereof. [0089] 76. The
composition according to any one of items 73 to 75, wherein the
concentration of said at least one vitamin D is in the range of
0.05 to 15 .mu.M. [0090] 77. The composition according to item 76,
wherein the concentration of said at least one vitamin D is in the
range of 0.1 to 10 .mu.M, such as in the range of 0.1 to 1 .mu.M.
[0091] 78. The composition according to any one of items 67 to 77,
wherein said composition comprises at least one hypoxia inducing
compound. [0092] 79. The composition according to item 78, wherein
said hypoxia inducing compound is at least one RAR-related orphan
receptor alpha (ROR-alpha) ligand. [0093] 80. The composition
according to item 79, wherein said at least one RAR-related orphan
receptor alpha (ROR-alpha) ligand is selected from the group
consisting of CGP52608, CGP52608 analogs, melatonin, melatonin
analogs, cholesterol, cholesterol derivatives, and combinations
thereof. [0094] 81. The composition according to item 79 or 80,
wherein said at least one RAR-related orphan receptor alpha
(ROR-alpha) ligand is CGP52608 or a CGP52608 analog. [0095] 82. The
composition according to any one of items 78 to 81, wherein the
concentration of said at least one hypoxia inducing compound is in
the range of 0.05 to 50 .mu.M. [0096] 83. The composition according
to item 82, wherein the concentration of said at leat one hypoxia
inducing compound is in the range of 0.1 to 10 .mu.M, such as in
the range of 2.5 to 7.5 .mu.M. [0097] 84. The composition according
to any one of items 67 to 83, wherein said composition comprises a
sphingosine or sphingosine derivative. [0098] 85. The composition
according to item 84, wherein said sphingosine is
D-erythro-sphingosine. [0099] 86. The composition according to item
85, wherein said sphingosine derivative is shingosine-1-phosphate.
[0100] 87. The composition according to item 85, wherein said
sphingosine derivative is a sphingolipid. [0101] 88. The
composition according to item 87, wherein said sphingolipid is a
ceramide or a ceramide analog. [0102] 89. The composition according
to item 88, wherein said ceramide is
N-palmitoyl-D-erythro-sphingosine. [0103] 90. The composition
according to item 88, wherein said ceramide analoge is L-erythro
MAPP or D-erythro MAPP. [0104] 91. The composition according to any
one of items 84 to 90, wherein the concentration of said a
sphingosine or sphingosine derivative is in the range of such as
0.05 to 15 .mu.M. [0105] 92. The composition according to item 91,
wherein the concentration of said a sphingosine or sphingosine
derivative is in the range of 0.1 to 10 .mu.M, such as in the range
of 0.1 to 1 .mu.M. [0106] 93. The composition according to any one
of items 67 to 92, wherein said composition comprises at least one
activator of peroxisome proliferator-activated receptors (PPARs).
[0107] 94. The composition according to item 93, wherein said at
least one activator of peroxisome proliferator-activated receptors
is selected from the group consisting of thiazolidinediones, free
fatty acids (FFAs), eicosanoids including eicosanoid precursors and
eicosanoid analog, and combinations thereof. [0108] 95. The
composition according to item 93 or 94, wherein said at least one
activator of peroxisome proliferator-activated receptors is at
least one unsaturated fatty acid selected from the group consisting
of 10Z-heptadecenoic acid, arachidonic acid (AA),
9(Z),11(E)-Conjugated Linoleic Acid, eicosadienoic acid,
eicosatrienoic acid (ETE), eicosapentaenoic acid (EPA),
docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), linoleic
acid, gamma-linolenic acid, dihomo-gamma-linolenic acid,
docosadiennoic acid, adrenic acid, mead acid, ricinoleic acid,
docosatrienoic acid, and combinations thereof. [0109] 96. The
composition according to any one of items 93 to 95, wherein said
composition comprises 10Z-heptadecenoic acid. [0110] 97. The
composition according to any one of items 93 to 96, wherein said
composition comprises arachidonic acid (AA). [0111] 98. The
composition according to any one of items 93 to 97, wherein said
composition comprises Docosahexaenoic acid (DHA). [0112] 99. The
composition according to any one of items 93 to 98, comprising
10Z-heptadecenoic acid and arachidonic acid (AA). [0113] 100. The
composition according to item 93 or 94, wherein said at least one
activator of peroxisome proliferator-activated receptors is at
least one saturated fatty acid selected from the group consisting
of dodecanoic acid, tridecanoic acid, tetradecanoic acid,
pentadecanoic acid, hexadecanoic acid, heptadecanoic acid,
eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic
acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid,
and combinations thereof. [0114] 101. The composition according to
any one of items 93 to 100, wherein said composition comprises
tetradecanoic acid. [0115] 102. The composition according to item
93 or 94, wherein said at least one activator of peroxisome
proliferator-activated receptors is at least one eicosanoid,
eicosanoid precursor or eicosanoid analog. [0116] 103. The
composition according to item 102, wherein said at least one
activator of peroxisome proliferator-activated receptors is at
least an eicosanoid, eicosanoid precursor or eicosanoid analog
selected from the group consisting of Diacylglycerol,
Eicosapentaenoic acid, Dihomo-gamma-linolenic acid, Arachidonic
acid, ETYA (5,8,11,14-eicosatetraynoic acid), members of the
hydroxyeicosatetraenoic acid (HETE) family, including 5-HETE and
15-HETE, members of the hydroxyoctadecadieonic acid (HODE) family,
incuding 9-HODE and 13-HODE, classic eicosanoids, and non-classic
eicosanoids. [0117] 104. The composition according to item 103,
wherein said classic eicosanoids are selected from the group
consisting of prostaglandins, prostacyclines, leukotriens, eoxins,
thromboxanes, and analogs thereof. [0118] 105. The composition
according to item 104, wherein said prostaglandins are selected
from the group consisting of pgd.sub.2, pgd.sub.3, pge.sub.1,
pge.sub.2, pge.sub.3, pgf.sub.1c, pgf.sub.2a, pgf.sub.3, and
pgj.sub.2. [0119] 106. The composition according to item 104,
wherein said prostacyclins are selected from the group consisting
of pgi.sub.2 and pgi.sub.3. [0120] 107. The composition according
to item 104, wherein said leukotriens are selected from the group
consisting of Lta.sub.4, Lta.sub.5, Ltb.sub.4, Ltb.sub.5,
Ltc.sub.4, Ltc.sub.5, Ltd.sub.4, Ltd.sub.5, Lte.sub.4, and
Lte.sub.5. [0121] 108. The composition according to item 104,
wherein said eoxins are selected from the group consisting of
14,15-leukotriene A4, 14,15-leukotriene C4, 14,15-leukotriene D4,
and 14,15-leukotriene E4. [0122] 109. The composition according to
item 104, wherein said thromboxanes are selected from the group
consisting of Txa.sub.1, Txa.sub.2, and Txa.sub.3. [0123] 110. The
composition according item 103, wherein said nonclassic eicosanoids
are selected from the group consisting of endocannabinoids,
hepoxilins, resolvins, isofurans, isoprastanes, lipoxins,
epi-lipoxins, epoxyeicosatrieonic acids (EETs). [0124] 111. The
composition according to item 110, wherein said endocannabionoids
are selected from the group consisting of anandamides, WIN55,
212-2, palmitylethanolamide, mead ethanolamid, R-mathandamide,
BML-190, N-arachidonylglycine, and arachidonamide. [0125] 112. The
composition according to any one of items 93 to 111, wherein the
concentration of said at least one activator of peroxisome
proliferator-activated receptors is in the range of 0.05 to 15
.mu.M. [0126] 113. The composition according to item 112, wherein
the concentration of said at least one activator of peroxisome
proliferator-activated receptors is in the range of 0.1 to 10
.mu.M, such as in the range of 0.1 to 1 .mu.M. [0127] 114. The
composition according to any one of items 67 to 113, wherein said
composition comprises at least one platelet-activating factor
(PAF). [0128] 115. The composition according to item 114, wherein
said at least one PAF is C16-PAF. [0129] 116. The composition
according to item 114 or 115, wherein the concentration of said at
least one platelet-activating factor (PAF) is in the range of 0.05
to 50 .mu.M. [0130] 117. The composition according to item 116,
wherein the concentration of said at least one platelet-activating
factor (PAF) is in the range of 0.1 to 10 .mu.M, such as in the
range of 2.5 to 7.5 .mu.M. [0131] 118. The composition according to
any one of items 67 to 117, wherein said composition comprises at
least one PKC inhibitor. [0132] 119. The composition according to
item 118, whereins said composition comprises at least one PKC
inhibitor selected from the group consisting of Bisindolylmaleimide
I, Bisindolylmaleimide II, Bisindolylmaleimide III,
Bisindolylmaleimide V, Bisindolylmaleimide VI, Bisindolylmaleimide
VII, Bisindolylmaleimide VIII, Bisindolylmaleimide X, HBDDE,
Rottlerin, Palmitoyl-DL-carnitine, R-Stearoyl Carnitine Chloride,
Piceatannol, H-9, H-8,
1-(5-Isoquinolinesulfonyl)-3-methylpiperazine, HA-100
dihydrochloride, HA-1004, HA-1077, 5-lodotubericidin, Ro-32-0432,
Ro-31-7549, Enzastaurin (LY317615), Sotrastaurin, Dequalinium
Chloride, Go 6976, Go 6983, Go 7874, Myricitrin,
4-Hydroxy-Tamoxifen,N-Desmethyltamoxifen HCl, Safingol, Phloretin,
UCN-01, 7-Oxostaurosporine, K-252a, K-252b, K-252c, Melittin,
Hispidin, Calphostin C, Ellagic acid, PKC Inhibitor Peptide 19-31,
PKC Inhibitor Peptide 19-36, PKC epsilon Translocation Inhibitor
II, EGF-R Fragment 651-658, PKC beta inhibitor (CAS 257879-35-9),
PKC 20-28, PKCpII/EGFR Inhibitor (CAS 145915-60-2), PKC6
Pseudosubstrate Inhibitor, PKC.theta./.delta. Inhibitor,
[Ala107]-MBP (104-118), [Ala113]-MBP (104-118), ZIP, C-1,
Bryostatin 1, LY 333531 hydrochloride, CGP 53353, Chelerythrine
Chloride, TCS 21311, CID 755673, Gossypol, ET-18-OCH3,
1-O-Hexadecyl-2-O-methyl-rac-glycerol, NPC-15437 dihydrochloride,
NGIC-I, MDL-27.032, DAPH-7, 7-Aminoindole, 5-Amino-2-methylindole,
rac-2-Methoxy-3-hexadecanamido-1-propylphosphocholine, Copper
bis-3,5-diisopropylsalicylate, D,L-3,4-Dihydroxymandelic Acid,
rac-3-Octadecanamido-2-Methoxypropan-1-ol Phosphocholine, KRIBB3,
Ilmofosine, rac-2-Methoxy-3-hexadecanamido-1-propylphosphocholine,
and combinations thereof. [0133] 120. The composition according to
item 118 or 119, wherein the concentration of said at least one PKC
inhibitor is in the range of 0.01 to 50 .mu.M. [0134] 121. The
composition according to item 120, wherein the concentration of
said at least one PKC inhibitor is in the range of about 0.5 to
about 10 .mu.M. [0135] 122. The composition according to any one of
items 67 to 121, comprising PP1, CGP52608, 10Z-heptadecenoic acid,
arachidonic acid (AA), cholecalciferol, calcitriol and
D-erythro-sphingosine. [0136] 123. The composition according to any
one of items 67 to 122, wherein said composition comprises at least
one extracellular matrix (ECM) component or ECM component mixture.
[0137] 124. The composition according to item 123, wherein said at
least one extracellular matrix (ECM) component or ECM component
mixture is selected from collagen, such as collagen I, II, III, IV,
V or VI, fibronectin, elastin, chondroitin sulfate proteoglycan,
dermatan sulfate proteoglycan, heparin proteoglycan, heparan
sulfate proteoglycan, such as glypicans, syndecans or perlecans,
glycosaminoglycans, nidogen/entactin, laminins, biglycan, tenascin,
hyaluronans, and combinations thereof. [0138] 125. The composition
according to item 123 or 124, wherein the composition comprises
collagen I and fibronectin. [0139] 126. The composition according
to item 125, wherein the concentration of collagen I is in the
range of about 2 to about 150 .mu.g/cm.sup.2 culture area. [0140]
127. The composition according to item 125 or 126, wherein the
concentration of fibronectin is in the range of about 2 to about 30
.mu.g/cm.sup.2 culture area. [0141] 128. A culture medium
comprising the composition according to any one of items 67 to 127.
[0142] 129. A kit comprising at least one maturation factor
selected from the group Src kinase inhibitors, vitamin D including
precursors, metabolites and analog thereof, hypoxia inducing
compounds, sphingosine and sphingosine derivatives, activators of
peroxisome proliferator-activated receptors (PPARs),
platelet-activating factor (PAF), PKC inhibitors, and combinations
thereof. [0143] 130. The kit according to item 129, comprising the
composition according to any one of items 67 to 127 or the culture
medium according to item 128. [0144] 131. The kit according to item
129 or 130 comprising at least one extracellular matrix (ECM)
component or ECM component mixture.
DETAILED DESCRIPTION OF THE INVENTION
[0145] The invention provides methods for maturing mammalian
hepatocytes, such as human hepatocytes, by exposing the cells to at
least one maturation factor selected from the group Src kinase
inhibitors, vitamin D including precursors, metabolites and analog
thereof, hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), PKC
inhibitors, and combinations thereof.
[0146] The methods may further comprise culturing of mammalian
hepatic progenitor cells, such as human hepatic progenitor cells,
in a supportive culture and differentiation medium to obtain said
hepatocytes where the cells are exposed to at least one maturation
factor selected from the group Src kinase inhibitors, vitamin D
including precursors, metabolites and analogs thereof, hypoxia
inducing compounds, sphingosine and sphingosine derivatives,
activators of peroxisome proliferator-activated receptors (PPARs),
platelet-activating factor (PAF), PKC inhibitors, and combinations
thereof.
[0147] The method for promoting the maturation of mammalian
hepatocytes, such as human hepatocytes, may thus be described as
comprising the step: [0148] Exposing said mammalian hepatocytes,
such as said human hepatocytes, to at least one maturation factor
selected from the group Src kinase inhibitors, vitamin D including
precursors, metabolites and analogs thereof, hypoxia inducing
compounds, sphingosine and sphingosine derivatives, activators of
peroxisome proliferator-activated receptors (PPARs),
platelet-activating factor (PAF), PKC inhibitors, and combinations
thereof.
[0149] The method for promoting the maturation of human hepatocytes
may further comprise the step of culturing mammalian hepatic
progenitor cells, such as human hepatic progenitor cells, under
differentiation conditions to obtain said hepatocytes.
[0150] The present invention also provides a method for producing
mammalian hepatocytes, such as human hepatocytes, whereby mammalian
hepatic progenitor cells, such as human hepatic progenitor cells,
are cultured under differentiation conditions to obtain
hepatocytes, and the obtained hepatocytes are exposed to at least
one maturation factor selected from the group Src kinase
inhibitors, vitamin D including precursors, metabolites and analogs
thereof, hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), PKC
inhibitors, and combinations thereof.
[0151] The method for producing mammalian hepatocytes, such as
human hepatocytes, may thus be described as comprising the
following steps: [0152] Culturing mammalian hepatic progenitor
cells, such as human hepatic progenitor cells, under
differentiation conditions to obtain hepatocytes, and [0153]
Exposing said hepatocytes to at least one maturation factor
selected from the group consisting of Src kinase inhibitors,
vitamin D including precursors, metabolites and analogs thereof,
hypoxia inducing compounds, sphingosine and sphingosine
derivatives, activators of peroxisome proliferator-activated
receptors (PPARs), platelet-activating factor (PAF), PKC
inhibitors, and combinations thereof.
[0154] Mammalian hepatic progenitor cells, such as human hepatic
progenitor cells, may thus be used as starting material according
to the invention. The hepatic progenitor starting material may, for
example, be an established cell line of hepatic progenitor cells,
hepatic progenitor cells de novo isolated from livers, such as
human livers, or may be prepared de novo, such as from mammalian
pluripotent stem (PS) cells, such as human pluripotent stem (hPS)
cells or mammalian definitive endoderm (DE) cells, such as human
definitive endoderm (DE) cells.
[0155] The differentiation and maturation of hepatocytes cells may
be divided into two phases, i.e. a first phase where the hepatic
progenitor cells differentiate into hepatocytes ("hepatic
progenitor phase"), and a second phase where the obtained
hepatocytes further mature (maturation phase). During the
maturation phase the obtained hepatocytes exhibit an increased gene
and protein expression of characteristic markers for mature
hepatocytes.
[0156] Suitable conditions for differentiating hepatic progenitor
cells into hepatocytes from human embryonic stem (hES) cells (Hay
et al., 2007; Hay et al., 2008; Brolen et al. 2010; Funakoshi et
al. 2011) and from human induced pluripotent stem (hiPS) cells
(U.S. Pat. No. 8,148,151B; Song et al. 2009; Sullivan et al. 2010;
Si-Tayeb et al. 2010; Chen et al. 2012) are known. WO 2009/013254
A1, for example, describes suitable basic protocols to obtain
hepatocytes from hepatic progenitor cells (Embodiments 1 to 4).
[0157] Generally, hepatic progenitor cells are cultured in a
differentiation medium comprising one or more growth factors, such
as HGF, and/or one or more differentiation inducer, such as
dimethylsulfoxide (DMSO), dexamethazone (DexM), omeprazole,
Oncostatin M (OSM), rifampicin, desoxyphenobarbital, ethanol or
isoniazide. The concentration of the one or more growth factors,
such as HGF, is usually in the range of about 10 to about 50 ng/ml,
such as about 10 to about 30 ng/ml. The concentration of the one or
more differentiation inducer may vary depending on the particular
compound used. The concentration of DMSO, for example, is usually
in the range of about 0.1 to about 1% v/v, such as about 0.25 to
about 0.75% v/v. The concentration of OSM, for example, is usually
in the range of about 1 to about 20 ng/ml, such as about 5 to about
15 ng/ml. The concentration of DexM, for example, is usually in the
range of about 0.05 to about 1 .mu.M, such as about 0.05 to about
0.2 .mu.M.
[0158] The differentiation medium may further comprise an albumin
source, such as FBS, FCS or BSA. The concentration of the albumin
source, if present, is usually in the range of about 0.1 to about
5% v/v, such as about 0.1 to about 1%, 0.2 to 3% v/v, about 0.5 to
about 2.5% v/v, about 0.5 to 1% v/v or about 1 to about 2.5%
v/v.
[0159] The differentiation medium may further comprise ascorbic
acid. The concentration of ascorbic acid, if present, is usually in
the range of about 0.01 to about 0.1 mg/ml, such as about 0.1 to
about 0.05 mg/ml.
[0160] The differentiation medium may further comprise
Hydrocortisone Hemisuccinate. The concentration of Hydrocortisone
Hemisuccinate, if present, is usually in the range of about 0.1 to
about 1 .mu.g/ml, such as about 0.5 to 0.8 .mu.g/ml.
[0161] The differentiation medium may further comprise transferrin.
The concentration of transferrin, if present, is usually in the
range of about 1 to 20 .mu.g/ml, such as about 5 to 15
.mu.g/ml.
[0162] The differentiation medium may further comprise Insulin. The
concentration of Insulin, if present, is usually in the range of
about 1 to about 10 .mu.g/ml, such as about 2.5 to about 7.5
.mu.g/ml.
[0163] The differentiation medium may further comprise epidermal
growth factor (EGF). The concentration of EGF, if present, is
usually in the range of about 0.001 to about 0.005 .mu.g/ml, such
as about 0.0025 to about 0.0035 .mu.g/ml.
[0164] The differentiation medium may further comprise other
supplements such as PEST and/or GlutaMAX. The concentration of PEST
is usually in the range of about 0.1 to about 0.5% v/v, such as
about 0.1 to about 0.25% v/v. The concentration of GlutaMAX is
usually in the range of about 0.5 to about 1.5% v/v, such as about
0.75 to 1.25% v/v, e.g. about 1% v/v.
[0165] The differentiation medium may further comprise at least one
activator of a retinoic acid responsive receptor, i.e. a compound
capable of binding to and activating a human retinoic acid receptor
(RAR) and/or human retinoid X receptor (RXR), such as, e.g., a
compound capable of binding to and activating both RAR and RXR. A
suitable activator of a retinoic acid responsive receptor for use
in the differentiation medium is retinoic acid, such as
9-cis-retinoic acid, 13-cis-retinoic acid or other retinoic acid
isomers, including all-trans-retinoic acid, 7-cis retinoic acid and
11-cis-retinoic acid, or an analogue of retinoic acid, such as
TTNPB, AM580, retilloic acid or CBS-211A, or a retinoid.
Accordingly, 9-cis-retinoic acid may be used as the activator of a
retinoic acid responsive receptor. Alternatively, or in addition,
13-cis-retinoic acid may also be used as the activator of a
retinoic acid responsive receptor. The concentration of the
activator of a retinoic acid responsive receptor, if present, is
usually in the range of about 0.1 to about 2.5 .mu.M, such as,
e.g., in the range of about 0.1 to about 0.5 .mu.M, such as, e.g.,
at about 0.2 .mu.M.
[0166] The differentiation medium may further comprise at least one
GSK-3 inhibitor and/or CDK inhibitor.
[0167] Suitable GSK-3 inhibitors for use in the invention are
9-Bromo-7,12-dihydro-indolo [3,2-d]-25 [1]benzazepin-6(5H)-one,
also known as Kenpaullone or NSC 664704; 1-Aza-Kenpaullone
(9-Bromo-7,12-dihydro-pyrido[3',2':2,3]azepino[4,5-b]indol-6(5H)-one);
Alsterpaullone
(9-Nitro-7,12-dihydroindolo-[3,2-d][1]benzazepin-6(5)-one);
4-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
also known as AT-7519;
N-(5-((5-tert-butyloxazol-2-yl)methylthio)thiazol-2-yl)piperidine-4-carbo-
xamide also known as SNS-032 (BMS-387032);
4-(1-isopropyl-2-methyl-1H-imidazol-5-yl)-N-(4-(methylsulfonyl)phenyl)pyr-
imidin-2-amine also known as AZD5438;
(2'Z,3')-6-Bromoindirubin-3'-oxime, also known as BIO (GSK3
Inhibitor IX); (2'Z,3'E)-6-Bromoindirubin-3'-acetoxime, also known
as BIO-Acetoxime (GSK3 Inhibitor X);
(5-Methyl-IH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine
(GSK3-Inhibitor XIII); Pyridocarbazole-cyclopenadienylruthenium
complex (GSK3 Inhibitor XV); TDZD-8
4-Benzyl-2-methyl-I,2,4-thiadiazolidine-3,5-dione (GSK3beta
Inhibitor 1); 2-Thio(3-iodobenzyl)-5-(I-pyridyl)-[I,3,4]-oxadiazole
(GSK3beta Inhibitor II); OTDZT
2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3beta Inhibitor
III); alpha-4-Dibromoacetophenone (GSK3beta Inhibitor VII);
N-(4-Methoxybenzyl)-N'-(5-nitro-I,3-thiazol-2-yl)urea, also known
as AR-AO 14418 (GSK-3beta Inhibitor VIII);
3-(I-(3-Hydroxypropyl)-IH-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrr-
ole-2,5-dione (GSK-3beta Inhibitor XI); TWSI 19 pyrrolopyrimidine
compound (GSK3beta Inhibitor XII); L803 H-KEAPPAPPQSpP-NH2 or its
myristoylated form (GSK3beta Inhibitor XIII);
2-Chloro-I-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3beta Inhibitor
VI); Aminopyrimidine CHIR99021;
3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione,
also known as SB216763; Indirubin-3'-monoxime; 3F8
(5-Ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]-isoquinoline-1,3-(2H)-dione),
A1070722, anorganic ions like Beryllium, Copper, Lithium, Mercury,
Tungstate (Wolfram), and Zinc, AR-A 014418, AZD2858, Axin GID-25
residues (peptide), bisindolylmaleimides, CHIR98014 (CT98014),
CHIR98023 (CT98023), FRATide-39 residues (peptide),
Halomethylketone derivatives, e.g. HMK-32, KT5720, L803-mts
(peptide) and variants, LY20900314, NP-12 (Tideglusib, NP031112),
NP00111, NP031115, Polyoxygenated bis-7-azaindolyl-maleimides,
R031-8220, SB415286 (maleimide), TC-G24, TCS2002, TCS21311, TDZD-8,
TOS119 and TWS119 (difluoroacetate). The GSK-3 inhibitor may, for
instance, be one chosen from Kenpaullone, 1-Aza-Kenpaullone,
Alsterpaullone, Aminopyrimidine CHIR99021 and
Indirubin-3'-monoxime.
[0168] Suitable CDK inhibitors for use in the invention are
9-Bromo-7,12-dihydro-indolo [3,2-d]-[1]benzazepin-6(5H)-one, also
known as Kenpaullone or NSC 664704;
(R)-2-(6-(benzylamino)-9-isopropyl-9H-purin-2-ylamino)butan-1-ol
also known as Roscovitine;
2-(2-chlorophenyl)-5,7-dihydroxy-8-((3S,4R)-3-hydroxy-1-methylpiperidin-4-
-yl)-4H-chromen-4-one also known as Flavopiridol;
4-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide
also known as AT-7519;
6-acetyl-8-cyclopentyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)py-
rido[2,3-d]pyrimidin-7(8H)-one hydrochloride also known as PD
0332991 HCl;
N-(5-((5-tert-butyloxazol-2-yl)methylthio)thiazol-2-yl)piperidine-4-carbo-
xamide also known as SNS-032 (BMS-387032); JNJ-7706621;
N-(6,6-dimethyl-5-(1-methylpiperidine-4-carbonyl)-1,4,5,6-tetrahydropyrro-
o[3,4-c]pyrazol-3-yl)-3-methylbutanamide also known as PHA-793887;
Dinaciclib (SCH727965);
(4-butoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)(2,6-difluoro-4-methylphenyl)met-
hanone also known as BMS-265246;
N,1,4,4-tetramethyl-8-(4-(4-methylpiperazin-1-yl)phenylamino)-4,5-dihydro-
-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide also known as
PHA-848125;
2-(pyridin-4-yl)-6,7-dihydro-1H-pyrrolo[3,2-c]pyridin-4(5H)-one
also known as PHA-767491; SCH 900776;
2-(2-chlorophenyl)-5,7-dihydroxy-8-((3S,4R)-3-hydroxy-1-methylpiperidin-4-
-yl)-4H-chromen-4-one hydrochloride also known as Flavopiridol HCl;
(4-amino-2-(1-(methylsulfonyl)piperidin-4-ylamino)pyrimidin-5-yl)(2,3-dif-
luoro-6-methoxyphenyl)methanone also known as R547;
(2S)-1-(5-(3-methyl-1H-indazol-5-yl)pyridin-3-yloxy)-3-phenylpropan-2-ami-
ne also known as A-674563;
4-(1-isopropyl-2-methyl-1H-imidazol-5-yl)-N-(4-(methylsulfonyl)phenyl)pyr-
imidin-2-amine also known as AZD5438;
N5-(6-aminohexyl)-N7-benzyl-3-isopropylpyrazolo[1,5-a]pyrimidine-5,7-diam-
ine hydrochloride also known as BS-181 HCl; CY-202; AG-024322;
P276-00; ZK 304709; GPC-286199; and BAY 80-3000, 2-hydroxybohemine,
A674563, Aminopurvanolol, BAY1000394, BMS-265246, BS-181
Butyrolactone, CR8 S-isomer, Diaciclib (SCH727965), JNJ-7706621,
N9-isopropyl-olomoucine, NU6140, NU6102, Olomoucine II, Oxindole I,
P276-00, PD332991, PHA-793887, PHA-767491, PHA-848125, PNU112455A,
Purvanolol A and B, R547, (R)-DRF053 and SCH900776 (MK-8776). The
GSK-3 inhibitor may, for instance, be one chosen from Kenpaullone,
1-Aza-Kenpaullone, Indirubin-3'-monoxime, Alsterpaullone,
SNS-032(BMS-387032), AT-7519 and AZD5438.
[0169] The concentration of the GSK3 inhibitor and/or CDK
inhibitor, if present, is usually in the range of about 0.01 to
about 10 .mu.M. In case that, for instance, Kenpaullone is employed
as the CDK inhibitor, the hepatocytes may be exposed to it at a
concentration in the range of about 0.05 to about 5 .mu.M, such as,
e.g., in the range of about 0.5 to about 1.5 .mu.M. Similar
concentrations may be used in case that, for instance,
1-Aza-Kenpaullone or Alsterpaullone is used.
[0170] The culture medium forming the basis for the differentiation
medium may be any culture medium suitable for culturing mammalian
hepatic progenitor cells such as such as RPMI 1640 medium, RPMI
1640 advanced medium, Iscove's Modified Dulbeccos Medium (IMDM),
Minimum Essential Medium (e.g., MEM, EMEM or GMEM), Dulbecco's
Modified Eagle Medium (e.g., DMEM or DMEM/F-12), Ham's medium
(e.g., Ham's F12 or Ham's F10), HCM medium, HBM medium, or Williams
E medium. Thus, the base medium may, for example, be RPMI 1640
medium or RPMI 1640 advanced medium. Alternatively, the base medium
may be Williams E medium.
[0171] The differentiation of mammalian hepatic progenitor cells,
such as human hepatic progenitor cells, and further maturation of
the obtained hepatocytes ("differentiation and maturation") may
take up to 35 days in total. Thus, in order to obtain hepatocytes,
the mammalian hepatic progenitor cells, such as human hepatic
progenitor cells, are cultured in differentiation medium for up to
35 days. For example, the mammalian hepatic progenitor cells, such
as human hepatic progenitor cells, may be cultured in
differentiation medium for any time between about 7 to about 35
days. They may thus also be cultured for about 10 to about 30 days.
They may also be cultured for about 10 to about 25 days.
Alternatively, they may be cultured for about 10 to about 20 days
or for about 10 to about 15 days. They may also be cultured for
about 15 to about 35 days. Thus, they may also be cultured for
about to about 30 days. Alternatively, they may be cultured for
about 15 to about 25 days. They may also be cultured for about 15
to about 20 days. During the culturing the differentiation medium
is usually exchanged for fresh medium every second or third
day.
[0172] Under the above described conditions, hepatocytes are
obtained from hepatic progenitor cells on or after 7 days of
culture. Thus, the differentiation and maturation of hepatocytes
may be divided into a hepatic progenitor phase of 7 days, whereby
hepatic progenitor cells differentiate into hepatocytes, and a
maturation phase lasting until the end of the total culture period
(e.g., until day 35), whereby the obtained hepatocytes further
mature.
[0173] The at least one maturation factor employed in the methods
of the invention may by any compound selected from the group
consisting of Src kinase inhibitors, vitamin D including
precursors, metabolites and analogs thereof, hypoxia inducing
compounds, sphingosine and sphingosine derivatives, activators of
peroxisome proliferator-activated receptors (PPARs),
platelet-activating factor (PAF), PKC inhibitors, and combinations
thereof.
[0174] Thus, the at least one maturation factor employed in the
methods of the invention may be at least one Src kinase inhibitor,
such as at least one (such as at least two) Src kinase inhibitor(s)
selected from the group consisting of PP1
(1-(1,1-Dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-a-
mine), PP2 (3-(4-chlorophenyl)
1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine), 1-NA
PP1 (1-Naphthyl PP1;
1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-am-
ine), 1-NM-PP1
(1-(1,1-dimethylethyl)-3-(1-naphthalenylmethyl)-1H-pyrazolo[3,4-d]pyrimid-
in-4-amine), Src Inhibitor-1 (Src-11;
6,7-Dimethoxy-N-(4-phenoxyphenyl)-4-quinazolinamine), Src Kinase
Inhibitor I (CAS 179248-59-0), Src Kinase Inhibitor II (CAS
459848-35-2), A-419529, A-770041, AZM 475271, bosutinib, CGP77675,
Damnacanthal, dasatinib, dasatinib monohydrate, ER 27319 maleate,
Fingolimod (FTY720), Geldanamycin, Herbimycin A, KB SRC 4, KX2-391,
KX1-004, Lavendustin A, Lavendustin C, LCK inhibitor 2, Lyn peptide
inhibitor, MLR-1023, MNS, N-Acetyl-O-phosphono-Tyr-Glu
Dipentylamide, N-Acetyl-O-phosphono-Tyr-Glu-Glu-Ile-Glu,
NVP-BHG712, PD 166285, PD173952, PD 180970, Piceatannol, pp60
c-src, quercetin, radicicol from Diheterospora chlamydosporia
solid, saracatinib, SU 6656, TC-S 7003, TG 100572, WH-4-023, ZM
306416, and combinations thereof.
[0175] Accordingly, the mammalian hepatocytes may be exposed to at
least PP1. The mammalian hepatocytes may be exposed to at least
PP2. The mammalian hepatocytes may be exposed to at least 1-NA PP1.
The mammalian hepatocytes may be exposed to at least 1-NM-PP1. The
mammalian hepatocytes may be exposed to at least Src Inhibitor-1.
The mammalian hepatocytes may be exposed to at least Src Kinase
Inhibitor I (CAS 179248-59-0). The mammalian hepatocytes may be
exposed to at least Src Kinase Inhibitor II (CAS 459848-35-2). The
mammalian hepatocytes may be exposed to at least A-419529. The
mammalian hepatocytes may be exposed to at least A-770041. The
mammalian hepatocytes may be exposed to at least AZM 475271. The
mammalian hepatocytes may be exposed to at least bosutinib. The
mammalian hepatocytes may be exposed to at least CGP77675. The
mammalian hepatocytes may be exposed to at least Damnacanthal. The
mammalian hepatocytes may be exposed to at least dasatinib. The
mammalian hepatocytes may be exposed to at least dasatinib
monohydrate. The mammalian hepatocytes may be exposed to at least
ER 27319 maleate. The mammalian hepatocytes may be exposed to at
least Fingolimod (FTY720). The mammalian hepatocytes may be exposed
to at least Geldanamycin. The mammalian hepatocytes may be exposed
to at least Herbimycin A. The mammalian hepatocytes may be exposed
to at least KB SRC 4. The mammalian hepatocytes may be exposed to
at least KX2-391. The mammalian hepatocytes may be exposed to at
least KX1-004. The mammalian hepatocytes may be exposed to at least
Lavendustin A. The mammalian hepatocytes may be exposed to at least
Lavendustin C. The mammalian hepatocytes may be exposed to at least
LCK inhibitor 2. The mammalian hepatocytes may be exposed to at
least Lyn peptide inhibitor. The mammalian hepatocytes may be
exposed to at least MLR-1023. The mammalian hepatocytes may be
exposed to at least MNS, N-Acetyl-O-phosphono-Tyr-Glu
Dipentylamide. The mammalian hepatocytes may be exposed to at least
N-Acetyl-O-phosphono-Tyr-Glu-Glu-Ile-Glu. The mammalian hepatocytes
may be exposed to at least NVP-BHG712. The mammalian hepatocytes
may be exposed to at least PD 166285. The mammalian hepatocytes may
be exposed to at least PD173952. The mammalian hepatocytes may be
exposed to at least PD 180970. The mammalian hepatocytes may be
exposed to at least Piceatannol. The mammalian hepatocytes may be
exposed to at least pp60 c-src. The mammalian hepatocytes may be
exposed to at least quercetin. The mammalian hepatocytes may be
exposed to at least radicicol from Diheterospora chlamydosporia
solid. The mammalian hepatocytes may be exposed to at least
saracatinib. The mammalian hepatocytes may be exposed to at least
SU 6656. The mammalian hepatocytes may be exposed to at least TC-S
7003. The mammalian hepatocytes may be exposed to at least TG
100572. The mammalian hepatocytes may be exposed to at least
WH-4-023. The mammalian hepatocytes may be exposed to at least ZM
306416.
[0176] The mammalian hepatocytes may be exposed to any combinations
of Src kinase inhibitors, such as any combination of the
afore-mentioned compounds. For example, the mammalian hepatocytes
may be exposed to at least PP1 and PP2.
[0177] Generally, the concentration of said at least one Src kinase
inhibitor, when employed in accordance with the present invention,
is in the range of about 0.05 to about 50 .mu.M, such as, e.g., in
the range of about 0.5 to about 10 .mu.M.
[0178] The mammalian hepatocytes, such as human hepatocytes, may
thus be exposed to said at least one Src kinase inhibitor at a
concentration in the range of about 0.05 to about 25 .mu.M. The
mammalian hepatocytes may be exposed to said at least one Src
kinase inhibitor at a concentration in the range of about 0.05 to
about 15 .mu.M. The mammalian hepatocytes may be exposed to said at
least one Src kinase inhibitor at a concentration in the range of
about 0.05 to about 10 .mu.M. The mammalian hepatocytes may be
exposed to said at least one Src kinase inhibitor at a
concentration in the range of about 0.05 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one Src
kinase inhibitor at a concentration in the range of about 0.1 to
about 25 .mu.M. The mammalian hepatocytes may be exposed to said at
least one Src kinase inhibitor at a concentration in the range of
about 0.1 to about 15 .mu.M. The mammalian hepatocytes may be
exposed to said at least one Src kinase inhibitor at a
concentration in the range of about 0.1 to about 15 .mu.M. The
mammalian hepatocytes may be exposed to said at least one Src
kinase inhibitor at a concentration in the range of about 0.1 to
about 10 .mu.M. The mammalian hepatocytes may be exposed to said at
least one Src kinase inhibitor at a concentration in the range of
about 0.1 to about 7.5 .mu.M. The mammalian hepatocytes may be
exposed said at least one Src kinase inhibitor at a concentration
in the range of about 0.5 to about 25 .mu.M. The mammalian
hepatocytes may be exposed to said at least one Src kinase
inhibitor at a concentration in the range of about 0.5 to about 15
.mu.M. The mammalian hepatocytes may be exposed to said at least
one Src kinase inhibitor at a concentration in the range of about
0.5 to about 10 .mu.M. The mammalian hepatocytes may be exposed to
said at least one Src kinase inhibitor at a concentration in the
range of about 0.5 to about 7.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one Src kinase inhibitor at a
concentration in the range of about 1 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least one Src
kinase inhibitor at a concentration in the range of about 1 to
about 7.5 .mu.M. The mammalian hepatocytes may be exposed to said
at least one Src kinase inhibitor at a concentration in the range
of about 1.5 to about 7.5 .mu.M. The mammalian hepatocytes may be
exposed to said at least one Src kinase inhibitor at a
concentration in the range of about 2.5 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one Src
kinase inhibitor at a concentration in the range of about 3.5 to
about 6.5 .mu.M. The mammalian hepatocytes may be exposed to said
at least one Src kinase inhibitor at a concentration in the range
of about 4 to about 6 .mu.M. The mammalian hepatocytes may be
exposed said at least one Src kinase inhibitor at a concentration
in the range of about 4.5 to about 5.5 .mu.M. For example, The
mammalian hepatocytes may be exposed to about 5 .mu.M of said at
least one Src kinase inhibitor.
[0179] In case that, for instance, PP1 is employed according to the
invention, it may be employed at a concentration in the range of
about 0.05 to about 50 .mu.M, such as, e.g., in the range of about
0.5 to about 10 .mu.M, such as, e.g., at about 5 .mu.M.
[0180] Similar concentrations may be used in case that, for
instance, PP2 is employed.
[0181] The at least one maturation factor employed in the methods
of the invention may also be at least one vitamin D, vitamin D
precursor, vitamin D metabolite or vitamin D analog, such as
vitamin D1, D2, D3, D4 or D5, including precursors, metabolites and
analogs thereof.
[0182] Accordingly, the mammalian hepatocytes may be exposed to at
least vitamin D3 (cholecalciferol), a vitamin D3 precursor (such as
7-dehydrocholesterol), a vitamin D3 metabolite (such as calcifediol
or calcitriol) and/or a vitamin D3 analog (such as calcipotriol,
tacalcitol, ZK191784 or ZK203278)
[0183] The mammalian hepatocytes may be exposed to at least one
vitamin D3 selected from the group consisting of cholecalciferol,
calcifediol, calcitriol, and combinations thereof. The mammalian
hepatocytes may be exposed to at least one vitamin D3 selected from
the group consisting of cholecalciferol, calcitriol, and
combinations thereof.
[0184] The mammalian hepatocytes may be exposed to at least
cholecalciferol. The mammalian hepatocytes may be exposed to at
least calcifediol. The mammalian hepatocytes may be exposed to at
least calcitriol.
[0185] The mammalian hepatocytes may be exposed to at least
cholecalciferol and calcifediol. The mammalian hepatocytes may be
exposed to at least cholecalciferol and calcitriol. The mammalian
hepatocytes may be exposed to at least calcifediol and calcitriol.
The mammalian hepatocytes may be exposed to at least
cholecalciferol, calcifediol and calcitriol.
[0186] The mammalian hepatocytes may be exposed to at least one
vitamin D3 precursor, such as at least 7-dehydrocholesterol.
[0187] The mammalian hepatocytes may be exposed to at least one
vitamin D3 analog selected from the group consisting of
22-oxacalcitriol (OCT), paricalcitol, doxercalciferol,
calcipotriol, tacalcitol, ZK191784 and ZK203278. The mammalian
hepatocytes may be exposed to at least calcipotriol.
[0188] Generally, the concentration of said at least one vitamin D,
vitamin D precursor, vitamin D metabolite or vitamin D analog, when
employed in accordance with the present invention, is in the range
of about 0.05 to about 15 .mu.M, such as, e.g., in the range of
about 0.1 to about 5 .mu.M.
[0189] The mammalian hepatocytes, such as human hepatocytes, may
thus be exposed to said at least one vitamin D, vitamin D
precursor, vitamin D metabolite or vitamin D analog at a
concentration in the range of about 0.05 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.05 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.05 to about 5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.1 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.1 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.1 to about 5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.1 to about 2.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.1 to about 1 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.1 to about 0.75 .mu.M. The
mammalian hepatocytes may be exposed said at least one vitamin D,
vitamin D precursor, vitamin D metabolite or vitamin D analog at a
concentration in the range of about 0.25 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.25 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.25 to about 5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.25 to about 2.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.25 to about 1 .mu.M. The
mammalian hepatocytes may be exposed to said at least one vitamin
D, vitamin D precursor, vitamin D metabolite or vitamin D analog at
a concentration in the range of about 0.25 to about 0.75 .mu.M.
[0190] In case that, for instance, cholecalciferol is employed
according to the invention, it may be employed at a concentration
in the range of about 0.05 to about 15 .mu.M, such as, e.g., in the
range of about 0.1 to about 5 .mu.M, about 0.1 to about 2.5 .mu.M,
about 0.1 to about 1 .mu.M or about 0.1 to about 0.5., such as,
e.g., at about 0.2 .mu.M.
[0191] In case that, for instance, calcifediol is employed
according to the invention, it may be employed at a concentration
in the range of about 0.05 to about 15 .mu.M, such as, e.g., in the
range of about 0.1 to about 5 .mu.M, about 0.1 to about 2.5 .mu.M,
about 0.1 to about 1 .mu.M or about 0.25 to about 0.75, such as,
e.g., at about 0.5 .mu.M.
[0192] In case that, for instance, calcitriol is employed according
to the invention, it may be employed at a concentration in the
range of about 0.05 to about 15 .mu.M, such as, e.g., in the range
of about 0.1 to about 5 .mu.M, about 0.1 to about 2.5 .mu.M, about
0.1 to about 1 .mu.M or about 0.25 to about 0.75, such as, e.g., at
about 0.5 .mu.M.
[0193] The at least one maturation factor employed in the methods
of the invention may be at least one hypoxia inducing compound,
such as, e.g, hypoxia inducing compound selected from the group
consisting of RAR-related orphan receptor alpha (ROR-alpha)
ligands, CoCl.sub.2, and NaN.sub.3.
[0194] Accordingly, the mammalian hepatocytes may be exposed to at
least one RAR-related orphan receptor alpha (ROR-alpha) ligand,
such as at least one RAR-related orphan receptor alpha (ROR-alpha)
ligand selected from the group consisting of CGP52608, CGP52608
analogs, melatonin, melatonin analogs, cholesterol, cholesterol
derivatives, and combinations thereof.
[0195] The mammalian hepatocytes may thus be exposed to at least
CGP52608 or a CGP52608 analog. The mammalian hepatocytes may thus
be exposed to at least CGP52608. The mammalian hepatocytes may also
be exposed to at least a CGP52608 analog, such as CGP53065, CGP
52528, CGP 53079, CGP58238, CGP 52113, CGP 52749, CGP 55644, CGP
55706, CGP 55707, CGP 56753, CGP 55066 or GP 50468.
[0196] The mammalian hepatocytes may thus be exposed to at least
melatonin or a melatonin analog. The mammalian hepatocytes may thus
be exposed to at least melatonin. The mammalian hepatocytes may
also be exposed to at least a melatonin analog, such as
6-methoxybenzoxazolinone, ramelteon
((S)--N-[2-(1,6,7,8-tetrahydro-2H-indeno[5,4-b]
furan-8-yl)-ethyl]propionamide), agomelatine
(N-(2-[7-methoxy-1-naphthalenyl]ethyl) acetamide) or synthetic
kynurenines, such as 2-acetamide-4-(3-methoxyphenyl)-4-oxobutyric
acid, 2-acetamide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid,
2-butyramide-4-(3-methoxy-phenyl)-4-oxobutyric acid or
2-butyramide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid.
[0197] The mammalian hepatocytes may thus be exposed to at least
cholesterol or a cholesterol derivative.
[0198] The mammalian hepatocytes may thus be exposed to at least
CoCl.sub.2 or NaN.sub.3
[0199] Generally, the concentration of said at least one hypoxia
inducing compound, and in particularly said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, when employed
in accordance with the present invention, is in the range of about
0.05 to about 50 .mu.M, such as, e.g., in the range of about 0.5 to
about 10 .mu.M.
[0200] The mammalian hepatocytes, such as human hepatocytes, may
thus be exposed to said at least one hypoxia inducing compound, and
in particularly to said at least one RAR-related orphan receptor
alpha (ROR-alpha) ligand, at a concentration in the range of about
0.05 to about 25 .mu.M. The mammalian hepatocytes may be exposed to
said at least one hypoxia inducing compound, and in particularly to
said at least one RAR-related orphan receptor alpha (ROR-alpha)
ligand, at a concentration in the range of about 0.05 to about 15
.mu.M. The mammalian hepatocytes may be exposed to said at least
one hypoxia inducing compound, and in particularly to said at least
one RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.05 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.05 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.1 to about 25 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.1 to about 15 .mu.M. The
mammalian hepatocytes may be exposed to said at least hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.1 to about 15 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.1 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.1 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.5 to about 25 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.5 to about 15 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.5 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 0.5 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 1 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 1 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 1.5 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 2.5 to about 7.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 3.5 to about 6.5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 4 to about 6 .mu.M. The
mammalian hepatocytes may be exposed said at least one hypoxia
inducing compound, and in particularly to said at least one
RAR-related orphan receptor alpha (ROR-alpha) ligand, at a
concentration in the range of about 4.5 to about 5.5 .mu.M. For
example, The mammalian hepatocytes may be exposed to about 5 .mu.M
of said at least one hypoxia inducing compound, and in particularly
of said at least one RAR-related orphan receptor alpha (ROR-alpha)
ligand.
[0201] In case that, for instance, CGP52608 is employed according
to the invention, it may be employed at a concentration in the
range of about 0.05 to about 50 .mu.M, such as, e.g., in the range
of about 0.5 to about 10 .mu.M, such as, e.g., at about 5
.mu.M.
[0202] The at least one maturation factor employed in the methods
of the invention may also be at least one sphingosine, such as
D-erythro-sphingosine or L-erythro-sphingosine, or a sphingosine
derivative, such as sphingosine-1-phosphate or a sphingolipid.
[0203] Accordingly, the mammalian hepatocytes may be exposed to at
least one sphingosine. More specifically, the mammalian hepatocytes
may be exposed to at least D-erythro-sphingosine. The mammalian
hepatocytes may be exposed to at least L-erythro-sphingosine.
[0204] The mammalian hepatocytes may be exposed to at least a
sphingosine derivative.
[0205] The mammalian hepatocytes may be exposed to at least a
sphingosine derivative selected from the group consisting of
sphingosine-1-phosphate, dihydrosphingosine, such as
DL-erythro-dihydrosphingosine, L-threo-sphingosine C-18,
Azido-erythro-sphingosine,
3-O-(tert-Butyldimethylsilyloxy)-erythro-sphingosine,
(2S,3R,4E)-2-Azido-3-(tert-butyldimethylsilyl)-1-pivaloyl-erythro-sphingo-
sine, 3-O-(tert-Butyldimethylsilyloxy)-2-Fmoc-erythro-sphingosine,
(2S,3R,4E)-2-Azido-3-(tert-butyldimethylsilyl)-erythro-sphingosine,
N-Boc-erythro-sphingosine, Safingol, and ceramides.
[0206] The mammalian hepatocytes may thus be exposed to at least
sphingosine-1-phosphate, such as D-erythro-sphingosine or
L-erythro-sphingosine. The mammalian hepatocytes may be exposed to
at least D-erythro-sphingosine-1-phosphate. The mammalian
hepatocytes may be exposed to at least
L-erythro-sphingosine-1-phosphate.
[0207] The mammalian hepatocytes may be exposed to at least a
sphingolipid, such as a ceramide or a ceramide analog. The ceramide
may, for instance, be a N--C.sub.2-24-ceramide, such as a N-C12-,
N-C14-, N-C16- or N-C18-ceramide. More specifically, the ceramide
may be a D-erythro-ceramide, such as a a N-C16-D-erythro-ceramide.
The ceramide may be a L-erythro-ceramide, such as a
N-C16-L-erythro-ceramide. The ceramide analog may be MAPP, such as
L-erythro MAPP or D-erythro MAPP.
[0208] The mammalian hepatocytes may thus be exposed to at least a
ceramide or ceramide analog. The mammalian hepatocytes may be
exposed to at least a ceramide. The mammalian hepatocytes may be
exposed to at least a ceramide analog. The mammalian hepatocytes
may thus be exposed to at least a N--C.sub.2-24-ceramide, such as a
N-C10-, N-C12-, N-C14-, N-C16-, N-C18- or N-C20-ceramide. The
mammalian hepatocytes may be exposed to at least a N-C16-ceramide,
such as N-C16-D-erythro-ceramide
(N-palmitoyl-D-erythro-sphingosine).
[0209] Generally, the concentration of said at least one
sphingosine or sphingosine derivative, when employed in accordance
with the present invention, is in the range of about 0.05 to about
15 .mu.M, such as, e.g., in the range of about 0.1 to about 5
.mu.M.
[0210] The mammalian hepatocytes, such as human hepatocytes, may
thus be exposed to said at least one sphingosine or sphingosine
derivative at a concentration in the range of about 0.05 to about
10 .mu.M. The mammalian hepatocytes may be exposed to said at least
one sphingosine or sphingosine derivative at a concentration in the
range of about 0.05 to about 7.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one sphingosine or sphingosine
derivative at a concentration in the range of about 0.05 to about 5
.mu.M. The mammalian hepatocytes may be exposed to said at least
one sphingosine or sphingosine derivative at a concentration in the
range of about 0.1 to about 10 .mu.M. The mammalian hepatocytes may
be exposed to said at least one sphingosine or sphingosine
derivative at a concentration in the range of about 0.1 to about
7.5 .mu.M. The mammalian hepatocytes may be exposed to said at
least one sphingosine or sphingosine derivative at a concentration
in the range of about 0.1 to about 5 .mu.M. The mammalian
hepatocytes may be exposed to said at least one sphingosine or
sphingosine derivative at a concentration in the range of about 0.1
to about 2.5 .mu.M. The mammalian hepatocytes may be exposed to
said at least one sphingosine or sphingosine derivative at a
concentration in the range of about 0.1 to about 1 .mu.M. The
mammalian hepatocytes may be exposed to said at least one
sphingosine or sphingosine derivative at a concentration in the
range of about 0.1 to about 0.75 .mu.M. The mammalian hepatocytes
may be exposed said at least one sphingosine or sphingosine
derivative at a concentration in the range of about 0.25 to about
10 .mu.M. The mammalian hepatocytes may be exposed to said at least
one sphingosine or sphingosine derivative at a concentration in the
range of about 0.25 to about 7.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one sphingosine or sphingosine
derivative at a concentration in the range of about 0.25 to about 5
.mu.M. The mammalian hepatocytes may be exposed to said at least
one sphingosine or sphingosine derivative at a concentration in the
range of about 0.25 to about 2.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one sphingosine or sphingosine
derivative at a concentration in the range of about 0.25 to about 1
.mu.M. The mammalian hepatocytes may be exposed to said at least
one sphingosine or sphingosine derivative at a concentration in the
range of about 0.25 to about 0.75 .mu.M.
[0211] In case that, for instance, sphingosine, such as
D-erythro-sphingosine, is employed according to the invention, it
may be employed at a concentration in the range of about 0.05 to
about 15 .mu.M, such as, e.g., in the range of about 0.1 to about 5
.mu.M, about 0.1 to about 2.5 .mu.M, about 0.1 to about 1 .mu.M or
about 0.25 to about 0.75, such as, e.g., at about 0.5 .mu.M.
[0212] In case that, for instance, a sphingosine-1-phosphate, such
as D-erythro-sphingosine-1-phosphate, is employed according to the
invention, it may be employed at a concentration in the range of
about 0.05 to about 15 .mu.M, such as, e.g., in the range of about
0.1 to about 5 .mu.M, about 0.1 to about 2.5 .mu.M, about 0.1 to
about 1 .mu.M or about 0.25 to about 0.75, such as, e.g., at about
0.5 .mu.M.
[0213] In case that, for instance, a ceramide, such as
N-C16-D-erythro-ceramide, is employed according to the invention,
it may be employed at a concentration in the range of about 0.05 to
about 15 .mu.M, such as, e.g., in the range of about 0.1 to about 5
.mu.M, about 0.1 to about 2.5 .mu.M, about 0.1 to about 1 .mu.M or
about 0.25 to about 0.75, such as, e.g., at about 0.5 .mu.M.
[0214] The at least one maturation factor employed in the methods
of the invention may also be at least one activator of peroxisome
proliferator-activated receptors (PPARs), such as at least one
activator of peroxisome proliferator-activated receptors (PPARs)
selected from the group consisting of thiazolidinediones, free
fatty acids (FFAs), eicosanoids including eicosanoid precursors and
eicosanoid analog, and combinations thereof.
[0215] Accordingly, the mammalian hepatocytes, such as human
hepatocytes, may be exposed to at least one thiazolidinedione, such
as at least one thiazolidinedione selected from the group
consisting of CGP52608, CGP52608 analogs, ciglitazone,
rosiglitazone, pioglitazone, lobeglitazone, troglitazone, TS5444,
and combinations thereof.
[0216] The mammalian hepatocytes, such as human hepatocytes, may be
exposed to at least one free fatty acid, such as a saturated or
unsaturated fatty acid.
[0217] The mammalian hepatocytes may be exposed to at least one
saturated fatty acid, such as at least one saturated fatty acid
selected from the group consisting of dodecanoic acid, tridecanoic
acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid,
heptadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic
acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid,
hexacosanoic acid, and combinations thereof.
[0218] The mammalian hepatocytes may be exposed, for instance, to
at least tetradecanoic acid.
[0219] The mammalian hepatocytes may be exposed to at least one
unsaturated fatty acid, such as at least one unsaturated fatty acid
selected from the group consisting of 10Z-heptadecenoic acid,
arachidonic acid (AA), 9(Z),11(E)-Conjugated Linoleic Acid,
eicosadienoic acid, eicosatrienoic acid (ETE), eicosapentaenoic
acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid
(DHA), linoleic acid, gamma-linolenic acid, dihomo-gamma-linolenic
acid, docosadiennoic acid, adrenic acid, mead acid, ricinoleic
acid, docosatrienoic acid, and combinations thereof.
[0220] The mammalian hepatocytes may be exposed, for instance, to
at least one unsaturated fatty acid selected from the group
consisting of 10Z-heptadecenoic acid, arachidonic acid (AA),
Docosahexaenoic acid (DHA), and combinations thereof. The mammalian
hepatocytes may thus be exposed to at least 10Z-heptadecenoic acid.
The mammalian hepatocytes may thus be exposed to at least
arachidonic acid (AA). The mammalian hepatocytes may be exposed to
at least Docosahexaenoic acid (DHA). The mammalian hepatocytes may
thus be exposed to at least 10Z-heptadecenoic acid and arachidonic
acid (AA). The mammalian hepatocytes may thus be exposed to at
least 10Z-heptadecenoic acid and Docosahexaenoic acid (DHA). The
mammalian hepatocytes may thus be exposed to at least arachidonic
acid (AA) and Docosahexaenoic acid (DHA). The mammalian hepatocytes
may thus be exposed to at least 10Z-heptadecenoic acid, arachidonic
acid (AA) and Docosahexaenoic acid (DHA).
[0221] The mammalian hepatocytes may be exposed to at least one
eicosanoid, eicosanoid precursor or eicosanoid analog, such as at
least one eicosanoid, eicosanoid precursor or eicosanoid analog
selected from the group consisting of Diacylglycerol,
Eicosapentaenoic acid, Dihomo-gamma-linolenic acid, Arachidonic
acid, ETYA (5,8,11,14-eicosatetraynoic acid), members of the
hydroxyeicosatetraenoic acid (HETE) family, including 5-HETE and
15-HETE, members of the hydroxyoctadecadieonic acid (HODE) family,
incuding 9-HODE and 13-HODE, classic eicosanoids, and non-classic
eicosanoids.
[0222] The mammalian hepatocytes may be exposed, for instance, to
at least one classic eicosanoid, such as at least one classic
eicosanoid selected from the group consisting of prostaglandins,
prostacyclines, leukotriens, eoxins, thromboxanes, and analogs,
precursors or derivatives thereof.
[0223] The mammalian hepatocytes may be exposed to at least one
prostaglandin, such as at least one prostaglandin selected from the
group consisting of pgd.sub.2, pgd.sub.3, pge.sub.1, pge.sub.2,
pge.sub.3, pgf.sub.1c, pgf.sub.2a, pgf.sub.3, and pgj.sub.2.
[0224] The mammalian hepatocytes may be exposed to at least one
prostacyclin, such as at least one prostacyclins selected from the
group consisting of pgi.sub.2 and pgi.sub.3.
[0225] The mammalian hepatocytes may be exposed to at least one
leukotriene, such as at least one are selected leukotriene from the
group consisting of Lta.sub.4, Lta.sub.5, Ltb.sub.4, Ltb.sub.5,
Ltc.sub.4, Ltc.sub.5, Ltd.sub.4, Ltd.sub.5, Lte.sub.4, and
Lte.sub.5.
[0226] The mammalian hepatocytes may be exposed to at least one
eoxin, such as at least one eoxin selected from the group
consisting of 14,15-leukotriene A4, 14,15-leukotriene C4,
14,15-leukotriene D4, and 14,15-leukotriene E4.
[0227] The mammalian hepatocytes may be exposed to at least one
thromboxane, such as at least one thromboxane selected from the
group consisting of Txa.sub.1, Txa.sub.2, and Txa.sub.3.
[0228] The mammalian hepatocytes may be exposed, for instance, to
at least one non-classic eicosanoid, such as at least one
nonclassic eicosanoid selected from the group consisting of
endocannabinoids, hepoxilins, resolvins, isofurans, isoprastanes,
lipoxins, epi-lipoxins, epoxyeicosatrieonic acids (EETs).
[0229] The mammalian hepatocytes may be exposed to at least one
endocannabionoid, such as at least one endocannabionoid selected
from the group consisting of anandamides, WIN55, 212-2,
palmitylethanolamide, mead ethanolamid, R-mathandamide, BML-190,
N-arachidonylglycine, and arachidonamide.
[0230] Generally, the concentration of said at least one activator
of peroxisome proliferator-activated receptors (PPARs), when
employed in accordance with the present invention, is in the range
of about 0.05 to about 50 .mu.M, such as, e.g., in the range of
about 0.5 to about 10 .mu.M.
[0231] The mammalian hepatocytes, such as human hepatocytes, may
thus be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.05 to about 10 .mu.M. The mammalian hepatocytes
may be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.05 to about 7.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.05 to about 5 .mu.M. The mammalian hepatocytes may
be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.1 to about 10 .mu.M. The mammalian hepatocytes may
be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.1 to about 7.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.1 to about 5 .mu.M. The mammalian hepatocytes may
be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.1 to about 2.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.1 to about 1 .mu.M. The mammalian hepatocytes may
be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.1 to about 0.75 .mu.M. The mammalian hepatocytes
may be exposed said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.25 to about 10 .mu.M. The mammalian hepatocytes
may be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.25 to about 7.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.25 to about 5 .mu.M. The mammalian hepatocytes may
be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.25 to about 2.5 .mu.M. The mammalian hepatocytes
may be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.25 to about 1 .mu.M. The mammalian hepatocytes may
be exposed to said at least one activator of peroxisome
proliferator-activated receptors (PPARs) at a concentration in the
range of about 0.25 to about 0.75 .mu.M.
[0232] In case that, for instance, at least one thiazolidinedione
is employed according to the invention, it may be employed at a
concentration in the range of about 0.05 to about 50 .mu.M, such
as, e.g., in the range of about 0.5 to about 10 .mu.M.
[0233] In case that, for instance, at least one saturated fatty
acid, such as tetradecanoic acid, is employed according to the
invention, it may be employed at a concentration in the range of
about 0.05 to about 50 .mu.M, such as, e.g., in the range of about
0.5 to about 10 .mu.M, about 1 to about 10 .mu.M. or about 2.5 to
about 7.5 .mu.M.
[0234] In case that, for instance, at least one unsaturated fatty
acid, is employed according to the invention, it may be employed at
a concentration in the range of about 0.05 to about 15 .mu.M, such
as, e.g., in the range of about 0.1 to about 5 .mu.M, about 0.1 to
about 2.5 .mu.M, about 0.1 to about 1 .mu.M or about 0.25 to about
0.75, such as, e.g., at about 0.5 .mu.M.
[0235] More specifically, in case that, for instance,
10Z-heptadecenoic acid, is employed according to the invention, it
may be employed at a concentration in the range of about 0.05 to
about 15 .mu.M, such as, e.g., in the range of about 0.1 to about 5
.mu.M, about 0.1 to about 2.5 .mu.M, about 0.1 to about 1 .mu.M or
about 0.25 to about 0.75, such as, e.g., at about 0.5 .mu.M.
[0236] In case that, for instance, arachidonic acid (AA), is
employed according to the invention, it may be employed at a
concentration in the range of about 0.05 to about 15 .mu.M, such
as, e.g., in the range of about 0.1 to about 5 .mu.M, about 0.1 to
about 2.5 .mu.M, about 0.1 to about 1 .mu.M or about 0.25 to about
0.75, such as, e.g., at about 0.5 .mu.M.
[0237] In case that, for instance, Docosahexaenoic acid (DHA), is
employed according to the invention, it may be employed at a
concentration in the range of about 0.05 to about 15 .mu.M, such
as, e.g., in the range of about 0.1 to about 5 .mu.M, about 0.1 to
about 2.5 .mu.M, about 0.1 to about 1 .mu.M or about 0.25 to about
0.75, such as, e.g., at about 0.5 .mu.M.
[0238] The at least one maturation factor employed in the methods
of the invention may also be at least one platelet-activating
factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF), such as
1-C.sub.1-24-alkyl-2-acetyl-sn-glycero-3-phosphocholine
(C.sub.1-24-PAF).
[0239] The mammalian hepatocytes, such as human hepatocytes, may be
exposed to at least one
1-C.sub.1-24-alkyl-2-acetyl-sn-glyero-3-phosphoholine
(C.sub.1-24-PAF), such as at least one
1-C.sub.1-24-alkyl-2-acetyl-sn-glycero-3-phosphocholine
(C.sub.12-20-PAF). The mammalian hepatocytes may, for example, be
exposed to 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine
(C16-PAF). The mammalian hepatocytes may, for example, be exposed
to 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C18-PAF).
[0240] Generally, the concentration of said at least one
platelet-activating factor, when employed in accordance with the
present invention, is in the range of about 0.05 to about 15 .mu.M,
such as, e.g., in the range of about 0.1 to about 5 .mu.M.
[0241] The mammalian hepatocytes, such as human hepatocytes, may
thus be exposed to said at least one platelet-activating factor at
a concentration in the range of about 0.05 to about 10 .mu.M. The
mammalian hepatocytes may be exposed to said at least
platelet-activating factor at a concentration in the range of about
0.05 to about 7.5 .mu.M. The mammalian hepatocytes may be exposed
to said at least one platelet-activating factor at a concentration
in the range of about 0.05 to about 5 .mu.M. The mammalian
hepatocytes may be exposed to said at least one platelet-activating
factor at a concentration in the range of about 0.1 to about 10
.mu.M. The mammalian hepatocytes may be exposed to said at least
one platelet-activating factor at a concentration in the range of
about 0.1 to about 7.5 .mu.M. The mammalian hepatocytes may be
exposed to said at least one platelet-activating factor at a
concentration in the range of about 0.1 to about 5 .mu.M. The
mammalian hepatocytes may be exposed to said at least one
platelet-activating factor at a concentration in the range of about
0.1 to about 2.5 .mu.M. The mammalian hepatocytes may be exposed to
said at least one platelet-activating factor at a concentration in
the range of about 0.1 to about 1 .mu.M. The mammalian hepatocytes
may be exposed to said at least one platelet-activating factor at a
concentration in the range of about 0.1 to about 0.75 .mu.M. The
mammalian hepatocytes may be exposed said at least one
platelet-activating factor at a concentration in the range of about
0.25 to about 10 .mu.M. The mammalian hepatocytes may be exposed to
said at least one platelet-activating factor at a concentration in
the range of about 0.25 to about 7.5 .mu.M. The mammalian
hepatocytes may be exposed to said at least one platelet-activating
factor at a concentration in the range of about 0.25 to about 5
.mu.M. The mammalian hepatocytes may be exposed to said at least
one platelet-activating factor at a concentration in the range of
about 0.25 to about 2.5 .mu.M. The mammalian hepatocytes may be
exposed to said at least one platelet-activating factor at a
concentration in the range of about 0.25 to about 1 .mu.M. The
mammalian hepatocytes may be exposed to said at least one
platelet-activating factor at a concentration in the range of about
0.25 to about 0.75 .mu.M.
[0242] In case that, for instance,
1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-PAF), is
employed according to the invention, it may be employed at a
concentration in the range of about 0.05 to about 15 .mu.M, such
as, e.g., in the range of about 0.1 to about 5 .mu.M, about 0.1 to
about 2.5 .mu.M, about 0.1 to about 1 .mu.M or about 0.25 to about
0.75, such as, e.g., at about 0.5 .mu.M.
[0243] The at least one maturation factor employed in the methods
of the invention may also be at least one protein kinase C (PKC)
inhibitor, such as at least one PKC inhibitor selected from the
group consisting of Bisindolylmaleimide I, Bisindolylmaleimide II,
Bisindolylmaleimide III, Bisindolylmaleimide V, Bisindolylmaleimide
VI, Bisindolylmaleimide VII, Bisindolylmaleimide VIII,
Bisindolylmaleimide X, HBDDE, Rottlerin, Palmitoyl-DL-carnitine,
R-Stearoyl Carnitine Chloride, Piceatannol, H-9, H-8,
1-(5-Isoquinolinesulfonyl)-3-methylpiperazine, HA-100
dihydrochloride, HA-1004, HA-1077, 5-lodotubericidin, Ro-32-0432,
Ro-31-7549, Enzastaurin (LY317615), Sotrastaurin, Dequalinium
Chloride, Go 6976, Go 6983, Go 7874, Myricitrin,
4-Hydroxy-Tamoxifen,N-Desmethyltamoxifen HCl, Safingol, Phloretin,
UCN-01, 7-Oxostaurosporine, K-252a, K-252b, K-252c, Melittin,
Hispidin, Calphostin C, Ellagic acid, PKC Inhibitor Peptide 19-31,
PKC Inhibitor Peptide 19-36, PKC epsilon Translocation Inhibitor
II, EGF-R Fragment 651-658, PKC beta inhibitor (CAS 257879-35-9),
PKC 20-28, PKC.beta.II/EGFR Inhibitor (CAS 145915-60-2), PKC6
Pseudosubstrate Inhibitor, PKC6/5 Inhibitor, [Ala107]-MBP
(104-118), [Ala113]-MBP (104-118), ZIP, C-1, Bryostatin 1, LY
333531 hydrochloride, CGP 53353, Chelerythrine Chloride, TCS 21311,
CID 755673, Gossypol, ET-18-OCH3,
1-O-Hexadecyl-2-O-methyl-rac-glycerol, NPC-15437 dihydrochloride,
NGIC-I, MDL-27.032, DAPH-7, 7-Aminoindole, 5-Amino-2-methylindole,
rac-2-Methoxy-3-hexadecanamido-1-propylphosphocholine, Copper
bis-3,5-diisopropylsalicylate, D,L-3,4-Dihydroxymandelic Acid,
rac-3-Octadecanamido-2-Methoxypropan-1-ol Phosphocholine, KRIBB3,
Ilmofosine,
rac-2-Methoxy-3-hexadecanamido-1-propylphosphocholine,andcombinationsther-
eof.
[0244] Generally, the concentration of said at least one protein
kinase C (PKC) inhibitor, when employed in accordance with the
present invention, is in the range of about 0.01 to about 50 .mu.M,
such as, e.g., in the range of about 0.5 to about 10 .mu.M.
[0245] The mammalian hepatocytes, such as human hepatocytes, may
not only be exposed to one maturation factor, but may be exposed to
a combination of maturation factors described herein, such as a
combination comprising at least two, such as at least three, at
least four, at least five, at least six, at least seven, or at last
eight, of the maturation factors described herein.
[0246] Accordingly, the mammalian hepatocytes, such as human
hepatocytes, may be exposed to a combination comprising at least
two maturation factors. The mammalian hepatocytes, such as human
hepatocytes, may be exposed to a combination comprising at least
three maturation factors. The mammalian hepatocytes, such as human
hepatocytes, may be exposed to a combination comprising at least
four maturation factors. The mammalian hepatocytes, such as human
hepatocytes, may be exposed to a combination comprising at least
five maturation factors. The mammalian hepatocytes, such as human
hepatocytes, may be exposed to a combination comprising at least
six maturation factors. The mammalian hepatocytes, such as human
hepatocytes, may be exposed to a combination comprising at least
seven maturation factors. The mammalian hepatocytes, such as human
hepatocytes, may be exposed to a combination comprising at least
eight maturation factors.
[0247] The mammalian hepatocytes, such as human hepatocytes, may be
exposed to any one of the combinations of the following items:
[0248] a) a combination comprising at least two maturation
factor(s) selected from the group Src kinase inhibitors, vitamin D
including precursors, metabolites and analog thereof, hypoxia
inducing compounds, sphingosine and sphingosine derivatives,
activators of peroxisome proliferator-activated receptors (PPARs),
platelet-activating factor (PAF), PKC inhibitors, and combinations
thereof;
[0249] b) a combination according to item a), comprising at least
one (such as at least two) Src kinase inhibitor;
[0250] c) a combination according to item a) or b), comprising at
least one (such as at least two) vitamin D, vitamin D precursor,
vitamin D metabolite or vitamin D analog;
[0251] d) a combination according to any one of items a) to c),
comprise at least one (such as at least two) sphingosine and
sphingosine derivative;
[0252] e) a combination according to any one of items a) to d),
comprising at least one (such as at least two) activator of
peroxisome proliferator-activated receptors (PPARs);
[0253] f) a combination according to any one of items a) to e),
comprising at least one (such as at least two) platelet-activating
factor (PAF);
[0254] g) a combination according to any one of items a) to f),
comprising at least one (such as at least two) PKC inhibitor.
[0255] The differentiating and maturing hepatic cells may, for
example, be exposed to the at least one maturation factor for up to
about 35 days. They may, for example, be exposed to the at least
one maturation factor for about 2 days to about 30 days. They may
be exposed to the at least one maturation factor for about 2 days
to about 25 days. They may be exposed to the at least one
maturation factor for about 2 days to about 20 days. They may be
exposed to the at least one maturation factor for about 2 days to
about 15 days. They may be exposed to the at least one maturation
factor for about 7 days to about 35 days. They may be exposed to
the at least one maturation factor for about 7 days to about 30
days. They may be exposed to the at least one maturation factor for
about 7 days to about 25 days. They may be exposed to the at least
one maturation factor for about 7 days to about 20 days.T hey may
be exposed to the at least one maturation factor for about 10 days
to about 35 days. They may be exposed to the at least one
maturation factor for about 10 days to about 30 days. They may be
exposed to the at least one maturation factor for about 10 days to
about 25 days. They may be exposed to the at least one maturation
factor for about 10 days to about 20 days.
[0256] In accordance with the invention, the at least one
maturation factor may be added to the differentiation medium at any
time point after initiation of culturing mammalian hepatic
progenitor cells under differentiation conditions to obtain said
mammalian hepatocytes, such as after 4 days of culturing. Thus, in
addition to exposing said mammalian hepatocytes to said at least
one maturation factor, said mammalian hepatic progenitor cells may
also be exposed to said at least one maturation factor. However,
usually, the at least one maturation factor is added to the
differentiation medium at a time t.gtoreq.day 7 after initiation of
culturing mammalian hepatic progenitor cells under differentiation
conditions to obtain said mammalian hepatocytes.
[0257] In addition to being exposed to at least one maturation
factors as described herein, the mammalian hepatocytes (and
optionally also said mammalian hepatic progenitor cells from which
said mammalian hepatocytes are derived) may optionally also be
exposed to an overlay of one or more components characteristic of
the mammalian extracellular matrix (matrix overlay). Thus, the
exposure to the at least one maturation factors is further combined
with the exposure to a matrix overlay.
[0258] Matrix overlays consisting of Collagen I or Matrigel (a
basement membrane mix extracted from the Engelbreth-Holm-Swarm
mouse sarcoma) have been used for culturing primary hepatocytes for
several decades (e.g. Dunn et al. 1991; Page et al. 2007), since it
was found that primary hepatocytes maintain a better functionality
and live longer in a so called sandwich configuration, with one
extracellular matrix (ECM) layer below the cells and one ECM layer
on top of the cells. Classically, Collagen I and Matrigel overlays
are thick, containing e.g. 125 .mu.g Matrigel/cm.sup.2 or 50 .mu.g
Collagen I/cm.sup.2. However, this is not reflecting the
physiological composition or thickness of the liver ECM (compare
e.g. Turner et al. 2011; Wang et al. 2011).
[0259] The matrix overlay employed in the methods of the invention
is a novel, more physiological combination of component present in
the ECM of the adult liver and comprises, or is composed of, one or
more ECM components, which form part of the normal mammalian
extracellular matrix environment. Suitable ECM components for use
as matrix overlay in the present invention are collagen, such as
collagen I, II, III, IV, V or VI, fibronectin, elastin, chondroitin
sulfate proteoglycan, dermatan sulfate proteoglycan, heparin
proteoglycan, heparan sulfate proteoglycan, such as glypicans,
syndecans or perlecans, glycosaminoglycans, nidogen/entactin,
laminins, biglycan, tenascin, hyaluronans, or other ECM components,
or ECM component mixtures comprising, or consisting of, e.g.,
collagens, laminin, fibronectin, tenascin, proteoglycans, and
glycosaminoglycans.
[0260] Accordingly, the mammalian hepatocytes (and optionally said
mammalian hepatic progenitor cells from which said mammalian
hepatocytes are derived) may be exposed to a matrix overlay
comprising, or composed of, one or more, such as two, three, four,
five, six, seven, eight, nine or ten, or up to 20 of the above
mentioned ECM components. Thus, the mammalian hepatocytes may be
exposed to a matrix overlay comprising, or composed of, two of the
above mentioned ECM components.
[0261] For example, the mammalian hepatocytes may be exposed to a
matrix overlay comprising, or composed of, collagen and fibronectin
(collagen-fibronectin-matrix overlay), such as a matrix overlay
comprising, or composed of, collagen I and fibronectin (collagen
I-fibronectin-matrix overlay).
[0262] The matrix overlay employed in the methods of the invention
is thin compared to the thick matrices so far used. The thickness
of the matrix overlay thereby correlates with the concentration of
the ECM components employed. Suitable concentrations for the matrix
overlay are e.g. 0.01-35 .mu.g, such as 0.01-20 .mu.g, ECM
component/cm.sup.2 culture area. However, it is also contemplated
that higher concentrations may be used.
[0263] Collagen I may, for example, be present in the matrix
overlay at a concentration from about 2 to about 150 .mu.g/cm.sup.2
culture area, such as from about 30 to 150 .mu.g/cm.sup.2 culture
area, such as about 31.25 .mu.g/cm.sup.2 culture area.
[0264] Fibronectin may, for example, be present in the matrix
overlay at a concentration from about 2 to about 30 .mu.g/cm.sup.2
culture area, such as from about 2 to about 10 .mu.g/cm.sup.2
culture area, such as about 6 .mu.g/cm.sup.2 culture area.
[0265] It is to be understood that the above concentrations in
".mu.g/cm.sup.2 culture area" are with respect to the respective
component in its dry state.
[0266] Further matrix overlays which can be employed in the methods
of the invention are described in WO2014/083132 (page 43, line 7 to
page 47, line 29), the content of which is hereby incorporated by
reference.
[0267] Generally, cells may be cultured on a coating as growth
support which covers the surface of the culture vessel. Gelatine or
fibronectin based coating are widely used as growth support. Thus,
the cells, in particular the hepatic progenitor cells and
hepatocyte, may be cultured on a gelatin or fibronectin based
coating. However, the cells may also be cultured on a coating which
has a composition similar or identical to a matrix overlay as
defined above. For example, when a matrix overlay is to be
employed, the cells may be cultured on a coating which has a
composition which is identical to that of the employed matrix
overlay. Accordingly, a so-called "sandwich" type culture
environment is provided. The cells may, for example, be cultured on
Collagen I-fibronectin-based coating. Such Collagen
I-Fibronectin-based coating may have a concentration of about 2 to
about 10 .mu.g, such as about 2 .mu.g, Fibronectin and about 2 to
about 12 .mu.g, such as about 10 .mu.g, Collagen I per cm.sup.2
culture area.
[0268] The mammalian cells employed in the present invention may,
for instance, be human cells, primate cells, mouse cells, rat
cells, canine cells, feline cells, porcine cells, bovine cells or
equine cells. Thus, the methods of the present invention may be
based on and directed to human cells.
[0269] As an optional pre-step, the mammalian hepatic progenitor
cells used in the methods of the invention may initially be derived
from mammalian pluripotent stem (PS) cells, such as from mammalian
embryonic stem (ES) cells or mammalian artificial pluripotent stem
cells, such as mammalian induced pluripotent stem (iPS) cells. The
methods of the invention may thus further comprise as an initial
step the culturing of mammalian PS cells under differentiation
conditions to obtain said hepatic progenitor cells. In accordance
thereto, mammalian PS cells are initially differentiated into said
hepatic progenitor cells. This step is referred to herein as
initial hepatic differentiation.
[0270] The mammalian pluripotent stem cells employed in the present
invention may, for instance, be human pluripotent stem cells,
primate pluripotent stem cells, mouse pluripotent stem cells, rat
pluripotent stem cells, canine pluripotent stem cells, feline
pluripotent stem cells, porcine pluripotent stem cells, bovine
pluripotent stem cells or equine pluripotent stem cells.
[0271] Especially, the mammalian pluripotent stem cells employed in
the present invention may be any type of human pluripotent stem
cells, such as human embryonic stem (hES) cells or human artificial
pluripotent stem cells, such as human induced pluripotent stem
(hiPS) cells.
[0272] As indicated above, pluripotent stem cells which may also be
used as starting material to obtain endodermal and/or hepatic
progenitor cells may be embryonic stem cells, such as human
embryonic stem cells. Various techniques for obtaining ES cells,
such as hES cells, are known to the skilled person. For example,
hES cells for use according to the invention may be derived (or
obtained) by employing the single blastomere removal technique
described in e.g. Chung et al (2008), further described by Mercader
et al. in Essential Stem Cell Methods (First Edition, 2009).
Alternatively, established and publically available stem cells
lines may be used. Suitable hES cell lines for use are those
established by Klimanskaya et al. (2006), such as cell lines MA01
and MA09, and Chung et al. (2008), such as cell lines MA126, MA127,
MA128 and MA129, which all are listed with the International Stem
Cell Registry (assigned to Advanced Cell Technology, Inc.
Worcester, Mass., USA). Other suitable hES cell lines for use are,
for example, the cell lines SA167, SA181, SA461 (Takara Bio Europe
AB, Goteborg, Sweden).
[0273] Alternatively, the pluripotent stem cells which may be used
as starting material to obtain the definitive endodermal cells
and/or hepatic progenitor cells may be artificial pluripotent stem
cells. Various techniques for obtaining artificial pluripotent stem
cells are are known to the skilled person, and include artificial
reprogramming methods such as somatic nuclear transfer (SCNT), ES
cell fusion-mediated reprogramming (FMR), chemical stimulation of
oocytes (parthenogenetic stem cells) and induced pluripotency
(iPS). Techniques for obtaining hiPS cells are, for example,
described in Takahashi et al. (2007); Zhou et al. (2009); Yu and
Thomson in Essentials of Stem Cell Biology (2.sup.nd Edition].
[0274] It is also envisaged that the endodermal and/or hepatic
progenitor cells may also be derived from other pluripotent stem
cells such as adult stem cells, cancer stem cells or from other
embryonic, fetal, juvenile or adult sources. For example, hepatic
progenitor cells used in accordance with the invention may be
hepatic progenitor derived from the liver, so-called LDPCs. Such
LDPCs have been shown to be capable of hepatic differentiation both
in vitro and in vivo.
[0275] Suitable conditions for differentiating mammalian
pluripotent stem cells, especially hPS cells, into hepatic
progenitor cells are known (see, e.g., Hay 2008, Brolen 2010 and
Duan 2010). WO 2009/013254 A1, for example, describes suitable
protocols to obtain cells of the hepatic progenitor cells from hPS
cells (Embodiments 1 to 4).
[0276] The mammalian pluripotent stem cells, such as hPS cells, are
cultured for up to 14 days in suitable differentiation medium in
order to obtain hepatic progenitor cells. For example, the
mammalian pluripotent stem cells, especially hPS cells, may be
cultured in suitable differentiation medium for about 6 to about 14
days, such as for about 7 to 11 days.
[0277] The initial hepatic differentiation may be defined by
including a pre-endodermal step, i.e. the culturing of the
mammalian pluripotent stem cells, such as hPS cells, under
differentiation conditions to obtain cells of the definitive
endoderm (DE cells), which is followed by a pre-hepatic step, i.e.
the culturing of the obtained DE cells under differentiation
conditions to obtain the hepatic progenitor cells. Accordingly, hPS
cells are first differentiated into definitive endoderm, followed
by the further differentiation of the definitive endoderm into
hepatic progenitor cells.
[0278] Suitable conditions for differentiating mammalian
pluripotent stem cells, especially hPS cells, into DE cells are
known (see, e.g., D'Amour 2005; Siller 2015). WO 2009/013254 A1,
for example, describes suitable protocols to obtain cells of the
definitive endoderm from hPS cells (Embodiments 1 to 4). An
alternative protocol for differentiating mammalian pluripotent stem
cells, especially hPS cells, into DE cells without using activin is
described by Siller et al (2015).
[0279] Generally, in order to obtain DE cells, mammalian
pluripotent stem cells, such as hPS cells, may be cultured in one
or more differentiation media comprising one or more of activin,
such as activin A or B, an albumin source, such as FBS, FCS, N2,
B27 or BSA, a GSK3-inhibitor, such as, e.g., CHIR99021. One or more
differentiation media may comprise activin, such as activin A. One
or more differentiation medium may include a histone deacetylase
(HDAC) inhibitor, such as Sodium Butyrate (NaB), Phenylbutyrate
(PB), valproate, trichostatin A, Entinostat or Panobinstat. One or
more differentiation media may comprise one or more growth factors,
such as FGF1, FGF2 and FGF4. The differentiation media may comprise
an albumin source, such as FBS, FCS, N2, B27 or BSA. One or more
differentiation media may comprise a GSK3-inhibitor, such as, e.g.,
CHIR99021, or an activator of Wnt signalling, such as Wnt3A. One or
more differentiation media may comprise a PI3K (Phosphoinositide
3-kinase) inhibitor, such as LY294002.
[0280] The concentration of activin, if present, is usually in the
range of about 50 to about 150 ng/ml, such as about 80 to about 120
ng/ml. Activin may, for example, be present in the differentiation
medium at a concentration of about 50 ng/ml or about 100 ng/ml. The
concentration of the HDAC inhibitor, if present, is usually in the
range of about 0.5 to about 2 mM. The HDAC inhibitor may, for
example, be present in the differentiation medium at a
concentration of about 0.5 mM or about 1 mM. The concentration of
the one or more growth factors, if present, may vary depending on
the particular compound used. The concentration of FGF2, for
example, is usually in the range of about 2 to about 50 ng/ml, such
as about 2 to about 10 ng/ml. FGF2 may, for example, be present in
the differentiation medium at a concentration of about 4 or about 5
ng/ml. The concentration of FGF1, for example, is usually in the
range of about 50 to about 200 ng/ml, such as about 80 to about 120
ng/ml. FGF1 may, for example, be present in the differentiation
medium at a concentration of about 100 ng/ml. The concentration of
FGF4, for example, is usually in the range of about 20 to about 40
ng/ml. FGF4 may, for example, be present in the differentiation
medium at a concentration of about 30 ng/ml. The concentration of
the albumin source, if present, is usually in the range of about
0.1 to about 2% v/v, such as about 0.1 to about 0.5%, about 0.2 to
about 1.5% v/v, about 0.2 to about 1% v/v, about 0.5 to 1% v/v or
about 0.5 to about 1.5% v/v. The albumin source may, for example,
be present in the differentiation medium at a concentration of
about 0.2% v/v, about 0.5% v/v or about 1% v/v. The concentration
of the GSK3 inhibitor, if present, is usually in the range of about
0.1 to about 10 .mu.M, such as about 0.05 to about 5 .mu.M. The
concentration of the activator of Wnt signalling, if present, is
usually in the range of about 0.05 to about 10 ng/ml, such as about
0, 5 to about 5 .mu.M. The concentration of the PI3K inhibitor, for
example, is usually in the range of about 0.1 to 10 .mu.M, such as
about 1 to 5 .mu.M.
[0281] The differentiation medium may further comprise other
supplements such as PEST and/or GlutaMAX. The differentiation
medium may also further comprise a ROCK inhibitor. The
concentration of PEST is usually in the range of about 0.1 to about
0.5% v/v, such as about 0.1 to about 0.25% v/v. The concentration
of GlutaMAX is usually in the range of about 0.5 to about 1.5% v/v,
such as about 0.75 to 1.25% v/v, e.g. about 1% v/v. The
differentiation medium may also further comprise a ROCK inhibitor.
The concentration of the ROCK inhibitor is usually in the range of
about 1 to about 10 .mu.M, such as about 2.5 to about 7.5 .mu.M,
e.g., about 5 .mu.M.
[0282] The culture medium forming the basis for the differentiation
medium may be any culture medium suitable for culturing hPS cells
such as such as such as RPMI 1640 medium, RPMI 1640 advanced
medium, Iscove's Modified Dulbeccos Medium (IMDM), Minimum
Essential Medium (e.g., MEM, EMEM or GMEM), Dulbecco's Modified
Eagle Medium (e.g., DMEM or DMEM/F-12), Ham's medium (e.g., Ham's
F12 or Ham's F10), HCM medium, HBM medium, or Williams E medium.
Thus, the base medium may, for example, be RPMI 1640 medium or RPMI
1640 advanced medium. Alternatively, the base medium may be
Williams E medium.
[0283] The differentiating mammalian pluripotent stem cells, such
as hPS cells, may be exposed to a DNA demethylating agent. Cells
may be exposed to (or treated with) said agent at any stage between
pluripotent stem cell stage and definitive endodermal stage. Thus,
the exposure to said DNA demethylating agent may take place during
the differentiation of the PS cells into DE cells, i.e. during the
pre-endodermal step. The cells are then cultured through endodermal
stage until hepatic progenitor stage is reached, i.e. until hepatic
progenitor cells are obtained, at which point the further
differentiation and maturation into hepatocytes, including the
exposure to the at least one maturation factor, is carried out.
[0284] The DNA demethylating agent may be any compound that
interferes with DNA methyltransferase enzyme activity. Suitable DNA
demethylating agents are ones of the nucleoside-analog type, such
as cytidine analogues, e.g. 5-aza-2-deoxycytidine (decitabine),
5-azacytidine (azacitidine) or zebularine, and of the
non-nucleoside type, such as procaine, RG108,
S-5-adenosyl-L-homocysteine, Caffeic acid, Chlorogenic acid,
Epogallocatechin gallate, Hydralazine hydrochloride, Procainamide
hydrochloride or Psammaplin A.
[0285] The differentiating mammalian PS cells may generally be
exposed to the DNA demethylating agent at a concentration in the
range of about 1 nM to about 10 .mu.M, such as in the range of
about 1 nM to about 5 .mu.M. In case that, for instance,
5-aza-2-deoxycytidine is employed as the DNA demethylating agent,
the differentiating PS cells may be exposed to it at a
concentration in the range of 1 nM to about 1 .mu.M, such as in the
range of 1 nM to 50 nM, such as at about 10 nM.
[0286] For endodermal differentiation, mammalian pluripotent stem
cells, such as hPS cells, are normally cultured for up to 10 days
in an activin containing differentiation medium as described above.
The mammalian pluripotent stem cells, such as hPS cells, may, for
example, be cultured in said differentiation medium for about 2 to
about 10 days, such as for about 7 to about 9 days.
[0287] Instead of de novo preparation of DE cells from pluripotent
stem cells, DE cells obtainable from commercial sources may be
employed and used as starting material in accordance with the
invention. Human definitive endoderm cells may, for example, be
obtained upon request from Takara Bio Europe AB, Arvid Wallgrens
Backe 20, 41346 Gothenburg, Sweden.
[0288] In order to obtain hepatic progenitor cells, DE cells are
generally cultured in a differentiation medium comprising DMSO.
Alternatively, the DE cells may be cultured in a differentiation
medium comprising one or more growth factors, such as FGF1, FGF2
and FGF4, and optionally one or more bone morphogenic proteins,
such as BMP2 and BMP4. The differentiation medium may further
comprise HGF, EGF and/or serum.
[0289] The concentration of DMSO is usually in the range of about
0.1% to about 2% v/v, such as about 0.5% to about 1.5% v/v. DMSO
may, for example, be present in the differentiation medium at a
concentration of about 1%. The concentration of the one or more
growth factors may vary depending on the particular compound used.
The concentration of FGF2, for example, is usually in the range of
about 2 to about 50 ng/ml, such as about 2 to about ng/ml. FGF2
may, for example, be present in the differentiation medium at a
concentration of 4 or 5 ng/ml. The concentration of FGF1, for
example, is usually in the range of about 50 to about 200 ng/ml,
such as about 80 to about 120 ng/ml. FGF1 may, for example, be
present in the differentiation medium at a concentration of about
100 ng/ml. The concentration of FGF4, for example, is usually in
the range of about 20 to about 40 ng/ml. FGF4 may, for example, be
present in the differentiation medium at a concentration of about
30 ng/ml. The concentration of HGF, if present, is usually in the
range of about 10 to about 30 ng/ml. HGF may, for example, be
present in the differentiation medium at a concentration of about
20 ng/ml. The concentration of EGF, if present is usually in the
range of about 5 to about 15 ng/ml. EGF may, for example, be
present in the differentiation medium at a concentration of about
10 ng/ml. The concentration of serum, if present, is usually in the
range of about 0.1 to about 2% v/v, such as such as about 0.1 to
about 0.5%, about 0.2 to about 1.5% v/v, about 0.2 to about 1% v/v,
about 0.5 to 1% v/v or about 0.5 to about 1.5% v/v. Serum may, for
example, be present in the differentiation medium at a
concentration of about 0.2% v/v, about 0.5% v/v or about 1%
v/v.
[0290] The differentiation medium may further comprise other
supplements such as PEST and/or GlutaMAX. The concentration of PEST
is usually in the range of about 0.1 to about 0.5% v/v, such as
about 0.1 to about 0.25% v/v. The concentration of GlutaMAX is
usually in the range of about 0.5 to about 1.5% v/v, such as about
0.75 to 1.25% v/v, e.g. about 1% v/v.
[0291] The differentiation medium may further comprise other
supplements such as Knockout-Serum Replacement, non-essential amino
acids (NEAA) and/or beta-mercaptoethanol. The concentration of
Knockout-Serum Replacement is usually in the range of about 10 to
about 30% v/v, such as about 15 to about 25% v/v, e.g., about 20%
v/v. The concentration of non-essential amino acids (NEAA) is
usually in the range of about 0.5 to about 1.5% v/v, such as about
0.75 to 1.25% v/v, e.g. about 1% v/v. The concentration of
non-essential amino acids (NEAA) is usually in the range of about
0.1 to about 0.5% v/v, such as about 0.1 to 0.3% v/v, e.g. about
0.2% v/v.
[0292] The culture medium forming the basis for the differentiation
medium may be any culture medium suitable for culturing human
endodermal cells such as such as such as RPMI 1640 medium, RPMI
1640 advanced medium, Iscove's Modified Dulbeccos Medium (IMDM),
Minimum Essential Medium (e.g., MEM, EMEM or GMEM), Dulbecco's
Modified Eagle Medium (e.g., DMEM or DMEM/F-12), Ham's medium
(e.g., Ham's F12 or Ham's F10), HCM medium, HBM medium, or Williams
E medium. Thus, the base medium may, for example, be RPMI 1640
medium or RPMI 1640 advanced medium. Alternatively, the base medium
may be Williams E medium.
[0293] For differentiation into hepatic progenitor cells, DE cells
are normally cultured for up to 7 days in differentiation medium as
described above. The DE cells may, for example, be cultured in
differentiation medium for about 4 to about 7 days.
[0294] Basic, non-limiting culture conditions for obtaining DE
cells, hepatic progenitor cells and hepatocyte are also provided in
Example 2 herein.
[0295] Further, the hepatocyte of the present invention may be
obtained under xeno-free conditions. As such, the starting material
employed in the methods of the invention may thus be xeno-free,
such as xeno-free PS cells or cell lines, or xeno-free hepatic
progenitor cells or cell lines which have been obtained or
established under animal-free conditions.
[0296] Moreover, throughout the methods of the invention cells may
be cultured completely under xeno-free conditions, giving rise to
truly xeno-free hepatocyte. Such cells or cell line would be better
suited to therapeutic or regenerative medicine applications and
could be distinguished from a non-xeno free composition by the
presence in non-xeno free cells of the non-human sialic acid Neu5Gc
or other non-human markers (Martin et al 2005).
[0297] As a result of the methods of the present invention,
mammalian hepatocytes are obtained with more mature and functional
features compared to currently available state of the art
methods.
[0298] The mammalian hepatocytes obtained by employing the methods
of the invention show elevated expression of hepatocyte-associated
genes such as e.g. CYP1A, CYP3A4, CYP2C9, CYP2C19, CYP2B6 and/or
CYP2D6 (see FIGS. 1 to 4). The mammalian hepatocyte obtained by
employing the methods of the invention and principles as laid out
in present invention may be used to a multitude of purposes
comprising drug discovery processes, toxicity test, for studying
drug transporters, drug metabolizing enzyme, as in vitro models for
studying hepatogenesis, such as, e.g., early hepatogenesis, for
studying human hepatoregenerative disorders, for in vitro
hepatotoxicity testing.
[0299] Further the mammalian hepatocyte obtained by employing the
methods of the invention may be used for therapeutic purposes
comprising: in a medicament, for the manufacture of a medicament or
medicinal product for the prevention and/or treatment of
pathologies and/or diseases caused by tissue degeneration, such as,
e.g., the degeneration of liver tissue. The mammalian hepatocytes
of the present invention may also be used for the manufacture of a
medicament or medicinal product for the treatment of liver
disorders. Liver disorders are, for example, auto immune disorders
including primary biliary cirrhosis; metabolic disorders including
dyslipidemia; liver disorders caused by e.g. alcohol abuse;
diseases caused by viruses such as, e.g., hepatitis B, hepatitis C,
and hepatitis A; liver necrosis caused by acute toxic reactions to
e. g. pharmaceutical drugs; and tumour removal in patients
suffering from e. g. hepatocellular carcinoma.
[0300] Alternatively, the mammalian hepatocytes obtained by
employing the methods of the invention may be used for the
manufacture of a medicament or medicinal product for the treatment
and/or prevention of metabolic pathologies and/or diseases. The
medicament or medicinal product may, for example, be in the form of
a replacement tissue or cell injection.
[0301] The differentiation and maturation of mammalian hepatocytes
in accordance to the invention may be useful for obtaining
metabolically improved hepatocytes, for studying maturation towards
hepatocytes or for screening a compound for its ability to modulate
hepatocellular function, comprising exposing in vitro derived
hepatocytes obtained according to the directions provided herein to
the compound, determining any phenotypic or metabolic changes in
the cells that result from contact with the compound, and
correlating the change with an ability to modulate hepatocellular
function.
[0302] The present invention also provides compositions and kits.
Such composition or kits are particularly useful in carrying out
the methods of the invention, e.g, for maturing mammalian
hepatocytes in accordance with the invention.
[0303] A composition of the invention comprises at least one
maturation factor as described above. Thus, a composition of the
invention comprises at least one (such as at least two) maturation
factor(s) selected from the group Src kinase inhibitors, vitamin D
including precursors, metabolites and analog thereof, hypoxia
inducing compounds, sphingosine and sphingosine derivatives,
activators of peroxisome proliferator-activated receptors (PPARs),
platelet-activating factor (PAF), PKC inhibitors, and combinations
thereof.
[0304] A composition of the invention may thus be any one of the
following items:
[0305] a) A composition of the invention comprises at least one
(such as at least two) maturation factor(s) selected from the group
Src kinase inhibitors, vitamin D including precursors, metabolites
and analog thereof, hypoxia inducing compounds, sphingosine and
sphingosine derivatives, activators of peroxisome
proliferator-activated receptors (PPARs), platelet-activating
factor (PAF), PKC inhibitors, and combinations thereof;
[0306] b) a composition according to item a), comprising at least
one (such as at least two) Src kinase inhibitor;
[0307] C) a composition according to item a) or b), comprising at
least one (such as at least two) vitamin D, vitamin D precursor,
vitamin D metabolite or vitamin D analog;
[0308] d) a composition according to any one of items a) to c),
comprise at least one (such as at least two) sphingosine and
sphingosine derivative;
[0309] e) a composition according to any one of items a) to d),
comprising at least one (such as at least two) activator of
peroxisome proliferator-activated receptors (PPARs);
[0310] f) a composition according to any one of items a) to e),
comprising at least one (such as at least two) platelet-activating
factor (PAF);
[0311] g) a composition according to any one of items a) to f),
comprising at least one (such as at least two) PKC inhibitor.
[0312] It is understood that all details given above with respect
to the maturation factors employed in the methods of the invention,
including all combinations and embodiments, especially type of
maturation factor and respective concentrations, also apply to
maturation factors comprised by the composition of the
invention.
[0313] Optionally, the composition may further comprise at least
one (such as at least two) extracellular matrix (ECM) component or
ECM component mixture as described above. The composition may, for
instance, further comprise collagen I and fibronectin.
[0314] The present invention also provides a culture medium
comprising a composition of the invention. Such culture medium is
particularly useful in carrying out the methods of the invention,
e.g, for maturing mammalian hepatocytes in accordance with the
invention.
[0315] The culture medium may be based on any suitable culture
medium such as such as such as RPMI 1640 medium, RPMI 1640 advanced
medium, Iscove's Modified Dulbeccos Medium (IMDM), Minimum
Essential Medium (e.g., MEM, EMEM or GMEM), Dulbecco's Modified
Eagle Medium (e.g., DMEM or DMEM/F-12), Ham's medium (e.g., Ham's
F12 or Ham's F10), HCM medium, HBM medium, or Williams E medium.
Thus, the base medium may, for example, be RPMI 1640 medium or RPMI
1640 advanced medium. Alternatively, the base medium may be
Williams E medium.
[0316] The culture medium, besides comprising a composition of the
invention, may optionally comprise additional components, such as
those optional components described above in the context of the
differentiation medium for obtaining hepatocytes from hepatic
progenitor cells.
[0317] The present invention further provides kits. Such kits are
particularly useful in carrying out the methods of the invention,
e.g, for maturing human hepatocyte-like cells in accordance with
the invention. A kit according to the invention comprises at least
one (such as at least two) maturation factor(s) selected from the
group Src kinase inhibitors, vitamin D including precursors,
metabolites and analog thereof, hypoxia inducing compounds,
sphingosine and sphingosine derivatives, activators of peroxisome
proliferator-activated receptors (PPARs), platelet-activating
factor (PAF), PKC inhibitors, and combinations thereof.
[0318] It is understood that all details given above with respect
to the maturation factors employed in the methods of the invention,
including all combinations and embodiments, especially type of
maturation factor and respective concentrations, also apply to
maturation factors comprised by the kit of the invention.
[0319] A kit according to the present invention may comprise a
composition of the present invention.
[0320] A kit according to the invention may comprise a culture
medium of the present invention.
[0321] A kit according to the present invention may comprise the
least one (such as at least two) maturation factor(s) at a
concentration which is about to 2 to about 100 fold, such as about
to about 50 fold, higher than the concentration employed in the
methods of the invention. In such case, the concentration may have
to be adjusted to the actual concentration prior to use, such as by
dilution.
[0322] A kit of the invention may further comprise mammalian
definitive endoderm cells (DE cells), such as human DE cells. The
DE cells may suitably be provided as a cell suspension, or may be
provided in a frozen state.
[0323] A kit of the invention may further comprise mammalian
pluripotent stem cells, such as human pluripotent stem cells.
Hence, a kit of the invention may comprise mammalian embryonic stem
cells or mammalian induced pluripotent stem cells. The mammalian
pluripotent stem cells may suitably be provided as a cell
suspension, or may be provided in a frozen state.
[0324] The components of a kit of the invention may be provided in
the same or separate containers. For instance, the at least one
(such as at least two) maturation factor(s) may be provided in the
same container. If an at least one extracellular matrix (ECM)
component or ECM component mixture is also comprised by the kit,
such ECM component or ECM component mixture may be generally
provided in a separate container.
[0325] Likewise, if mammalian definitive endoderm cells or
mammalian pluripotent stem cells are comprised by a kit, DE cells
or pluripotent stem cells are generally provide in a container
which is different from the container(s) containing the other
components.
Definitions
[0326] As used herein, "pluripotent" or "pluripotency" refers to
the potential to form all types of specialized cells of the three
germ layers (endoderm, mesoderm, and ectoderm); and is to be
distinguished from "totipotent" or "totipotency", that is the
ability to form a complete embryo capable of giving rise to
offsprings.
[0327] As used herein, "pluripotent stem cells" (PSC) refers to
cells that have the capacity, under appropriate conditions, to
self-renew as well as the ability to form any type of specialized
cells of the three germ layers (endoderm, mesoderm, and ectoderm).
PS cells may have the ability to form a teratoma in 8-12 week old
SCID mice and/or the ability to form identifiable cells of all
three germ layers in tissue culture. Included in the definition of
pluripotent stem cells are embryonic cells of various types
including human embryonic stem (hES) cells, (see, e.g., Thomson et
al. (1998), Heins et.al. (2004), as well as induced pluripotent
stem cells [see, e.g. Takahashi et al., (2007); Zhou et al. (2009);
Yu and Thomson in Essentials of Stem Cell Biology (2.sup.nd
Edition]. The various methods described herein may utilise PS cells
from a variety of sources. For example, PS cells, and especially
human PS cells, suitable for use may have been obtained from
developing embryos by use of a non-destructive technique such as by
employing the single blastomere removal technique described in e.g.
Chung et al (2008), further described by Mercader et al. in
Essential Stem Cell Methods (First Edition, 2009). Additionally or
alternatively, suitable PS cells may be obtained from established
cell lines or may be adult stem cells.
[0328] As used herein "iPS cells" refers to induced pluripotent
stem cells. iPS cells are a type of pluripotent stem cells derived
from non-pluripotent cells--typically adult somatic cells--by
induction of the expression of genes associated with pluripotency,
such as SSEA-3, SSEA-4,TRA-1-60,TRA-1-81, Oct-4, Sox2, Nanog and
Lin28.
[0329] As used herein "hiPS cells" refers to human induced
pluripotent stem cells. hiPS cells are a type of pluripotent stem
cells derived from non-pluripotent cells--typically adult somatic
cells--by induction of the expression of genes associated with
pluripotency, such as SSEA-3, SSEA-4,TRA-1-60,TRA-1-81,Oct-4, Sox2,
Nanog and Lin28.
[0330] As used herein "definitive endoderm (DE)" and "definitive
endoderm cells (DE cells)" refers to cells exhibiting protein
and/or gene expression as well as morphology typical to cells of
the definitive endoderm or a composition comprising a significant
number of cells resembling the cells of the definitive endoderm.
The definitive endoderm is the germ cell layer which gives rise to
cells of the intestine, pancreas, liver and lung. DE cells may
generally be characterized, and thus identified, by a positive gene
and protein expression of the endodermal markers FOXA2, CXCR4,
HHEX, SOX17, GATA4 and GATA6. The two markers SOX17 and CXCR4 are
specific for DE and not detected in hPSC, hepatic progenitor cells
or hepatocytes. Lastly, DE cells do not exhibit gene and protein
expression of the undifferentiated cell markers Oct4, SSEA-3,
SSEA-4, TRA-1-60, TRA-1-81, but can show low Nanog expression.
[0331] As used herein, "hepatic progenitors" or "hepatic progenitor
cells" refers to cells which have entered the hepatic cell path and
give rise to hepatocyte. "Hepatic progenitors" are thus
distinguished from "endodermal cells" in that they have lost the
potential to develop into cells of the intestine, pancreas and
lung. "Hepatic progenitors" may generally be characterized, and
thus identified, by a positive gene and protein expression of the
early hepatic markers EpCAM, c-Met (HGF-receptor), AFP, CK19, HNF6,
C/EBPa and p. They do not exhibit gene and protein expression of
the DE-markers CXCR4 and SOX17. Lastly, "hepatic progenitors" do
not exhibit gene and protein expression of the undifferentiated
cell markers Oct4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 nor the
mature hepatic markers CYP1A2, CYP2C9, CYP19, CYP3A4, CYP2B6 and
PXR.
[0332] As used herein, "hepatocytes" refers to fully differentiated
hepatic cells. "Hepatocytes" may generally be described, and thus
identified, by a positive gene and protein expression of the mature
hepatic markers CYP1A2, CYP3A4, CYP2C9, CYP2C19, CYP2B6, GSTA1-1,
OATP-2, NTCP, Albumin, PXR, CAR, and HNF4a (isoforms 1+2) among
others. Further, "hepatocytes" do not exhibit gene and protein
expression of the undifferentiated cell markers Oct4, SSEA-3,
SSEA-4, TRA-1-60 and TRA-1-81. Compared to DE cells, "hepatocytes"
do not exhibit gene and protein expression of the DE cell markers
SOX17 and CXCR4. Compared to "hepatic progenitors", "hepatocytes"
do not exhibit gene and protein expression of the hepatic
progenitor markers Cytokeratin 19 and AFP.
[0333] As meant herein, a gene or protein shall be interpreted as
being "expressed", if in an experiment measuring the expression
level of said gene or protein, the determined expression level is
higher than three times the standard deviation of the
determination, wherein the expression level and the standard
deviation are determined in 10 separate determinations of the
expression level. The determination of the expression level in the
10 separate determinations is preferably corrected for
background-signal.
[0334] As used herein HDAC inhibitors refers to Histone deacetylase
inhibitors, such as Sodium Butyrate ("NaB"), Phenyl Butyrate
("PB"), Trichostatin A and Valproic Acid ("VA").
[0335] As used herein, "GSK inhibitor" refers to a compound which
inhibits GSK (especially GSK3, including GSK3alpha or
GSK3beta).
[0336] As used herein, a DNA demethylating agent is intended to
mean a compound that interferes with DNA methyltransferase enzyme
activity, such as nucleoside analogues, like cytidine analogs,
notably 5-aza-2-deoxycytidine (decitabine) and 5-azacytidine
(azacitidine), and non-nucleoside types, such as RG108,
S-5-Adenosyl-L-homocysteine, and procaine.
[0337] As used herein "CYP" is intended to mean Cytochrome P, and
more specifically Cytochrome P 450, the major phase I metabolizing
enzyme of the liver constituting of many different isoenzymes, such
as CYP1A1, CYP1A2, CYP1B1, CYP2A6/2A7/2A13, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7 and CYP7A1.
[0338] As used herein, the term "GST" is intended to mean
glutathione transferase, and examples of subtypes thereof are GST
A1-1, GST M1-1, and GST P1-1.
[0339] As used herein the term "UGT" is intended to mean uridine
diphosphoglucuronosyl transferase, which is a group of liver
enzymes catalyzing glucuronidation activities.
[0340] As used herein the term "NTCP" is taken to mean
Na+-taurocholate cotransporting polypeptide, a Sodium/bile acid
co-transporter encoded by the gene SLC10A1
[0341] As used herein the term "CAR" is taken to mean Constitutive
androstane receptor
[0342] The term "functional drug metabolising enzymes" is intended
to mean functional enzymes belonging to the phase I and phase
enzymes that perform chemical modifications of xenobiotics and
drugs, so called drug or xenobiotic metabolism.
[0343] As used herein, the term "functional activity" means
effective measurable hepatic cell function, such as a measurable
transportation of drugs for drug transporters and a measurable
metabolism of enzymes for the Cytochrome P450s (CYPs), commonly
detected in primary human hepatocytes.
[0344] As used herein, the term "extraembryonic endoderm (ExE)" is
intended to mean the differentiated endodermal cells that, as to
the opposite of the definitive endoderm, will constitute the
compartments outside the embryo in the human development, such as
the yolk sac.
[0345] As used herein, the term "AAT" is intended to mean the liver
marker alpha-anti-trypsin.
[0346] As used herein, the term "AFP" is intended to mean the liver
marker alpha-fetoprotein.
[0347] As used herein, the term "BSEP" is intended to mean the bile
transporter bile salt export pump.
[0348] As used herein, the term "CK" is intended to mean the liver
marker cytokeratin (used interchangeably) with different subtypes
such as Cytokeratin 18 (CK18/KRT18), Cytokeratin 19 (CK19/KRT19),
Cytokeratin 8 (CK8) and Cytokeratin 7 (CK7).
[0349] As used herein, the term "FGF" means fibroblast growth
factor, preferably of human and/or recombinant origin, and subtypes
belonging thereto are e.g. "bFGF" (means basic fibroblast growth
factor, sometimes also referred to as FGF2) and FGF4. "aFGF" means
acidic fibroblast growth factor (sometimes also referred to as
FGF1).
[0350] As used herein, the term "BMP" means Bone Morphogenic
Protein, preferably of human and/or recombinant origin, and
subtypes belonging thereto are e.g. BMP4 and BMP2.
[0351] As used herein, the term "HGF" means Hepatocyte Growth
Factor, preferably of human and/or recombinant origin.
[0352] As used herein, the term "EGF" means Epidermal Growth
Factor, preferably or human and/or recombinant origin.
[0353] As used herein, the "HNF4alpha", or "HNF4a", used
interchangeably are intended to mean hepatocyte nuclear factor 4
also known as NR2A1 (nuclear receptor subfamily 2, group A, member
1), a transcription factor regulating gene expression in endodermal
derived tissue, e.g. the liver, pancreatic islets, and adipocytes.
The encoded protein controls the expression of several genes,
including hepatocyte nuclear factor 1 alpha.
[0354] As used herein, the term "MDR" is intended to mean
multi-drug resistance transporter. MDR 1 and 3 are members of the
ATP-binding cassette (ABC) family of transporters and both are drug
efflux transporters. MDR 1 is important in regulating the traffic
of drugs, peptides and xenobiotics into the body and in protecting
the body against xenobiotic insults and drug toxicity, while MDR 3
is essential for phospholipid secretion into bile.
[0355] As used herein, the term "Activin" is intended to mean a
TGF-beta family member that exhibits a wide range of biological
activities including regulation of cellular proliferation and
differentiation such as "Activin A" or "Activin B". Activin belongs
to the common TGF-beta superfamiliy of ligands.
[0356] As used herein, the term "activator of a retinoic acid
responsive receptor" is intended to mean a compound capable of
binding to and activating a human retinoic acid receptor (RAR)
and/or retinoid X receptor (RXRs).
[0357] As used herein, the term "retinoic acid receptor" or "RAR"
is intended to mean a member of the family of retinoic acid
receptors, in particular RAR-alpha, RAR-beta, and RAR-gamma, which
are encoded by the RARA, RARB, RARG genes, respectively. Each
receptor isoform has several splice variants: two for alpha, four
for beta, and two for gamma. These isoforms are also included in
the definition of a "retinoic acid receptor".
[0358] As used herein, the term "retinoic acid" is intended to mean
a retinoic acid isomer, including but not limited to
all-trans-retinoic acid, 7-cis-retinoic acid, 9-cis retinoic acid,
11-cis-retinoic acid and 13-cis retinoic.
[0359] As used herein, the term "inhibitor of a cyclin dependent
kinase" or "CDK inhibitor" is intended to mean a compound capable
of inhibiting the function (e.g., the activity) of a cyclin
dependent kinase, such as cyclin dependent kinase 2 (CDK2).
[0360] As used herein, the term "ROCK inhibitor" is intended to
mean an inhibitor of ROCK Rho-associated protein kinase
activity
[0361] As used herein, the term "matrix" is intended to refer to
any component, either isolated or in combination, which forms part
of the normal mammalian extracellular matrix environment. Such
matrix components include, but are not limited to, collagen,
fibronectin, and laminin and may be from natural or synthetic
sources.
[0362] As used herein, the term "overlay" is intended to refer to a
layer of, e.g., extracellular matrix components, which is applied
on top of the cultured cells.
[0363] As used herein, the term "coating" is intended to refer to a
layer of, e.g., extracellular matrix components, which covers the
surface of a culture vessel and on which the cells are
cultured.
[0364] As used herein the term "xeno-free" is intended to mean
complete circumvention of direct or in-direct exposure to non-human
animal components.
[0365] As used herein, the term "hepatocellular toxicity" indicates
cellular responses such as necrotic toxicity, apoptosis,
mitochondrial toxicity, phospholipidosis, steatosis and bile acid
transport.
BRIEF DESCRIPTION OF THE DRAWINGS
[0366] FIGS. 1A-1U: Effect of treatment with 10Z-Heptadecenoic acid
(A,B), Arachidonic acid (C,D), Calcifediol (E,F), Calcitriol (E,F),
Cholecalciferol (G,H), CGP 52608 (1,J), Docosahexaenoic acid (K,L),
PP1 (M,N,O,P), PP2 (Q,R), D-erythro-Sphingosine (S,T), and
Tetradecanoic acid (U) on CYP activities of hiPSC-derived
hepatocytes on day 29 and/or 36 of the differentiation
protocol.
[0367] Abbreviations: CGP=CGP 52608; CYP=Cytochrome P450;
Sphingosine=D-erythro-Sphingosine.
[0368] FIGS. 2A-2H: Effect of treatment with 10Z-Heptadecenoic acid
(A,B), Arachidonic acid (A,B), Calcitriol (E,F), Cholecalciferol
(A,B), CGP 52608 (A,B), PP1 (A,B), D-erythro-Sphingosine (A,B), 13
cis-RA (C,D), L-erythro MAPP (C,D), PAF C16 (C,D), C16 Ceramide
(C,D), and combinations of these compounds (A,B,E,F,G,H) on CYP
activities of hiPSC-derived hepatocytes on day 29 and/or 36 of the
differentiation protocol.
[0369] FIGS. 2I-2K: Effect of treatment with 10Z-Heptadecenoic acid
(I,J) and a combination of CGP 52608, PP1, Cholecalciferol,
Arachidonic acid, 10Z-Heptadecenoic acid, and D-erythro-Sphingosine
(K) on mRNA expression levels of the nuclear receptor PXR (I,K) and
the transporter protein OATP1E1 (J).
[0370] Abbreviations: 10Z=10Z-Heptadecenoic acid; 13 cis=13 cis
retinoic acid; Ara=Arachidonic acid; Caltr=Calcitriol; Ceramide=C16
Ceramide; CGP=CGP 52608; Chole=Cholecalciferol; CYP=Cytochrome
P450; MAPP=L-erythro MAPP; PAF=platelet activating factor;
OATP11=organic anion-transporting polypeptide 1B1; PXR=pregnane X
receptor; Sph=D-erythro-Sphingosine.
[0371] FIGS. 3A-3D: Effect of treatment with 10Z-Heptadecenoic
acid, Arachidonic acid, Calcitriol, Cholecalciferol, CGP 52608,
PP1, and D-erythro-Sphingosine starting at different time points of
the differentiation protocol on CYP activities of hiPSC-derived
hepatocytes on day 29 (A,B) and 36 (C,D) of the differentiation
protocol.
[0372] Abbreviations: 10Z=10Z-Heptadecenoic acid; Ara=Arachidonic
acid; Caltr=Calcitriol; CGP=CGP 52608; Chole=Cholecalciferol;
CYP=Cytochrome P450; Sph=D-erythro-Sphingosine.
[0373] FIGS. 4A-4N: Effect of treatment of hepatocytes derived from
the hiPS cell lines ChiPSC4 (A,B), ChiPSC6b (C,D), ChiPSC22 (E,F),
P11015 (G,H), P11021 (1,J), P11032 (K,L), and the hES cell line
SA121 (M,N) with 10Z-Heptadecenoic acid, Arachidonic acid,
Calcitriol, Cholecalciferol, CGP 52608, PP1, and
D-erythro-Sphingosine on day 29 of the differentiation
protocol.
[0374] Abbreviations: 10Z=10Z-Heptadecenoic acid; Ara=Arachidonic
acid; Caltr=Calcitriol; CGP=CGP 52608; Chole=Cholecalciferol;
CYP=Cytochrome P450; Sph=D-erythro-Sphingosine.
[0375] FIGS. 5A-5B: Effect of treatment with 10Z-Heptadecenoic
acid, Arachidonic acid, Calcitriol, Cholecalciferol, CGP 52608,
PP1, and D-erythro-Sphingosine, and with or without Oncostatin M
and/or HGF on CYP1A, 3A, and 2C9 (A) and CYP2B6, 2D6, and 2C19 (B)
activities of hiPSC-derived hepatocytes on day 31 of the
differentiation protocol.
[0376] Abbreviations: CYP=Cytochrome P450; HGF=hepatocyte growth
factor; OSM=Oncostatin M.
EXAMPLES
[0377] Examples of general culturing and passaging techniques are
disclosed in applications WO2004/099394, WO2003/055992,
WO/2007/042225, WO2007/140968 and WO2011116930.
[0378] As laid out in the following examples, the starting material
may comprise any hepatic progenitor cell type, particularly one
derived through an initial differentiation towards a definitive or
extraembryonic lineage from a mammalian pluripotent stem cell, such
as a human pluripotent stem cell. The starting material may also be
any cell of hepatic progenitor lineage.
Example 1: Maintenance of hPS Cell Types
[0379] All hPS cells (as defined above) can be used as staring
material for this invention. For the examples below in particular
hepatocytes were derived in vitro from undifferentiated human
embryonic stem cells (hESC) established on mEF feeder cells (Heins
et al 2004) and maintained under feeder-free conditions. The cell
lines used for this experiment could be, but are not limited to the
hES cell lines SA121, SA167, SA181, SA461 (Cellartis AB, Goteborg,
Sweden) and they can be propagated as described by Heins et al.
2004 and Caisander et al. 2006.
[0380] Along with hPS obtained from hESC, hiPS (human induced
pluripotent stem) cells have also been used for the derivation of
hepatocytes for the examples of this invention.
[0381] The hiPSC line ChiPSC4 used in this invention was derived as
followed: Human dermal fibroblasts (CRL2429, ATCC) were maintained
in DMEM supplemented with 10% fetal bovine serum, 1.times.
glutamax, 5 U/ml penicillin and 5 .mu.g/ml streptomycin at
37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in air.
Fibroblasts were tranduced with recombinant lentiviruses encoding
mouse Oct4, Sox2, Klf4 and c-myc and cultured for 5 days. The
transduced cells were then dispersed with trypsin and seeded onto
mitomycin C treated human dermal fibroblast feeder cells at a
density of 5.times.10.sup.3 cells/cm.sup.2 in their normal growth
medium. After 24 hours the medium was replaced with knockout DMEM
supplemented with 20% knockout serum replacement, 1.times.
non-essential amino acids, 1.times. glutamax, 5 U/ml penicillin, 5
.mu.g/ml streptomycin, 100 .mu.M 2-mercaptoethanol and 30 ng/ml
bFGF at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in
air. Half of the volume of medium was replaced every day and
colonies of iPS cells emerged after approximately 30 days. iPS
colonies were picked, expanded in DEF-CS.TM., and cell banks
prepared. The banked cells were then characterised to check for the
expression of endogenous Oct4, Sox2, Klf4 and c-Myc, silencing of
transgenes, potential to differentiate into cell types
representative of all three germ layers in vitro, and to confirm
their authenticity by STR profiling and comparison with the
parental fibroblast cell line (ATCC).
[0382] The hiPSC line ChiPSC6b used in this invention was derived
from fibroblast line P11031 (Cellectis SA) by transfecting with
episomal vectors encoding OCT4, SOX2, KLF4, LIN28, and L-MYC using
electroporation (Neon transfection system; Invitrogen).
[0383] All other hiPSC lines used in this invention were obtained
from Cellectis SA and were derived from either peripheral blood
cells (P11021) or adult dermal fibroblasts (ChiPSC18, ChiPSC22,
P11015, P11021, P11032) using either episomal (P11021) or
retroviral reprogramming (ChiPSC18, ChiPSC22, P11015, P11021,
P11032) with OCT4, SOX2, KLF4, LIN28, and L-MYC. These 6 hiPSC
lines were initially established in a Matrigel-based culture system
and subsequently transferred to the DEF-CS.
[0384] Alternatively to reprogramming using lentivirus, retrovirus
and episomal vectors, hiPSC lines can also be reprogrammed using
Sendai virus, adenovirus, proteins and mRNAs or other techniques.
Other suitable cell lines for use are those established by Chung et
al. (2008), such as cell lines MA126, MA127, MA128 and MA129
(Advanced Cell Technology, Inc. Worcester, Mass., USA), which all
are listed with the International stem cell registry. These cell
lines have been derived (or obtained) without destruction of the
human embryo by employing a single blastomere removal
technique.
[0385] All hPSC lines used in this invention were cultured under
standard conditions in the DEF-CS with continuous passaging twice a
week and were immuno-positive for OCT4, TRA1-60, TRA1-81, and
SSEA-4, and immuno-negative for SSEA-1. Pluripotency was confirmed
by in vitro differentiation. Karyotyping as described by Caisander
et al. 2006 showed a normal chromosomal profile.
Example 2: Differentiation of hPS Cell Types to Produce
Hepatocytes
[0386] Hepatocytes may be derived from hPS cells by employing the
following exemplary basic protocols A and B:
[0387] Protocol A:
[0388] Undifferentiated hPS cells are dissociated and seeded
directly in day 0-medium onto a Fibronectin-based coating. The
different mediums were prepared freshly and added day 0, 1, 2, 3,
4, 5, 7 and then every second or third day during the pre-hepatic
phase, and differentiation and maturation phase.
[0389] Day 0
[0390] Pre-treatment medium
[0391] 3 .mu.M CHIR99021
[0392] 5 .mu.M ROCK inhibitor
[0393] Day 1
[0394] RPMI 1640 (+0.1% PEST+1% Glutamax)
[0395] 1.times.B27
[0396] 50 ng/ml Activin A
[0397] 3 .mu.M CHIR99021
[0398] 5 .mu.M LY294002
[0399] 3 .mu.M CHIR99021
[0400] Day 2
[0401] RPMI 1640 (+0.1% PEST+1% Glutamax)
[0402] 1.times.B27
[0403] 50 ng/ml Activin A
[0404] 5 .mu.M LY294002
[0405] 10 nM 5-aza-2-deoxycytidine
[0406] Day 3
[0407] RPMI 1640 (+0.1% PEST+1% Glutamax)
[0408] 1.times.B27
[0409] 50 ng/ml Activin A
[0410] Day 4-7
[0411] RPMI 1640 (+0.1% PEST+1% Glutamax)
[0412] 1.times.B27
[0413] 50 ng/ml Activin A
[0414] The pre-treatment medium is available from Takara Bio Europe
AB (Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden) upon
request.
[0415] On day 7 the cells are passaged. The cells are incubated for
3-7 minutes with TrypLE Select at 37.degree. C., the same volume of
BasHES is added and the cell suspension is centrifuged at 200-300
g, 5-6 min. Thereafter, the cells are replated onto a
Fibronectin-based coating at a cell density of 50 000-350 000
cells/cm.sup.2 such as e.g. 75 000-300 000 cells/cm.sup.2,
preferably 100 000 cells/cm.sup.2. The Fibronectin-based coating
has a concentration of 7.5 .mu.g Fibronectin per cm.sup.2 culture
area. To prepare the coating, 50 .mu.l of a 1 mg/ml Fibronectin
stock is added per ml DPBS, and 150 .mu.l of this coating solution
is added per cm.sup.2 culture area.
[0416] Day 7-14 (pre-hepatic)
[0417] Knockout-DMEM+1% PEST+1% Glutamax
[0418] 20% Knockout-Serum Replacement
[0419] 1% non-essential amino acids (NEAA)
[0420] 0.2% beta-mercaptoethanol
[0421] 1% DMSO
[0422] Day 14-45 (differentiation and maturation)
[0423] WME+1% Glutamax+0.1% PEST
[0424] 0.55 mg/mL BSA-FAF
[0425] 0.025 mg/mL Ascorbic Acid
[0426] 0.67 .mu.g/mL Hydrocortisone Hemisuccinate
[0427] 10 .mu.g/mL Transferrin
[0428] 5 .mu.g/mL Insulin
[0429] 0.003 .mu.g/mL EGF
[0430] 0.1 .mu.M DexM
[0431] 10 ng/ml OsM
[0432] ng/ml HGF
[0433] 0.5% DMSO
[0434] 1.4 .mu.M BIO
[0435] 0.5 .mu.M Kenpaullone
[0436] 0.2 .mu.M 9cis retinoic acid
[0437] On day 14 and 16, matrix overlays are performed. To this
end, 53 .mu.l of a 1 mg/ml Fibronectin stock and 9 .mu.l of a 3
mg/ml Collagen I stock are added per ml day 14-45 medium (RT), the
medium is mixed well and then a regular medium change is performed.
The addition of the matrix components corresponds to the addition
of 25 .mu.g Fibronectin and 12.5 .mu.g Collagen I per cm.sup.2
culture area per overlay addition.
[0438] Protocol B:
[0439] Undifferentiated hPS cells are dissociated and seeded
directly in day 0-medium onto a Fibronectin-based coating. The
different mediums were prepared freshly and added day 0, 1, 2, 3,
4, 5, 7 and then every second or third day during the pre-hepatic
phase, and differentiation and maturation phase.
[0440] Day 0
[0441] Pre-treatment medium
[0442] 3 .mu.M CHIR99021
[0443] 5 .mu.M ROCK inhibitor
[0444] Day 1
[0445] RPMI 1640 (+0.1% PEST+1% Glutamax)
[0446] 1.times.B27
[0447] 50 ng/ml Activin A
[0448] 3 .mu.M CHIR99021
[0449] 5 .mu.M LY294002
[0450] 3 .mu.M CHIR99021
[0451] Day 2
[0452] RPMI 1640 (+0.1% PEST+1% Glutamax)
[0453] 1.times.B27
[0454] 50 ng/ml Activin A
[0455] 5 .mu.M LY294002
[0456] 10 nM 5-aza-2-deoxycytidine
[0457] Day 3
[0458] RPMI 1640 (+0.1% PEST+1% Glutamax)
[0459] 1.times.B27
[0460] 50 ng/ml Activin A
[0461] Day 4-7
[0462] RPMI 1640 (+0.1% PEST+1% Glutamax)
[0463] 1.times.B27
[0464] 50 ng/ml Activin A
[0465] The pre-treatment medium is available from Takara Bio Europe
AB (Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden) upon
request.
[0466] On day 7 the cells are passaged. The cells are incubated for
3-7 minutes with TrypLE Select at 37.degree. C., the same volume of
BasHES is added and the cell suspension is centrifuged at 200-300
g, 5-6 min. Thereafter, the cells are replated onto a Collagen
I-Fibronectin-based coating at a cell density of 50 000-350 000
cells/cm.sup.2 such as e.g. 75 000-300 000 cells/cm.sup.2,
preferably 100 000 cells/cm.sup.2. The Collagen I-Fibronectin-based
coating has a concentration of 2 .mu.g Fibronectin and 10 .mu.g
Collagen I per cm.sup.2 culture area. To prepare the coating, 12
.mu.l of a 1 mg/ml Fibronectin stock and 21.5 .mu.l of a 3 mg/ml
Collagen I stock is added per ml DPBS, and 150 .mu.l of this
coating solution is added per cm.sup.2 culture area.
[0467] Day 7-14 (pre-hepatic)
[0468] Knockout-DMEM+1% PEST+1% Glutamax
[0469] 20% Knockout-Serum Replacement
[0470] 1% non-essential amino acids (NEAA)
[0471] 0.2% beta-mercaptoethanol
[0472] 1% DMSO
[0473] Day 14-45 (differentiation and maturation)
[0474] WME+1% Glutamax+0.1% PEST
[0475] 0.55 mg/mL BSA-FAF
[0476] 0.025 mg/mL Ascorbic Acid
[0477] 0.67 .mu.g/mL Hydrocortisone Hemisuccinate
[0478] 10 .mu.g/mL Transferrin
[0479] 5 .mu.g/mL Insulin
[0480] 0.003 .mu.g/mL EGF
[0481] 0.1 .mu.M DexM
[0482] 10 ng/ml OsM
[0483] 20 ng/ml HGF
[0484] 0.5% DMSO
[0485] 1.4 .mu.M BIO
[0486] 0.5 .mu.M Kenpaullone
[0487] 0.2 .mu.M 9cis retinoic acid
[0488] On day 14 and 16, matrix overlays are performed. To this
end, 12 .mu.l of a 1 mg/ml Fibronectin stock solution and 21.5
.mu.L of a 3 mg/ml Collagen I stock solution are added per ml day
14-45 medium (RT), the medium is mixed well and then a regular
medium change is performed. The addition of the matrix components
corresponds to the addition of 6 .mu.g Fibronectin and 31.25 .mu.g
Collagen I per cm.sup.2 culture area per overlay addition. Example
3: Effect of treatment of hiPSC-derived hepatocytes with
10Z-Heptadecenoic acid, Arachidonic acid, Calcifediol, Calcitriol,
Cholecalciferol, CGP 52608, Docosahexaenoic acid, PP1, PP2,
D-erythro-Sphingosine, and Tetradecanoic acid.
[0489] Procedure:
[0490] Following the basic protocol A, hiPS cell derived
hepatocytes cultured on a Fibronectin-based coating were treated
with 0.5 or 5 .mu.M 10Z-Heptadecenoic acid, 0.5 or 5 .mu.M
Arachidonic acid, 5 .mu.M Calcifediol, 5 .mu.M Calcitriol, 0.2 or
0.5 .mu.M Cholecalciferol, 5 or 50 .mu.M CGP 52608, 0.5 or 5 .mu.M
Docosahexaenoic acid, 0.5, 1.25, 2.5, 5, or 10 .mu.M PP1, 0.5 or 5
.mu.M PP2, 0.5 or 5 .mu.M D-erythro-Sphingosine, or 0.5 or 5 .mu.M
Tetradecanoic acid from day 21 of the differentiation protocol and
onwards (FIG. 1).
[0491] On day 29 and 36 of the differentiation protocol, the cell
cultures are subjected to a CYP activity assay according to the
following protocol: Cells are washed twice with warm Williams's
medium E w/o phenol red (+0.1% PEST). Then CYP activity assay,
consisting of warm Williams medium E w/o phenol red (+0.1% PEST), 2
mM L-Glutamine, 25 mM HEPES, 10 .mu.M Phenacetin (model substrate
for CYP1A), 10 .mu.M Bupropion (model substrate for 2B6), 10 .mu.M
Diclofenac (model substrate for CYP2C9), 10 .mu.M Bufuralol (model
substrate for 2D6), and 5 .mu.M Midazolam (model substrate for
CYP3A), is added to the cells (e.g. 110 .mu.l/cm.sup.2) and
incubated for 16 hr at 37.degree. C. Then 100 .mu.l of the
supernatant is transferred into a 96 well plate which is sealed
with a tight seal tape and stored at -80.degree. C. until
LC/MS-analysis of metabolite formation: Acetaminophen (Paracetamol)
for CYP1A, OH-Bupropion for CYP2B6, OH-Diclofenac for CYP2C9,
OH-Bufuralol for CYP2D6, and OH-Midazolam for CYP3A.
[0492] Results:
[0493] FIGS. 1A-1U) 10Z-Heptadecenoic acid, Arachidonic acid,
Calcifediol, Calcitriol, Cholecalciferol, CGP 52608,
Docosahexaenoic acid, PP1, PP2, D-erythro-Sphingosine, and
Tetradecanoic acid increase CYP activity levels in hiPS
cell-derived hepatocytes both on day 29 and 36 of the
differentiation protocol. In some cases, 2B6 activity levels were
below the detection level.
[0494] These eleven compounds increase CYP activity levels in hiPS
cell-derived hepatocytes. Therefore the skilled person wishing to
improve CYP activity may select from these compounds according to
their interest.
Example 4: Effect of Treatment of hiPSC-Derived Hepatocytes with
10Z-Heptadecenoic Acid, Arachidonic Acid, Calcitriol,
Cholecalciferol, CGP 52608, PP1, D-Erythro-Sphingosine, 13 Cis-RA,
L-Erythro MAPP, PAF C16, C16 Ceramide, and Combinations of these
Compounds
[0495] Procedure:
[0496] Following the basic protocol B, ChiPSC4-derived hepatocytes
cultured on a Collagen I-Fibronectin-based coating were treated
with 0.5 .mu.M 10Z-Heptadecenoic acid, 0.5 .mu.M Arachidonic acid,
0.5 .mu.M Calcitriol, 5 .mu.M CGP 52608, 0.2 .mu.M Cholecalciferol,
5 .mu.M PP1, 0.5 .mu.M D-erythro-Sphingosine, 0.2 .mu.M 13 cis-RA,
0.5 .mu.M L-erythro MAPP, 0.5 .mu.M PAF C16, 0.5 .mu.M C16
Ceramide, or different combinations of these compounds from day 21
of the differentiation protocol and onwards (FIG. 2).
[0497] On day 29 of the differentiation protocol, the cell cultures
are subjected to a CYP activity assay according to the protocol
described in Example 3. In some experiments, 50 .mu.M Mephenytoin
(model substrate for 2C19) was included additionally to the
substrates mentioned above and then the formation of the metabolite
OH-Mephenytoin was measured by LC/MS in order to determine CYP2C19
activity.
[0498] Results:
[0499] FIGS. 2A-2H) 10Z-Heptadecenoic acid (10Z), Arachidonic acid
(Ara), Calcitriol (Caltr), Cholecalciferol (Chole), CGP 52608
(CGP), PP1, D-erythro-Sphingosine (Sph), 13 cis-RA (13 cis),
L-erythro MAPP (MAPP), PAF C16 (PAF), C16 Ceramide (Cera), or
different combinations of these compounds increase CYP activity
levels in hiPS cell-derived hepatocytes on day 29 of the
differentiation protocol. In some cases, 2B6 activity levels were
below the detection level.
[0500] FIGS. 2I-2J) Protocol B and protocol B plus
10Z-Heptadecenoic acid increase OATP1B1 and PXR mRNA levels in hiPS
cell-derived hepatocytes on day 36 of the differentiation protocol
compared to protocol A.
[0501] FIG. 2K) Protocol B plus CGP 52608, PP1, Cholecalciferol,
Arachidonic acid, 10Z-Heptadecenoic acid, and Sphingosine increase
PXR mRNA levels in hiPS cell-derived hepatocytes on day 29 and 36
of the differentiation protocol compared to protocol A.
[0502] These eleven compounds increase CYP activity levels in hiPS
cell-derived hepatocytes. Therefore the skilled person wishing to
improve CYP activity may select from these compounds according to
their interest.
Example 5: Effect of Treatment of hiPSC-Derived Hepatocytes with
10Z-Heptadecenoic Acid, Arachidonic Acid, Calcitriol,
Cholecalciferol, CGP 52608, PP1, and D-Erythro-Sphingosine Starting
at Different Time Points of the Differentiation Protocol
[0503] Procedure:
[0504] Following the basic protocol B, ChiPSC4-derived hepatocytes
cultured on a Collagen I-Fibronectin-based coating were treated
with 0.5 .mu.M 10Z-Heptadecenoic acid, 0.5 .mu.M Arachidonic acid,
0.5 .mu.M Calcitriol, 5 .mu.M CGP 52608, 0.2 .mu.M Cholecalciferol,
5 .mu.M PP1, and 0.5 .mu.M D-erythro-Sphingosine starting on day
11, 14, 21, 25 and 28, respectively, of the differentiation
protocol and onwards (FIG. 3).
[0505] On day 29 and 36 of the differentiation protocol, the cell
cultures are subjected to a CYP activity assay according to the
following described in Example 3.
[0506] Results:
[0507] FIGS. 3A-3D) Treatment with 10Z-Heptadecenoic acid (10Z),
Arachidonic acid (Ara), Calcitriol (Caltr), Cholecalciferol
(Chole), CGP 52608 (CGP), PP1, and D-erythro-Sphingosine (Sph)
starting between day 11 and 28 of the differentiation protocol
increases CYP activity levels in hiPS cell-derived hepatocytes on
day 29 and 36 of the differentiation protocol.
[0508] The treatment with the seven compounds starting between day
11 and 28 of the differentiation protocol increases CYP activity
levels in hiPS cell-derived hepatocytes. Therefore the skilled
person wishing to improve CYP activity may select from these
different time points to start the treatment according to their
interest.
Example 6: Effect of Treatment of Hepatocytes Derived from Several
hiPS and hES Cell Lines with 10Z-Heptadecenoic Acid, Arachidonic
Acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and
D-Erythro-Sphingosine
[0509] Procedure:
[0510] Following the basic protocol B, hepatocytes derived from the
hiPS cell lines ChiPSC4, ChiPSC6b, ChiPSC22, P11015, P11021, P11032
and the hES cell line SA121 were cultured on a Collagen
I-Fibronectin-based coating and treated with a combination of 0.5
.mu.M 10Z-Heptadecenoic acid, 0.5 .mu.M Arachidonic acid, 0.5 .mu.M
Calcitriol, 5 .mu.M CGP 52608, 0.2 .mu.M Cholecalciferol, 5 .mu.M
PP1, and 0.5 .mu.M D-erythro-Sphingosine from day 21 of the
differentiation protocol and onwards (FIG. 4).
[0511] On day 29 of the differentiation protocol, the cell cultures
are subjected to a CYP activity assay according to the following
protocol described in Example 3. In some experiments, 50 .mu.M
Mephenytoin (model substrate for 2C19) was included additionally to
the substrates mentioned above and then the formation of the
metabolite OH-Mephenytoin was measured by LC/MS in order to
determine CYP2C19 activity.
[0512] Results:
[0513] FIGS. 4A-4N) The treatment with 10Z-Heptadecenoic acid
(10Z), Arachidonic acid (Ara), Calcitriol (Caltr), Cholecalciferol
(Chole), CGP 52608 (CGP), PP1, and D-erythro-Sphingosine (Sph)
increases CYP activity levels in hepatocytes derived from the hiPS
cell lines ChiPSC4 (A,B), ChiPSC6b (C,D), ChiPSC22 (E,F), P11015
(G,H), P11021 (1,J), P11032 (K,L) and the hES cell line SA121 (M,N)
on day 29 of the protocol.
[0514] These seven compounds increase CYP activity levels in both
hiPS and hES cell-derived hepatocytes. Therefore the skilled person
wishing to improve CYP activity may select from these compounds
according to their interest.
Example 7: Effect of Treatment of hiPSC-Derived Hepatocytes with
10Z-Heptadecenoic Acid, Arachidonic Acid, Calcitriol,
Cholecalciferol, CGP 52608, PP1, and D-Erythro-Sphingosine with or
without the Addition of Oncostatin M and/or HGF
[0515] Procedure:
[0516] Following the basic protocol B, hepatocytes derived from the
hiPS cell line ChiPSC18 were cultured on a Collagen
I-Fibronectin-based coating and treated with a combination of 0.5
.mu.M 10Z-Heptadecenoic acid, 0.5 .mu.M Arachidonic acid, 0.5 .mu.M
Calcitriol, 5 .mu.M CGP 52608, 0.2 .mu.M Cholecalciferol, 5 .mu.M
PP1, and 0.5 .mu.M D-erythro-Sphingosine and with or without
Oncostatin M and/or HGF from day 21 of the differentiation protocol
and onwards (FIG. 5).
[0517] On day 31 of the differentiation protocol, the cell cultures
are subjected to a CYP activity assay according to the following
protocol described in Example 3. 50 .mu.M Mephenytoin (model
substrate for 2C19) was included additionally to the substrates
mentioned above and the formation of the metabolite OH-Mephenytoin
was measured in order to determine CYP2C19 activity.
[0518] Results:
[0519] FIGS. 5A-5B) The treatment with 10Z-Heptadecenoic acid,
Arachidonic acid, Calcitriol, Cholecalciferol, CGP 52608, PP1, and
D-erythro-Sphingosine, and with or without Oncostatin M and/or HGF
results in similar CYP activity levels in hepatocytes derived from
the hiPS cell line ChiPSC18 on day 31 of the protocol.
[0520] These seven compounds increase CYP activity levels in both
hiPS and hES cell-derived hepatocytes independent of the presence
of Oncostatin M and/or HGF. Therefore, the skilled person wishing
to improve CYP activity may select from these compounds according
to their interest and include or exclude Oncostatin M and/or
HGF.
REFERENCES
[0521] Brolen, G. et al. (2010) Hepatocyte-like cells derived from
human embryonic stem cells specifically via definitive endoderm and
a progenitor stage. J Biotechnol. 1; 145(3):284-94 [0522] Chen, Y.
F. et al. (2012) Rapid generation of mature hepatocyte-like cells
from human induced pluripotent stem cells by an efficient
three-step protocol. Hepatology. 2012 55(4):1193-203 [0523] Chung,
Y. et al. (2008) Human Embryonic Stem Cell Lines Generated without
Embryo Destruction. doi: 10.1016/j.stem.2007.12.013 [0524] Duan, Y.
et al. Differentiation and characterization of metabolically
functioning hepatocytes from human embryonic stem cells. Stem
Cells. 28(4):674-86 [0525] Dunn, J et al. (1991) Long-term in Vitro
function of adult hepatocytes in a collagen sandwich configuration.
Biotechnol. Prog. 7:237-245 [0526] D'Amour K. A. et al. (2005)
Efficient differentiation of human embryonic stem cells to
definitive endoderm. Nat Biotechnology. 23(12):1534-41. [0527]
Funakoshi, N. et al. (2011) Comparison of hepatic-like cell
production from human embryonic stem cells and adult liver
progenitor cells: CAR transduction activates a battery of
detoxification genes. Stem Cell Rev. 7(3):518-31 [0528] Hay, D. et
al (2007) Direct differentiation of human embryonic stem cells to
hepatocyte-like cells exhibiting functional activities. Cloning
Stem Cells. 2007 Spring; 9(1):51-62. Erratum in: Cloning Stem
Cells. 2009 March; 11(1):209. [0529] Hay, D. et al (2008) Efficient
differentiation of hepatocytes from human embryonic stem cells
exhibiting markers recapitulating liver development in vivo. Stem
Cells. April; 26(4):894-902. [0530] Heins, N. et al (2004)
Derivation, characterization, and differentiation of human
embryonic stem cells. Stem Cells. 22(3):367-76. [0531] Klimanskaya,
I. et al (2006) Human embryonic stem cell lines derived from single
blastomeres. Nature, November 23; 444(7118):481-5. Epub 2006 Aug.
23. Erratum in: Nature. 2006 Nov. 23; 444(7118):512. Nature. 2007
Mar. 15; 446(7133):342. [0532] Martin M. et al (2005) Human
embryonic stem cells express an immunogenic nonhuman sialic acid.
Nat Med. February; 11(2):228-32. [0533] Mercader, A. et al (2009)
Human Embryo Culture. Essential Stem Cell Methods, Chapter 16,
Academic Press, 1.sup.st Edition, Eds. Lanza, R. and Klimanskaya,
I. [0534] Page, J et al. (2007) Gene expression profiling of
extracellular matrix as an effector of human hepatocyte phenotype
in primary cell culture. Tox.Sci. 97(2):384-397 [0535] Si-Tayeb, K.
et al. (2010) Highly efficient generation of human hepatocyte-like
cells from induced pluripotent stem cells Hepatology.
51(1):297-305. [0536] Song. Z. et al. (2009) Efficient generation
of hepatocyte-like cells from human induced pluripotent stem cells.
Cell Res. 19(11):1233-42 [0537] Sullivan, G. J. et al. (2010)
Generation of functional human hepatic endoderm from human induced
pluripotent stem cells. Hepatology. 51(1):329-35. [0538] Siller, R.
et al. (2015) Small-molecule-driven hepatocyte differentiation of
human pluripotent stem cells. Stem Cell Reports 4(5):939-52. [0539]
Takahashi, K. et al (2007) Induction of pluripotent stem cells from
adult human fibroblasts by defined factors. Cell November 30;
131(5):861-72. [0540] Thomson, J. et al. (1998) Embryonic stem cell
lines derived from human blastocysts. Science. November 6;
282(5391):1145-7. Erratum in: Science 1998 Dec. 4; 282(5395):1827.
[0541] Turner, R. et al. (2011) Human hepatic stem cell and
maturational liver lineage biology. Hepatology. 53(3):1035-45
[0542] Wang, Y. et al. (2011) Lineage restriction of human hepatic
stem cells to mature fates is made efficient by tissue-specific
biomatrix scaffolds. Hepatology. 53(1):293-305. [0543] Yu, J. and
Thomson, J. (2009) Induced Puripotent Stem Cell Derivation.
Essentials of Stem Cell Biology, Chapter 37, Academic Press,
2.sup.nd Edition (2009), Eds. Lanza, R. et al. [0544] Zhou H. et al
(2009). Generation of induced pluripotent stem cells using
recombinant proteins. Cell Stem Cell. 4(5):381-4.
* * * * *