U.S. patent application number 16/346188 was filed with the patent office on 2019-10-03 for liver organoid compositions and methods of making and using same.
The applicant listed for this patent is Children's Hospital Medical Center. Invention is credited to Masaki Kimura, Hiroyuki Koike, Tadahiro Shinozawa, Takanori Takebe.
Application Number | 20190298775 16/346188 |
Document ID | / |
Family ID | 62076362 |
Filed Date | 2019-10-03 |
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United States Patent
Application |
20190298775 |
Kind Code |
A1 |
Takebe; Takanori ; et
al. |
October 3, 2019 |
LIVER ORGANOID COMPOSITIONS AND METHODS OF MAKING AND USING
SAME
Abstract
Disclosed are methods of inducing formation of a liver organoid
from precursor cells, such as iPSC cells. The disclosed liver
organoids may be used for screening for a serious adverse event
(SAE), such as liver failure and/or drug induced liver injury
(DILL), and/or drug toxicity. The disclosed liver organoids may
also be used to treat an individual having liver damage, or for
identifying a preferred therapeutic agent.
Inventors: |
Takebe; Takanori;
(Cincinnati, OH) ; Shinozawa; Tadahiro; (Fujisawa,
JP) ; Koike; Hiroyuki; (Tokyo, JP) ; Kimura;
Masaki; (Cincinnati, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Hospital Medical Center |
Cincinnati |
OH |
US |
|
|
Family ID: |
62076362 |
Appl. No.: |
16/346188 |
Filed: |
November 3, 2017 |
PCT Filed: |
November 3, 2017 |
PCT NO: |
PCT/US17/59845 |
371 Date: |
April 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62517414 |
Jun 9, 2017 |
|
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62417371 |
Nov 4, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0696 20130101;
A61P 1/16 20180101; C12N 2500/36 20130101; A61K 9/0029 20130101;
A61K 45/06 20130101; C12N 5/0672 20130101; G01N 33/5008 20130101;
C12N 2506/45 20130101; A61K 35/407 20130101; A61K 31/575 20130101;
C12N 5/0671 20130101; C12N 5/0697 20130101; C12N 2501/119 20130101;
C12N 15/01 20130101 |
International
Class: |
A61K 35/407 20060101
A61K035/407; C12N 5/074 20060101 C12N005/074; C12N 5/071 20060101
C12N005/071; A61P 1/16 20060101 A61P001/16 |
Claims
1. A method of inducing formation of a liver organoid from iPSC
cells, comprising the steps of a) contacting definitive endoderm
(DE) derived from said iPSC cells with a FGF pathway activator and
a GSK3 inhibitor, for a period of time sufficient to form posterior
foregut spheroids; b) incubating said posterior foregut spheroids
of step a in the presence of retinoic acid (RA) for a period of
time sufficient to form said liver organoid.
2. The method of claim 1, wherein said stem cells are human
iPSCs.
3. The method of claim 1, wherein said foregut spheroids are
embedded in a basement membrane matrix.
4. The method of claim 1, wherein said HLOs are characterized by
expression of alpha-fetoprotein (AFP), albumin (ALB), retinol
binding protein (RBP4), cytokeratin 19 (CK19), hepatocyte nuclear
factor 6 (HNF6), and cytochrome P450 3A4 (CYP3A4), HNF4a,
E-cadherin, DAPI, and Epcam.
5. The method of claim 1, wherein said HLOs have bile transport
activity.
6. A liver organoid derived from a stem cell, wherein said liver
organoid comprises a luminal structure comprising internalized
microvilli comprising mesenchymal cells; and wherein said luminal
structure is surrounded by polarized hepatocytes.
7. The liver organoid of claim 6 wherein said stem cell is a human
iPSC.
8. The liver organoid of claim 6 wherein said liver organoid
comprises functional stellate cells and functional Kupffer
cells.
9. The liver organoid of claim 6 wherein said liver organoid is
characterized by having one or more characteristics selected from
bile production capacity, bile transport activity, Complement
factor H expression of at least 50 ng/mL/1.times.e.sup.6 cells/24
hr, Complement factor B of at least 40 ng/mL/1.times.e.sup.6
cells/24 hr, C3 expression of at least 1000 ng/mL/1.times.e.sup.6
cells/24 hr; C4 expression of at least 1000 ng/mL/1.times.e.sup.6
cells/24 hr, fibrinogen production of at least 1,000
ng/mL/1.times.e.sup.6 cells/24 hr and albumin production of at
least 1,000 ng/mL/1.times.e.sup.6 cells/24 hr.
10. (canceled)
11. The liver organoid of claim 6, wherein said liver organoid
expresses one or more genes selected from PROX1, RBP4, CYP2C9,
CYP3A4, ABCC11, CFH, C3, C5, ALB, FBG, MRP2, ALCAM, CD68, CD34,
CD31.
12. The liver organoid of claim 6, wherein said HLO comprises a
drug metabolism cytochrome variant.
13. The liver organoid of claim 6, wherein said liver organoid does
not comprise inflammatory cells.
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. A method of treating an individual having liver damage,
comprising implanting a liver organoid into said individual.
19. The method of claim 18, wherein said liver damage is selected
from metabolic liver disease, end stage liver disease, or a
combination thereof.
20. A method of identifying a preferred therapeutic agent for an
individual, comprising contacting a liver organoid derived from an
iPSC of interest with a candidate compound.
21. (canceled)
22. (canceled)
23. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of U.S.
Provisional Patent Application 62/471,371, filed Nov. 4, 2016, and
62/517,414, filed Jun. 9, 2016, the contents of each are
incorporated by reference in their entirety for all purposes.
BACKGROUND
[0002] The liver is a vital organ that provides many essential
metabolic functions for life such as the detoxification of
exogenous compounds and coagulation as well as producing lipids,
proteins, ammonium, and bile. In vitro reconstitution of a
patient's liver may provide applications including regenerative
therapy, drug discovery and drug toxicity studies. Existing
methodology using liver cells exhibit extremely poor functionality,
largely due to a lack of essential anatomical structures, which
limits their practical use for the pharmaceutical industry.
[0003] Billions of dollars are lost annually from drug development
in the pharmaceutical industry due to the failures of drug
candidates identified in initial screens, and nearly a third of
drugs are withdrawn from the market due to such failures (Takebe
and Taniguchi, 2014). A failure of drug candidates results in a
tremendous loss of a patient's treatment opportunity. Preclinical
studies generally consist of an in vitro evaluation as a primary
efficacy screen to identify a "hit" compound, followed by safety
studies in vitro and in vivo to assess the mechanisms of metabolism
and toxicology. This inefficiency can be explained by the
substantial lack of physiologically relevant preclinical models
with high throughput in evaluating drug induced liver injury (DILI)
in humans and thus, an urgent need to develop an in vitro
humanistic screen model for the evaluation of the vast amounts of
continuously growing compound libraries.
[0004] Primary hepatocytes are a highly polarized metabolic cell
type, and form a bile canaliculi structure with microvilli-lined
channels, separating peripheral circulation from the bile acid
secretion pathway. The most upstream aspects of DILI include drug
(or their reactive metabolites) detoxification by hepatocytes and
excretion into bile canaliculi through transporters such as
multi-drug resistance-associated protein (MRP) transporters. This
suggests the need to reconstruct these uniquely organized
structures as a crucial property of hepatocytes in vivo for
predicting DILI pathology. However, there are considerable
differences in drug toxicity profiles between the current
simplified culture model with the use of isolated primary human
hepatocytes or hepatic cell lines, and in vivo physiology,
resulting in failed translation of drugs or drug discontinuation,
as in the case of Troglitazone, Nefazodone and Tolcapone
(https://livertox.nlm.nih.gov/index.html). The determination of
toxicological properties thus mainly relies on animals as an
essential step for drug development, however, due to the pronounced
differences in physiology between humans and animals, there is a
significant lack of fidelity to human outcomes (Leslie et al.,
2007; Yang et al., 2014). In addition, the onset of idiosyncratic
DILI (IDILI), which is very rare but nonetheless responsible for
about 10-15% of acute liver failures in the USA (Reuben et al.,
2010), is almost impossible to predict (Kullak-Ublick et al.,
2017). Collectively, effective human cell models are needed to
screen for compounds that test the detoxification and excretion of
proposed drugs.
[0005] Despite the progressive advancement of human hepatocyte
differentiation methods from pluripotent stem cell (PSC), clinical
trials in a dish using human stem cells remain `hype`. [Besides
drug screens for efficacy and/or toxicity, there is a need for
liver cell models for use in bio-artificial liver devices as a
bridge for transplant, for example, and for precision (personalized
medicine). The instant disclosure seeks to address one or more of
the aforementioned needs in the art.
BRIEF SUMMARY
[0006] Disclosed are methods of inducing formation of a liver
organoid from precursor cells, such as iPSC cells. The disclosed
liver organoids may be used for screening for a serious adverse
event (SAE), such as liver failure and/or drug induced liver injury
(DILI), and/or drug toxicity. The disclosed liver organoids may
also be used to treat an individual having liver damage, or for
identifying a preferred therapeutic agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Those of skill in the art will understand that the drawings,
described below, are for illustrative purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0008] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0009] FIG. 1. Generation of human liver organoid from iPSC with
luminal structure. A. Overview of the differentiation method for
liver organoid. B. Phase contrast image of human liver organoids C
Immunostaining for Albumin (ALB), Type IV collagen (Collagen IV)
and ZO-1 in organoids. Nuclei were stained with Haematoxylin
(blue). Bars, 50 .mu.m. D. Quantitative RT-PCR of Alpha-fetoprotein
(AFP), Albumin (ALB), Retinol-Binding Protein 4 (RBP4), Cytokeratin
19 (CK19), Hepatocyte nuclear factor 6 (HNF6) and Cytochrome P450
3A4 (CYP3A4) in undifferentiated iPS cells, organoids at Day 7, 11,
20 and 30 of differentiation and Primary hepatocytes (PH). Relative
expression values were compared with undifferentiated iPSCs (AFP,
ALB, RBP4 and CK19) or Day 7 organoids (HNF6) or Day 11 organoids
(CYP3A4). Bars represent the mean.+-.SD, n=3. E. Principal
component analysis based on RNA sequence data in undifferentiated
iPS cells (iPSC), Definitive endoderm (DE), Hepatic Specified cells
(HS), Hepatic Progenitor (HP), iPSC-Derived Cholangiocytes (iDC),
Normal Human Cholangiocyte (NHC), iPSC-derived Posterior foregut
(pFG), iPSC-derived human liver organoid, Primary hepatocytes,
Fetal liver tissue, Liver tissue and Right lobe of human Liver. F.
Albumin (ALB, n=10) and Fibrinogen (FBG, n=4) secretion levels from
organoids at Day 25-30. Bars represent the mean.+-.SEM. G.
Complement factors secretion level from organoids at Day 25-30. FH:
Factor H, FB: Factor B. Bars represent the mean.+-.SEM, n=5.
[0010] FIG. 2. Bile acid synthesis, uptake and excretion in human
iPSC liver organoid. A Immunostaining for Multidrug
resistance-associated protein 2 (MRP2) and Bile salt export pump
(BSEP) in a single organoid. Bars, 50 .mu.m. B. Transmission
electron micrograph of organoid showing microvilli (V)
intra-luminal surface; N: nuclei. Bars, 10 .mu.m. C. Quantitative
RT-PCR of ATP-binding cassette, sub-family B member 11 (ABCB11) and
Sodium taurocholate co-transporting polypeptide (NTCP) in
undifferentiated iPSCs, organoids at Day 20 (NTCP) and 30 (ABCB11)
of differentiation and Primary hepatocytes (PH). Relative
expression value was compared with undifferentiated iPS cells
(ABCB11) or Day 11 organoids (NTCP). Bars represent the mean.+-.SD,
n=3. D. Total bile acid secretion level inside organoids at day 27.
Bars represent the mean.+-.SEM, n=4. E. Bile acid uptake by
organoid after 30 min of culture in the presence of fluorescent
bile acid (CGamF). F. CLF transport activity on organoids derived
from 4 iPSC lines. T, W, 1 and F indicate clone name of iPS cell
lines. Green: CLF.
[0011] FIG. 3. Bosetan induced cholestasis is specific to CYP2C9*2
iPSC-liver organoids. A. Representative allele images of rs1799853
in CYP2C9*2 and rs4148323 in UGT1A1*6 show risk SNPs for DILI by
Bosentan and Irinotecan, respectively. The table indicates the
possession of risk alleles in each iPS cell line. B. Images of CLF
transport activity and inhibition by Bosentan. C. CLF intensity
levels in individual organoids derived from different 4 iPS cell
lines. *: p<0.01, **: p<1E-4, ****: p<1E-8,
Wilcoxon-Mann-Whitney Test. NS: not significant. In the box plots,
the top and bottom of the box represent the 75th and 25th
percentiles, the center line represents the median. Dot indicates
the data from each organoid.
[0012] FIG. 4. High fidelity drug induced cholestasis model using
organoids. A. Sequential images for efflux of fluorescein diacetate
from outside to inside of the organoids. B. Comparison of
fluorescein diacetate efflux transport. C. Quantification of
fluorescein diacetate efflux transport into organoid. Example left
image was quantified ratio of fluorescein intensity between inside
and outside the organoid. Right graph indicates the result of
validation study using control (DMSO), Cyclosporin A (CSA) and
Streptomycin (STP) as negative control. Bars represent the
mean.+-.SD, **: p<0.01, n=4. D. Image of fluorescein diacetate
transport inhibition after treatment of 9 training compounds for
24h. E. Quantification of transport inhibition after treatment of
training compounds, Bars represent the mean.+-.SD, *: p<0.05,
**: p<0.01, n=4-6. Quantification of MMP change after treatment
of training compounds, Bars represent the mean.+-.SD, *: p<0.05,
**: p<0.01, n=4-5. CON: Control sample, STP: Streptomycin, TOL:
Tolcapone, DICLO: Diclofenac, BOS: Bosentan, CSA: Cyclosporin
A.
[0013] FIG. 5. High-fidelity drug induced mitochondria-toxicity
screen using organoids. A. Image of mitochondria membrane potential
(MMP) on TMRM after treatment of 9 training compounds. Lower:
Quantification of transport inhibition after treatment of training
compounds, Bars represent the mean.+-.SD, *: p<0.05, **:
p<0.01, n=4-6. C. Quantification of MMP change after treatment
of training compounds, Bars represent the mean.+-.SD, *: p<0.05,
**: p<0.01, n=4-5. CON: Control sample, STP: Streptomycin, TOL:
Tolcapone, DICLO: Diclofenac, BOS: Bosentan, CSA: Cyclosporin A,
TRO: Troglitazone, NEFA: Nefazodone, ENTA: Entacapone, PIO:
Pioglitazone. B. Classification of the set of 9 training compounds
(TC) referred to Oorts., et al 2016 (Oorts et al., 2016). Class A
represents TCs with known reports on DILI in vivo, while those in
Class B are TCs with reports on drug-induced cholestasis in vivo.
The mechanism of toxicity based on literature data is also
provided. Class C compounds are generally considered safe regarding
DILI. C. Analysis between viability for 72h after treatment of
drugs and dual risk parameters, drug-induced cholestasis potential
and mitochondria toxicity potential. Cholestasis and Mitochondria
toxicity (Mito-tox) indexes were derived from data in FIG. 3. The
size of circles indicated the magnitude of viability decreases.
[0014] FIG. 6. Modeling drug-induced liver injury in vulnerable
conditions rescued by NAC exposure. A. Overview of evaluation of
drug-induced cytotoxicity on vulnerable organoid model. B.
Profiling of vulnerable model on lipid accumulation (Blue: nuclei,
Green: Lipid, Red: F-actin). C ROS production (Blue: nuclei, Green:
ROS) and D. mitochondria health (Blue: nuclei, Red: Mitochondria).
E. Image of organoids at 24h after drugs treatment. F. Viability
assessment on lipid accumulation-induced vulnerable organoid model.
Bars represent the mean.+-.SD, *: p<0.05, n=5-6. CON: control,
STP: Streptomycin, TRO: Troglitazone, NAC: N-acetylcysteine.
[0015] FIG. 7. Multiplexed liver organoid based screening for
predicting toxicity
[0016] FIG. 8. Optimization of retinoic acid treatment protocols A.
Scheme for timing and duration of retinoic acid treatment. RA:
retinoic acid, HCM: hepatocyte culture medium. B. Albumin secretion
level in organoids at day 25 in different duration of RA
treatment.
[0017] FIG. 9. The morphology of organoids at D20 Total number of
organoid at D20 were 305. Organoid with lumen: 216, Organoids
without lumen: 89.
[0018] FIG. 10. Conversion formula to determine the number of cells
in organoids A. Phase contrast image of single organoids. B. The
diameter and cell number of each single organoid. C. Correlation
between diameter and cell number in single organoid.
[0019] FIG. 11--Supplementary FIG. 4 The generation of organoids
from multiple PSC lines. Phase contrast image and albumin secretion
level of different iPS cell line (317D6 and 1383D6)-derived
organoid.
[0020] FIG. 12. The cell viability at 24h after treatment of 10
compounds. Viability assessment on lipid accumulation-induced
vulnerable organoid model. CON: Control sample, STP: Streptomycin,
TOL: Tolcapone, DICLO: Diclofenac, AMIO: Amiodarone, BOS: Bosentan,
CSA: Cyclosporin A, TRO: Troglitazone, NEFA: Nefazodone, ENTA:
Entacapone, PIO: Pioglitazone. Bars represent the mean.+-.SD,
n=4-6.
[0021] FIG. 13. ROS production and the morphological change of
mitochondria in lipotoxic liver organoid. A. The ratio of cell
number producing ROS in total cells on lipid accumulation-induced
vulnerable organoid model by treatment of 800 .mu.M oleic acid
(OA). B. Image of mitochondria in organoid on vulnerable organoid
model. Red: mitochondria, Purple: F-actin, Blue: Nucleus. C. The
number and size of mitochondria on vulnerable organoid model. Bars
represent the mean.+-.SD, *: p<0.05, n=5-6.
[0022] FIG. 14. Schematic of Cell Matrigel-Free Method. Shown is a
schematic for a liver organoid generation method that does not use
matrigel for generating organoids.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Unless otherwise noted, terms are to be understood according
to conventional usage by those of ordinary skill in the relevant
art.
[0024] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, e.g., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, or up to 10%,
or up to 5%, or up to 1% of a given value. Alternatively,
particularly with respect to biological systems or processes, the
term can mean within an order of magnitude, preferably within
5-fold, and more preferably within 2-fold, of a value. Where
particular values are described in the application and claims,
unless otherwise stated the term "about" meaning within an
acceptable error range for the particular value should be
assumed.
[0025] As used herein, the term "totipotent stem cells" (also known
as omnipotent stem cells) are stem cells that can differentiate
into embryonic and extra-embryonic cell types. Such cells can
construct a complete, viable organism. These cells are produced
from the fusion of an egg and sperm cell. Cells produced by the
first few divisions of the fertilized egg are also totipotent.
[0026] As used herein, the term "pluripotent stem cells (PSCs)"
encompasses any cells that can differentiate into nearly all cell
types of the body, i.e., cells derived from any of the three germ
layers (germinal epithelium), including endoderm (interior stomach
lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone,
blood, urogenital), and ectoderm (epidermal tissues and nervous
system). PSCs can be the descendants of inner cell mass cells of
the preimplantation blastocyst or obtained through induction of a
non-pluripotent cell, such as an adult somatic cell, by forcing the
expression of certain genes. Pluripotent stem cells can be derived
from any suitable source. Examples of sources of pluripotent stem
cells include mammalian sources, including human, rodent, porcine,
and bovine.
[0027] As used herein, the term "induced pluripotent stem cells
(iPSCs)," also commonly abbreviated as iPS cells, refers to a type
of pluripotent stem cells artificially derived from a normally
non-pluripotent cell, such as an adult somatic cell, by inducing a
"forced" expression of certain genes. hiPSC refers to human
iPSCs.
[0028] As used herein, the term "embryonic stem cells (ESCs)," also
commonly abbreviated as ES cells, refers to cells that are
pluripotent and derived from the inner cell mass of the blastocyst,
an early-stage embryo. For purpose of the present invention, the
term "ESCs" is used broadly sometimes to encompass the embryonic
germ cells as well.
[0029] As used herein, the term "precursor cell" encompasses any
cells that can be used in methods described herein, through which
one or more precursor cells acquire the ability to renew itself or
differentiate into one or more specialized cell types. In some
embodiments, a precursor cell is pluripotent or has the capacity to
becoming pluripotent. In some embodiments, the precursor cells are
subjected to the treatment of external factors (e.g., growth
factors) to acquire pluripotency. In some embodiments, a precursor
cell can be a totipotent (or omnipotent) stem cell; a pluripotent
stem cell (induced or non-induced); a multipotent stem cell; an
oligopotent stem cells and a unipotent stem cell. In some
embodiments, a precursor cell can be from an embryo, an infant, a
child, or an adult. In some embodiments, a precursor cell can be a
somatic cell subject to treatment such that pluripotency is
conferred via genetic manipulation or protein/peptide
treatment.
[0030] In developmental biology, cellular differentiation is the
process by which a less specialized cell becomes a more specialized
cell type. As used herein, the term "directed differentiation"
describes a process through which a less specialized cell becomes a
particular specialized target cell type. The particularity of the
specialized target cell type can be determined by any applicable
methods that can be used to define or alter the destiny of the
initial cell. Exemplary methods include but are not limited to
genetic manipulation, chemical treatment, protein treatment, and
nucleic acid treatment.
[0031] Pluripotent Stem Cells Derived from Embryonic Cells
[0032] In some embodiments, one step is to obtain stem cells that
are pluripotent or can be induced to become pluripotent. In some
embodiments, pluripotent stem cells are derived from embryonic stem
cells, which are in turn derived from totipotent cells of the early
mammalian embryo and are capable of unlimited, undifferentiated
proliferation in vitro. Embryonic stem cells are pluripotent stem
cells derived from the inner cell mass of the blastocyst, an
early-stage embryo. Methods for deriving embryonic stem cells from
blastocytes are well known in the art. Human embryonic stem cells
H9 (H9-hESCs) are used in the exemplary embodiments described in
the present application, but it would be understood by one of skill
in the art that the methods and systems described herein are
applicable to any stem cells.
[0033] Additional stem cells that can be used in embodiments in
accordance with the present invention include but are not limited
to those provided by or described in the database hosted by the
National Stem Cell Bank (NSCB), Human Embryonic Stem Cell Research
Center at the University of California, San Francisco (UCSF); WISC
cell Bank at the Wi Cell Research Institute; the University of
Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC);
Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg,
Sweden); ES Cell International Pte Ltd (Singapore); Technion at the
Israel Institute of Technology (Haifa, Israel); and the Stem Cell
Database hosted by Princeton University and the University of
Pennsylvania. Exemplary embryonic stem cells that can be used in
embodiments in accordance with the present invention include but
are not limited to SA01 (SA001); SA02 (SA002); ES01 (HES-1); ES02
(HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6);
BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14);
TE06 (16); UCO1 (HSF1); UCO6 (HSF6); WA01 (H1); WA07 (H7); WA09
(H9); WA13 (H13); WA14 (H14).
[0034] More details on embryonic stem cells can be found in, for
example, Thomson et al., 1998, "Embryonic Stem Cell Lines Derived
from Human Blastocysts," Science 282 (5391):1145-1147; Andrews et
al., 2005, "Embryonic stem (ES) cells and embryonal carcinoma (EC)
cells: opposite sides of the same coin," Biochem Soc Trans
33:1526-1530; Martin 1980, "Teratocarcinomas and mammalian
embryogenesis,". Science 209 (4458):768-776; Evans and Kaufman,
1981, "Establishment in culture of pluripotent cells from mouse
embryos," Nature 292(5819): 154-156; Klimanskaya et al., 2005,
"Human embryonic stem cells derived without feeder cells," Lancet
365 (9471): 1636-1641; each of which is hereby incorporated herein
in its entirety.
[0035] Induced Pluripotent Stem Cells (iPSCs)
[0036] In some embodiments, iPSCs are derived by transfection of
certain stem cell-associated genes into non-pluripotent cells, such
as adult fibroblasts. Transfection is typically achieved through
viral vectors, such as retroviruses. Transfected genes include the
master transcriptional regulators Oct-3/4 (Pouf51) and Sox2,
although it is suggested that other genes enhance the efficiency of
induction. After 3-4 weeks, small numbers of transfected cells
begin to become morphologically and biochemically similar to
pluripotent stem cells, and are typically isolated through
morphological selection, doubling time, or through a reporter gene
and antibiotic selection. As used herein, iPSCs include but are not
limited to first generation iPSCs, second generation iPSCs in mice,
and human induced pluripotent stem cells. In some embodiments, a
retroviral system is used to transform human fibroblasts
intopluripotent stem cells using four pivotal genes: Oct3/4, Sox2,
Klf4, and c-Myc. In alternative embodiments, a lentiviral system is
used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.
Genes whose expression are induced in iPSCs include but are not
limited to Oct-3/4 (e.g., Pou5fl); certain members of the Sox gene
family (e.g., Sox1, Sox2, Sox3, and Sox15); certain members of the
Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of
the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, and
LIN28.
[0037] In some embodiments, non-viral based technologies are
employed to generate iPSCs. In some embodiments, an adenovirus can
be used to transport the requisite four genes into the DNA of skin
and liver cells of mice, resulting in cells identical to embryonic
stem cells. Since the adenovirus does not combine any of its own
genes with the targeted host, the danger of creating tumors is
eliminated. In some embodiments, reprogramming can be accomplished
via plasmid without any virus transfection system at all, although
at very low efficiencies. In other embodiments, direct delivery of
proteins is used to generate iPSCs, thus eliminating the need for
viruses or genetic modification. In some embodiment, generation of
mouse iPSCs is possible using a similar methodology: a repeated
treatment of the cells with certain proteins channeled into the
cells via poly-arginine anchors was sufficient to induce
pluripotency. In some embodiments, the expression of pluripotency
induction genes can also be increased by treating somatic cells
with FGF2 under low oxygen conditions.
[0038] More details on embryonic stem cells can be found in, for
example, Kaji et al., 2009, "Virus free induction of pluripotency
and subsequent excision of reprogramming factors," Nature
458:771-775; Woltjen et al., 2009, "piggyBac transposition
reprograms fibroblasts to induced pluripotent stem cells," Nature
458:766-770; Okita et al., 2008, "Generation of Mouse Induced
Pluripotent Stem Cells Without Viral Vectors," Science
322(5903):949-953; Stadtfeld et al., 2008, "Induced Pluripotent
Stem Cells Generated without Viral Integration," Science
322(5903):945-949; and Zhou et al., 2009, "Generation of Induced
Pluripotent Stem Cells Using Recombinant Proteins," Cell Stem Cell
4(5):381-384; each of which is hereby incorporated herein in its
entirety.
[0039] In some embodiments, exemplary iPS cell lines include but
not limited to iPS-DF19-9; iPS-DF19-9; iPS-DF4-3; iPS-DF6-9;
iPS(Foreskin); iPS(IMR90); and iPS(IMR90).
[0040] More details on the functions of signaling pathways relating
to DE development can be found in, for example, Zorn and Wells,
2009, "Vertebrate endoderm development and organ formation," Annu
Rev Cell Dev Biol 25:221-251; Dessimoz et al., 2006, "FGF signaling
is necessary for establishing gut tube domains along the
anterior-posterior axis in vivo," Mech Dev 123:42-55; McLin et al.,
2007, "Repression of Wnt/.beta.-catenin signaling in the anterior
endoderm is essential for liver and pancreas development.
Development," 134:2207-2217; Wells and Melton, 2000, Development
127:1563-1572; de Santa Barbara et al., 2003, "Development and
differentiation of the intestinal epithelium," Cell Mol Life Sci
60(7): 1322-1332; each of which is hereby incorporated herein in
its entirety.
[0041] Any methods for producing definitive endoderm from
pluripotent cells (e.g., iPSCs or ESCs) are applicable to the
methods described herein. Any method for producing definitive
endoderm from pluripotent cells (e.g., iPSCs or ESCs) are
applicable to the methods described herein. Exemplary methods are
disclosed in, for example, "Methods and systems for converting
precursor cells into intestinal tissues through directed
differentiation," U.S. Pat. No. 9,719,068B2 to Wells et al., and
"Methods and systems for converting precursor cells into gastric
tissues through directed differentiation," US20170240866A1, to
Wells et al. In some embodiments, pluripotent cells are derived
from a morula. In some embodiments, pluripotent stem cells are stem
cells. Stem cells used in these methods can include, but are not
limited to, embryonic stem cells. Embryonic stem cells can be
derived from the embryonic inner cell mass or from the embryonic
gonadal ridges. Embryonic stem cells or germ cells can originate
from a variety of animal species including, but not limited to,
various mammalian species including humans. In some embodiments,
human embryonic stem cells are used to produce definitive endoderm.
In some embodiments, human embryonic germ cells are used to produce
definitive endoderm. In some embodiments, iPSCs are used to produce
definitive endoderm. Additional methods for obtaining or creating
DE cells that can be used in the present invention include but are
not limited to those described in U.S. Pat. No. 7,510,876 to
D'Amour et al.; U.S. Pat. No. 7,326,572 to Fisk et al.; Kubol et
al., 2004, "Development of definitive endoderm from embryonic stem
cells in culture," Development 131:1651-1662; D'Amour et al., 2005,
"Efficient differentiation of human embryonic stem cells to
definitive endoderm," Nature Biotechnology 23:1534-1541; and Ang et
al., 1993, "The formation and maintenance of the definitive
endoderm lineage in the mouse: involvement of HNF3/forkhead
proteins," Development 119:1301-1315.
[0042] Applicant has discovered methods for producing 3D liver
structures using human iPSCs. The structures comprise micro-liver
architectures, including polarized hepatic epithelium, stellate
cells, and canalicula structures. The disclosed compositions
display improvements in hepatic functions, bile transport activity,
and durability compared to existing models. The 3D structures model
may be used as a new and robust model for drug screening tests
and/or drug toxicity screening, transplantation, production of
serum protein products, and development of personalized therapy. In
one particular application, the compositions and methods may be
used to screen drug compounds for liver toxicity.
[0043] While 3D aggregated liver cells have been reported, the
disclosed compositions have very high functional activity such as
albumin production (up to a 10-fold increase compared with
conventional highest standard models using iPSC-derived
hepatocytes), and allow for improved oxygen and/or nutrition supply
due to the internal luminal structure, which allows for much longer
culture (at least over 60 days) and a long-term testing platform
useful for drug testing. The disclosed compositions may also be
useful for production of plasma products like albumin, coagulation
factor products for treatment of hypoalbuminemia, and for
therapeutic transplantation, in which the human iPSC-derived
miniature livers can be transplanted to treat disorders in vivo.
Lastly, the disclosed compositions may be used for personalized
medicine (therapy personalization).
[0044] In one aspect, a method of inducing formation of a liver
organoid from iPSC cells is disclosed. The method may comprise the
steps of
[0045] a) contacting definitive endoderm (DE) derived from iPSC
cells with a FGF pathway activator and a GSK3 inhibitor, for a
period of time sufficient to form posterior foregut spheroids,
preferably for a period of time of from about 1 day to about 3 days
and b) incubating the resulting posterior foregut spheroids of step
a in the presence of retinoic acid (RA) for a period of time
sufficient to form a liver organoid, preferably for a period of
time of from about 1 to about 5 days, preferably about 4 days.
[0046] Fibroblast growth factors (FGFs) are a family of growth
factors involved in angiogenesis, wound healing, and embryonic
development. The FGFs are heparin-binding proteins and interactions
with cell-surface associated heparan sulfate proteoglycans have
been shown to be essential for FGF signal transduction. Suitable
FGF pathway activators will be readily understood by one of
ordinary skill in the art. Exemplary FGF pathway activators
include, but are not limited to: one or more molecules selected
from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11,
FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20,
FGF21, FGF22, and FGF23. In some embodiments, siRNA and/or shRNA
targeting cellular constituents associated with the FGF signaling
pathway may be used to activate these pathways.
[0047] In some embodiments, DE culture is treated with the one or
more molecules of the FGF signaling pathway described herein at a
concentration of 10 ng/ml or higher; 20 ng/ml or higher; 50 ng/ml
or higher; 75 ng/ml or higher; 100 ng/ml or higher; 120 ng/ml or
higher; 150 ng/ml or higher; 200 ng/ml or higher; 500 ng/ml or
higher; 1,000 ng/ml or higher; 1,200 ng/ml or higher; 1,500 ng/ml
or higher; 2,000 ng/ml or higher; 5,000 ng/ml or higher; 7,000
ng/ml or higher; 10,000 ng/ml or higher; or 15,000 ng/ml or higher.
In some embodiments, concentration of signaling molecule is
maintained at a constant throughout the treatment. In other
embodiments, concentration of the molecules of a signaling pathway
is varied during the course of the treatment. In some embodiments,
a signaling molecule in accordance with the present invention is
suspended in media comprising DMEM and fetal bovine serine (FBS).
The FBS can be at a concentration of 2% and more; 5% and more; 10%
or more; 15% or more; 20% or more; 30% or more; or 50% or more. One
of skill in the art would understand that the regiment described
herein is applicable to any known molecules of the signaling
pathways described herein, alone or in combination, including but
not limited to any molecules in the FGF signaling pathway.
[0048] Suitable GSK3 inhibitors will be readily understood by one
of ordinary skill in the art. Exemplary GSK3 inhibitors include,
but are not limited to: Chiron/CHIR99021, for example, which
inhibits GSK3r3. One of ordinary skill in the art will recognize
GSK3 inhibitors suitable for carrying out the disclosed methods.
The GSK3 inhibitor may be administered in an amount of from about 1
uM to about 100 uM, or from about 2 uM to about 50 uM, or from
about 3 uM to about 25 uM. One of ordinary skill in the art will
readily appreciate the appropriate amount and duration.
[0049] In one aspect, the stem cells may be mammalian, or human,
iPSCs.
[0050] In one aspect, the foregut spheroids may be embedded in a
basement membrane matrix, such as, for example, the commercially
available basement membrane matrix sold under the tradename
Matrigel.
[0051] In one aspect, the liver organoids may be characterized in
that the liver organoids may express alpha-fetoprotein (AFP),
albumin (ALB), retinol binding protein (RBP4), cytokeratin 19
(CK19), hepatocyte nuclear factor 6 (HNF6), and cytochrome P450 3A4
(CYP3A4), HNF4a, E-cadherin, DAPI, and Epcam. Such expression may
occur, for example, at day 40 to day 50. The expression level may
be similar to that observed in human liver cells, for example, that
of an adult liver cell.
[0052] In one aspect, the liver organoid may be characterized in
that the liver organoid has bile transport activity.
[0053] In one aspect, the liver organoid may be derived from a stem
cell and may comprise a luminal structure further containing
internalized microvilli and mesenchymal cells. The luminal
structure may be surrounded by polarized hepatocytes and a basement
membrane. The liver organoid may comprise functional stellate cells
and functional Kupffer cells.
[0054] The liver organoid may, in certain aspects, be characterized
by having one or more of the following: bile production capacity,
bile transport activity, Complement factor H expression of at least
50 ng/mL/1.times.e.sup.6 cells/24 hr, Complement factor B of at
least 40 ng/mL/1.times.e.sup.6 cells/24 hr, C3 expression of at
least 1000 ng/mL/1.times.e.sup.6 cells/24 hr; C4 expression of at
least 1000 ng/mL/1.times.e.sup.6 cells/24 hr, fibrinogen production
of at least 1,000 ng/mL/1.times.e.sup.6 cells/24 hr and albumin
production of at least 1,000 ng/mL/1.times.e.sup.6 cells/24 hr. In
one aspect, the liver organoid may be characterized by having total
hepatic protein expression of at least 10,000 ng/mL 1.times.e.sup.6
cells/24 hours. The liver organoid may be characterized in that it
may express one or more genes selected from PROX1, RBP4, CYP2C9,
CYP3A4, ABCC11, CFH, C3, C5, ALB, FBG, MRP2, ALCAM, CD68, CD34,
CD31. In one aspect, the liver organoid may comprise cells
comprising a drug metabolism cytochrome variant, such as, for
example, a CY2C9*2 variant. The liver organoid may comprise a
vasculature, such as that described in US 20160177270.
[0055] In one aspect, the liver organoid may be characterized in
that the liver organoid does not comprise inflammatory cells, for
example T-cells or other inflammatory secreted proteins.
[0056] In one aspect, a method of screening for a serious adverse
event (SAE) is disclosed. The SAE may be liver failure and/or drug
induced liver injury (DILI). The method may include the step of
contacting a drug of interest, of which toxicity is of interest,
with a liver organoid as described herein. In one aspect, the
method may comprise the step of measuring intake and/or efflux of
fluorescein diacetate (FD), wherein impaired efflux indicates that
said drug is likely to induce a serious adverse event. The toxicity
of a drug of interest may be determined by measurement of a
parameter selected from mitochondria membrane potential,
measurement of ROS, swelling of liver mitochondria, and
combinations thereof, wherein injury to said mitochondria indicates
that said drug is likely to induce a serious adverse event. In one
aspect, the method comprises the step of assaying organoid
viability, wherein impaired or decreased organoid viability
indicates that said a drug of interest is likely to induce a
serious adverse event.
[0057] In one aspect, a method of treating an individual having
liver damage is disclosed, wherein the method may comprise the step
implanting a liver organoid as described herein into an individual
in need thereof. The liver damage may include, for example,
metabolic liver disease, end stage liver disease, or a combination
thereof.
[0058] In one aspect, methods for identifying a preferred
therapeutic agent for an individual are disclosed. In this aspect,
the method may include the step of contacting a liver organoid
derived from an iPSC of interest with a candidate compound, such as
wherein the iPSC of interest comprises one or more mutations found
in said individual, or such as wherein said iPSC of interest is
derived from the same ethic background of said individual, or
further, wherein said iPSC of interest is derived from said
individual.
EXAMPLES
[0059] In the present study, Applicant tested bile transport
activity using Fluorescein Diacetate, which was excreted by MRP2
across the canalicular membrane into the bile canalicular networks
(Tian et al., 2004). It has previously been reported that
Troglitazone and Cyclosporin inhibit the MRP2 (Chang et al., 2013;
Lechner et al., 2010). In addition, the efflux transporter MRP2
mediates export of Bosentan (Fahrmayr et al., 2013). Although the
inhibition of MRP2 by Nefazodone was not reported, mitochondria
stress by Nefazodone may be related to a decrease of the bile
transport activity, efflux of Fluorescein Diacetate, because MRP2
is an ATP-dependent bile salt transporter for canalicular excretion
of bile acids in hepatocytes.
[0060] Preclinical detection of risk compounds for drug induced
liver injury (DILI) remains a significant challenge in drug
development, highlighting a need for a predictive human system.
Here, Applicant developed a human liver organoid (HLO) model for
analyzing clinical DILI pathology at organoid resolution.
Differentiated HLO from human iPSC contain polarized hepatocytes
with an internal lumen lined by bile canaliculi-like architecture,
establishing the unidirectional bile acid transport pathway.
Applicant has leveraged the organoid's structural features by
modeling DILI using live organoid imaging, called LoT (Liver
organoid-based Toxicity screen). LoT is functionally validated with
10 marketed drugs and 5 different donors based on cholestatic
and/or mitochondrial toxicity. Bosentan-induced cholestasis is
specific to CYP2C9 poor metabolizer donor-derived HLO.
Interestingly, steatotic organoids were vulnerable to Rosiglitazone
toxicity as suggested in clinics, followed by chemical rescue from
massive organoid death. Thus, LoT is a high-fidelity organoid model
that can be used to analyze drug safety, and is further a
cost-effective platform, facilitates compound optimization,
provides mechanistic studies, and produces personalized medicine as
well as anti-DILI therapy screening applications.
[0061] Billions of dollars are lost annually from drug development
in the pharmaceutical industry due to the failures of drug
candidates identified in initial screens, and nearly (a third or
one-third) of drugs are withdrawn from the market (Takebe and
Taniguchi, 2014). Despite the promising efficacy, a failure of drug
candidates results in a tremendous loss of a patient's treatment
opportunity. Preclinical studies generally consist of an in vitro
evaluation as a primary efficacy screen to identify a "hit"
compound, followed by safety studies in vitro and in vivo to assess
the mechanisms of metabolism and toxicology. This inefficiency can
be explained by the substantial lack of physiologically relevant
preclinical models in evaluating drug induced liver injury (DILI)
in humans and thus, an urgent need to develop an in vitro
humanistic screen model for the evaluation of the vast amounts of
continuously growing compound libraries.
[0062] Primary hepatocytes are a highly polarized metabolic cell
type, and form a bile canaliculi structure with microvilli-lined
channels, separating peripheral circulation from the bile acid
secretion pathway. The most upstream aspects of DILI include drug
(or their reactive metabolites) detoxification by hepatocytes and
excretion into bile canaliculi through transporters such as
multi-drug resistance-associated protein (MRP) transporters. This
suggests the need to reconstruct these uniquely organized
structures as a crucial property of hepatocytes in vivo for
predicting DILI pathology. However, there are considerable
differences in drug toxicity profiles between the current
simplified culture model with the use of isolated primary human
hepatocytes or hepatic cell lines, and in vivo physiology,
resulting in failed translation of drugs or drug discontinuation,
as in the case of Troglitazone, Nefazodone and Tolcapone
(https://livertox.nlm.nih.gov/index.html). The determination of
toxicological properties thus mainly relies on animals as an
essential step for drug development, however, due to the pronounced
differences in physiology between humans and animals, there is a
significant lack of fidelity to human outcomes (Leslie et al.,
2007; Yang et al., 2014). In addition, the onset of idiosyncratic
DILI (IDILI), which is very rare but nonetheless responsible for
about 10-15% of acute liver failures in the USA (Reuben et al.,
2010), is almost impossible to predict (Kullak-Ublick et al.,
2017). Collectively, effective human cell models are eagerly
anticipated to screen for compounds that test the detoxification
and excretion of proposed drugs.
[0063] Despite the progressive advance of human hepatocyte
differentiation methods from pluripotent stem cell (PSC), clinical
trials in a dish using human stem cells remains `hype`. To a
certain degree, this can be explained by challenges in previous
cell-based approaches including: (1) overcoming lot-differences,
(2) minimization of experimental batch-differences, (3) enhancement
of assay throughput and (4) improvement in relevance to clinical
trial data. Applicant addresses these issues by developing a
relatively simple and robust organoid based testing platform using
stably expandable human stem cells, i.e. iPSC. Applicant first
directed the human PSCs into posterior foregut organoids, followed
by progressive hepatocyte differentiation through a polarization
culture with defined factors and matrix. Generated human liver
organoids possessed intraluminal structure surrounded by polarized
hepatocytes, and have shown to be capable of performing critical
human hepatocyte functions including protein and bile acid
production and transport functions. Interestingly, Applicant found
that live image based dynamic detection of fluorescent diacetate
uptake and excretion precisely models cholestasis induced by arrays
of DILI drugs characterized as inhibitors for bile excretion with a
high level of reproducibility. Separately, mitochondrial membrane
potential assessment enabled an independent risk assessment for
each of the compound, reflecting a conventional classification of
DILI drugs established by clinical trials. Furthermore, Applicant
extended the approach to model conditions induced by lipotoxic
stress and confirmed enhanced DILI potential through reactive
oxygen species (ROS) production. Organoid-based viability
assessment confirmed the reversal of DILI by N-acetylcysteine,
highlighting the potential of our approach for anti-DILI drug
screening. Taken together, this robust assay, named Liver Organoid
based Toxicity screen (LoT), is believed to be the first functional
readout developed in human liver organoids, and will facilitate
diagnosis, functional studies, drug development and personalized
medicine.
[0064] Results
[0065] Generation and characterization of polarized liver organoids
from multiple human iPSC
[0066] Applicant first established a new liver organoid
differentiation method by using human iPSC-derived foregut
spheroids (Spence et al., 2011) (FIG. 1A). As a first step,
Applicant used BMP and Activin A to promote differentiation into
definitive endoderm as previously described (D'Amour et al., 2005).
In addition, FGF4, and a GSK3 inhibitor (CHIR99021) were used to
induce foregut spheroids and budded spheroids were observed.
Organoids were embedded in Matrigel after delamination with
mesenchymal cells plated on the dish by gentle pipetting. It has
been reported that retinoic acid (RA) enhances cell polarity as
indicated by increased size and complexity of the bile canaliculi
and the pericanalicular sheaths (Falasca et al., 1998). To generate
polarized organoids suited for bile transport modeling, organoids
were treated with RA. To optimize the organoid generation method,
Applicant first varied the duration of RA treatment. The albumin
secretion levels of organoids were 1160, 1054, 3092, 4709 and 3865
ng/mL at D25, for 1, 2, 3, 4 and 5 days of RA treatment,
respectively, and the 4 day-RA treatment protocol tended to reach
the highest level (FIG. 8). Thus, duration of RA was set for 4 days
based on the level of albumin secretion. Morphologically, around 10
days after RA treatment, over 300 organoids covered with epithelial
cells were successfully generated, and the ratio of organoids with
lumenized structure was 71% (216/305) (FIG. 1, panel B and FIG. 9).
Immunohistochemistry analysis revealed that albumin was positive in
epithelial cells of organoids, and interestingly, Type IV collagen
was localized to the outer surface and ZO-1 (zonula occludens)
stained the intraluminal lining, suggesting that these organoids
have polarized characteristics (FIG. 1, panel C).
[0067] Quantitative polymerase chain reaction (qPCR) analysis
revealed that cells in organoids had a significant increase in
expression of hepatic marker genes such as alpha-fetoprotein (AFP),
albumin (ALB), retinol binding protein 4 (RBP4), Cytokeratin 19
(CK19), hepatocyte nuclear factor 6 (HNF6) which controls
cholangiocyte differentiation, and Cytochrome P450 3A4 (CYP3A4)
during differentiation (FIG. 1, panel D). However, the expression
level of the most hepatic genes extracted from bulk
organoid-derived RNA was lower in organoids than in primary
hepatocytes. Without intending to be limited by theory, it is
believed that these distinct mRNA profiles are partly due to the
presence of stromal lineages as approximately 30% of cells are
non-parenchymal cells which were identified by stromal cell markers
(Unpublished observation), making the organoids more similar to in
vivo liver tissue than primary hepatocytes. Applicant further
profiled the organoids by a comprehensive gene expression analysis
using RNA-sequence (RNA-seq). Principal component analysis
demonstrated that gene expression in organoids was not similar to
iPSC-derived cholangiocytes and normal human cholangiocyte (FIG. 1,
panel E). Additionally, hepatocyte specific proteins such as ALB,
fibrinogen (Fbg) and complement factors were confirmed in culture
supernatant by ELISA (FIG. 1, panels F-G). To quantitate the
hepatic functionality of the organoids, Applicant investigated the
albumin secretion level normalized by cell number (FIG. 10). The
albumin secretion level was 2133 ng/day/10.sup.6 cells (FIG. 1,
panel F) and higher than other experiments in 2D and 3D
differentiation of hPSCs into HLC (150-1000 ng/day/10.sup.6 cell)
relative to published iPSC-derived hepatocytes (Mild et al., 2011;
Song et al., 2015; Song et al., 2009; Vosough et al., 2013), while
primary hepatocytes produce 30-40 .mu.g/day/10.sup.6 cell in 3D
scaffolds (Davidson et al., 2016; Dvir-Ginzberg et al., 2003).
These results indicated that liver organoids contained hepatocytes
with reasonable albumin secretion activity compared to stem
cell-derived hepatocytes in the published literature. Importantly,
this organoid generation method is reproducible and therefore,
applicable to other PSC lines, as intra-luminal organoids were
generated from both 317D6 and 1383D6 iPS cell lines with albumin
secretion capacity (FIG. 11). Overall, Applicant established a
protocol for generating a large number of polarized liver organoids
with hepatocyte characteristics.
[0068] Micro-anatomical characterization of bile acid producing
human iPSC-liver organoids
[0069] Next, to test if liver organoids have bile transport
activity, Applicant first characterized organoids by staining key
proteins involved in bile synthesis and excretion function
Immunofluorescence staining of BSEP and MRP2 demonstrated that
these proteins preferentially localized in the intraluminal region
(FIG. 2, panel A). The bile canaliculus is the smallest
intrahepatic secretory channel and the canalicular lumen consists
of a space formed by a modified apical region of the opposing
plasma membranes of contiguous hepatocytes (Cutrin et al., 1996;
Tsukada et al., 1995). In addition, it is delimited by tight
junction complexes and the microvilli are located on the inside of
the canalicular lumen (Tsukada et al., 1995). ZO-1 staining is
known to stain canalicular region in liver, and FIG. 1, panel C
suggested that tight junctions were located inside of our liver
organoids. Transmission electron microscopy revealed organoids
contained microvilli directed towards the lumen (FIG. 2, panel B).
Consistent with these anatomical features, qRT-PCR analysis
revealed that organoids had gene expression of ABCB11 and
Na+-taurocholate co-transporting polypeptide (NTCP), yet the levels
were lower in organoids than primary hepatocytes (FIG. 2, panel C).
Therefore, the organoid contained polarized human hepatocytes
separated from internal lumen by tight junctions, which reflects a
unique micro-anatomical architecture resembling in vivo hepatic
canaliculi.
[0070] Next, in order to determine bile acid (BA) production
capacity, Applicant conducted ELISAs on intra-luminal fluid
collected from organoid culture. The level of total BA pool of
intra-luminal fluid was 26.7 .mu.g/day/10.sup.6 cells
(approximately 125 .mu.mon in an organoid with a 200 .mu.m
diameter) (FIG. 2, panel D) and, surprisingly, the BA concentration
was comparable to that in primary hepatocytes derived from sandwich
culture (approximately 40 .mu.g/day/10.sup.6 cells, 10 .mu.mon in
culture supernatant) in previous reports (Ni et al., 2016). Thus,
organoids do not merely have the canaliculi-like morphology but
possess bile acid production and secretion activity, suggesting
that the bile acids transport pathway is correctly constructed.
[0071] Dynamic visualization of bile acid intake and excretion in
human liver organoids
[0072] Bile acid excretion is the major determinant of bile flow,
therefore, defects in this system may result in impaired bile
secretion (cholestasis) associated with various liver disease
pathologies (Nishida et al., 1991). Efflux transport proteins
located in the apical (canaliculi) membranes of hepatocytes play an
important role in the hepatic elimination of many endogenous and
exogenous compounds, including drugs and metabolites (Kock and
Brouwer, 2012). BSEP and MRP2 mediate canaliculi bile salt
transport in humans. After demonstrating the positive expression of
key proteins for bile transport, the applicant next wondered if the
organoids can actively transport bile acid into its lumen. First,
to check the intake of bile acids into the organoid, applicant
challenged the organoids with cholylglycylamido-fluorescein
(CGamF), which is a bile salt analog (Mork et al., 2012). After
treatment of CGamF from outside, the accumulation of CGamF into the
intra-lumen of organoids was successfully confirmed (FIG. 2, panel
E). Similarly, the fluorescent bile acid cholyl-lysyl-fluorescein
(CLF) was found to be reproducibly excreted and accumulated into
organoids from multiple human iPSC lines (FIG. 2, panel F). To
determine the specificity of this assay, Applicant has developed an
iPSC line carrying a BSEP defunctionalized allele using the
CRISPR-Cas9 based gene editing approach. BSEP is responsible for
bile transport, and consistent with this, BSEP-KO iPSC-organoids
failed to accumulate fluorescent bile acid compared with parental
control organoids. Taken together, these data suggest that
organoids have the ability to uptake bile acid from the outside and
efflux them inside the organoids.
[0073] Bosentan induced cholestasis specific to CYP2C9*2 iPSC-liver
organoids
[0074] To test the clinical relevance of organoid based cholestatic
phenotyping method, Applicant employed pharmacogenomics insights
into our system to address the fidelity question. Specifically,
multiple iPSC lines have been collected, which carry a well-known
susceptibility gene variant (i.e. CYP2C9*2 for Bosentan, described
in, for example, Clin Pharmacol Ther. 2013 December; 94(6):678-86.
doi: 10.1038/clpt.2013.143. Epub 2013 Jul. 17. Association of
CYP2C9*2 with bosentan-induced liver injury.) (FIG. 3, panel A),
and compared their cholestatic potential in the presence of
Bosentan (FIG. 3, panel B). Interestingly, CLF excretion into
organoids was severely impaired in CY2C9*2 carrier organoids but
not in non-carrier organoids. This matches the clinical tendency to
cholestasis induced by Bosentan, shown in three-different iPSC
derived organoids in the absence of CYP2C9*2 as shown in FIG. 3,
panel C. In contrast, irinotecan based cholestasis was not specific
to CYP2C9*2 iPSC lines. These results indicated the organoid based
cholestasis assay predicts some aspects of human variation.
[0075] High-throughput drug induced cholestasis evaluation in
organoids
[0076] Considering the significant role of cholestasis in DILI
induced by drugs, the applicant next wondered if this organoid
model reflects the pathology of DILI in the presence of particular
compounds. Before testing a number of compounds, the applicant
first sought to develop a high-throughput fluorescence based assay
because both CLF and CGamF are not applicable for high-speed
imaging due to several issues: 1. background is strong,
necessitating a manual washing process; 2. signal intensity is
weak, requiring a careful acquisition setting. Alternatively, the
use of fluorescein diacetate (FD), a reportedly useful marker of
efflux transport in hepatocytes (Barth and Schwarz, 1982; Bravo et
al., 1998) is proposed. The polar fluorescent metabolite
fluorescein, is trapped in the cells until it is actively
transported from the cells into the canaliculi space (Malinen et
al., 2014). To determine if FD can be used for live assessment of
transport capacity without exchanging the medium and adjusting
exposure, chronological hepatobiliary transport activity was
further investigated with time-lapse imaging. Organoids were
incubated with fluorescein diacetate for 45 minutes and
intraluminal accumulation was observed inside of organoids at 20
minutes after treatment (FIG. 4, panels A, B). The opposite
directionality of this transport flow was determined by the
micro-injection of FD into organoids. After micro-injection of
diacetate into the lumen, fluorescein remained inside, and was
never observed outside the organoids (FIG. 4, panel C). In summary,
this FD based evaluation model has high-throughput potential to
assess unidirectional efflux bile transport in liver organoids by a
simple fluorescent live imaging analysis.
[0077] Next, the applicant validated the fidelity of a FD-based
assay by evaluating the viable dosing of 10 FDA approved drugs and
measured any secondary disturbance by cell damage. Applicant
successfully found an optimal dose for nine compounds with
acceptable viability. In contrast, amiodarone (AMIO) was
significantly toxic to organoids within the tested range, therefore
AMIO was excluded in further potential DILI assessment studies
(FIG. 12). Applicant investigated cholestasis potential in
organoids using FD with nine training compounds (TCs) which were
classified as one of three types based on DILI mechanism; DILI
compounds without cholestasis (Class A), DILI compounds with
cholestasis (Class B) and compounds not reported as DILI compounds
(Class C) (FIG. 4, panel D) (Oorts et al., 2016). To quantify the
inhibitory potential for FD excretion, Applicant developed a simple
but robust quantification method by determining the fluorescent
intensity ratio between outside and inside the organoid by image J
(FIG. 4, panel B). As a validation study, Applicant first confirmed
the ability to assess the inhibition ratio using Cyclosporin A
(CSA). At five minutes after treatment of FD, a significant
decrease (0.4 compared to control) was observed in the group
treated with CSA for 24h, compared to control (DMSO) (FIG. 4, panel
B). Applicant then screened nine TCs at multiple concentrations to
assess the fidelity of this approach. Interestingly, in this
screening system, at 24 hr after treatment of TCs, the efflux of FD
was significantly decreased (p<0.01 or 0.05) in Class B
compounds) Bosentan, CSA, Troglitazone and Nefazodone), similar to
clinical observations, while this inhibitory effect was not
observed in Class A and Class C compounds (FIG. 4, panel D upper
images and FIG. 4, panel E). These results suggested that the liver
organoid model is useful for classifying the bile transport
inhibition potency for candidate compounds in drug development with
a high relevance to human phenotypes.
[0078] Evaluating Mitochondrial Overload in Organoids
[0079] Further, Applicant investigated the mitochondria health
assessment because mitochondrial toxicity plays a central role in
DILI in multiple mechanisms associated with the onset of DILI
(Pessayre et al., 2012). In this study, to investigate
mitochondrial health in organoids, an index of mitochondrial
membrane potential (MMP) was used to monitor MMP of intact cells as
a direct readout of mitochondrial health (Li et al., 2014). After
treatment of TCs for 24h, dose-dependent increases of MMP were
observed with treatment of Tolcapone (2-8-fold change, p<0.01),
Diclofenac (7-13 Fold change, p<0.05 or 0.01), CSA (3-7-fold
change, p<0.01) and Nefazodone (4-42 fold change, p<0.01)
(FIG. 5, panel A lower images and graph). In addition, Troglitazone
also increased MMP in organoids (3-5-fold change, p<0.05),
although dose-dependence was not observed. On the other hand, after
treatment of Bosentan, Entacapone and Pioglitazone, increases of
MMP were not clearly observed even in multiple doses. These results
demonstrated that this live image based assay, named Liver
organoid-based Toxicity screen (LoT), discriminated between
compounds with and without mitochondrial toxicity.
[0080] Revisiting Mechanistic Classification of DILI Compounds by
LoT System
[0081] Severe manifestations of human DILI are multifactorial,
highly associated with combinations of drug potency specifically
related to known mechanisms of DILI such as mitochondrial and BSEP
inhibition (Aleo et al., 2014). However, current in vitro
functional models are difficult to assess such multi-factorial
contributions. Given the advantage of multiplexed and live
functional readouts in LoT system, Applicant attempted to analyze
the relationship among survival, cholestasis and mitochondrial
stress. Of note, drugs with dual action at 24 hours (cholestasis
and mitochondrial stress) such as CSA, TRO and NEFA significantly
lowered cell viability at 72 hours relative to TOL, DICLO and BOS.
These data are comparable to clinical data that shows that dual
toxicities were highly associated with the severity of DILI
consistent previous reports (Aleo et al., 2014) (FIG. 5, panels B,
C). Additionally, Applicant also noted that Entacapone treatment at
130 .mu.M decreased organoid viability (From 85% at 24h to 64% at
72h). Entacapone requires extensive binding to plasma proteins,
mainly to albumin, to induce DILI (Fisher et al., 2002). However,
based on available methods, it remains elusive how Entacapone is
toxic to the liver (Oorts et al., 2016). Taken together, the LoT
system is an advantageous human model system for the major
mechanistic classification of DILI, and a useful testing platform
for further delineating unknown complex mechanisms.
[0082] Assessing Vulnerability for DILI in Human Liver
Organoids
[0083] DILI incidence is known to be often confounded by a number
of host factors. Indeed, there is growing evidence that the risk of
hepatotoxicity from some drugs such as acetaminophen is greatly
increased due to obesity and NAFLD, both in rodents and humans
(APAP) (Fromenty, 2013; Michaut et al., 2016). Therefore, it is
important to predict DILI potential in such a "vulnerable"
condition with a patient even in the subclinical phase. In the
present study, Applicant established a lipotoxic organoid model by
co-exposure to an unsaturated fatty acid, oleic acid (FIG. 6, panel
A). At 3 days after oleic acid treatment to organoids, lipid
accumulation in organoids was intense (FIG. 6, panel B). The
oxidation of fatty acids is an important source of reactive oxygen
species (ROS), which leads to depletion of ATP and nicotinamide
dinucleotide, and induces DNA damage in fatty livers (Browning and
Horton, 2004). Consistent with this, ROS production was observed in
lipid treated organoids (FIG. 6, panel C and FIG. 13, panel A).
Additionally, fatty acids induced massive swelling of liver
mitochondria (FIG. 6, panel D and FIG. 13, panel B) similar to
published phenotypes (Zborowski and Wojtczak, 1963). As hepatic
mitochondrial dysfunction precedes the development of NAFLD in a
rat model (Rector et al., 2010), these results indicate that the
lipotoxic organoids model to some degree in vivo fatty liver
model.
[0084] Recognizing this lipotoxic organoid model as a vulnerable
condition with enhanced ROS production, Troglitazone (0-50 .mu.M)
was treated for 24h and cell viability in organoids was assessed.
By treatment of Troglitazone alone at 50 .mu.M, cell viability was
85% at 24 hours while it was decreased to 67% at 72h. However,
after treatment of Troglitazone on lipotoxic condition, massive
fragmentation of organoids was observed due to an organoid death.
Subsequent cell viability analysis confirmed this result (-40%
compared to control, p<0.05) (FIG. 6, panel E and 6, panel
F).
[0085] Next, Applicant investigated whether organoids can be
recovered from a DILI-like condition by a potential therapeutic
compound. The applicant used N-acetylcysteine (NAC), an
antioxidant, to inhibit ROS production because intravenous NAC
improves survival in patients with non-acetaminophen-related acute
liver failure (Lee et al., 2009) and diminished the
Troglitazone-induced cytotoxicity (Rachek et al., 2009). As
expected, cell viability was significantly improved by NAC,
suggesting that NAC rescued cell death in organoids even in
vulnerable conditions (FIG. 6, panel E and 6, panel F). In most
DILI cases, the only intervention is the removal of the causative
drug if identified (Polson and Lee, 2005) (Bohan et al., 2001;
Navarro and Senior, 2006). This LoT system may be a useful tool for
identifying causal drugs linked to multi-drug regimens as well as
drug discovery for treating DILI.
[0086] Serious adverse events (SAE) including liver failure are a
major cause of drug attrition during clinical development or
withdrawal of marketed pharmaceuticals. In particular, DILI is a
critical challenge in drug development, in which drug induced
cholestasis induced by inhibitions of transporter activity is one
major cause. Sandwich culture using human primary hepatocytes is
the current best choice in pharmaceuticals. Although recent reports
showed the promise of hepatocyte based cholestasis model using
transdifferentiated cells from human fibroblasts (Ni et al., 2016),
these assay platforms still have reproducibility challenges as well
as throughput problems because of variable and limited human
hepatocyte sources and the need for complex quantitation
algorithms. In addition, HepaRG cells, a human hepatoma cell line,
is also useful to evaluate cholestatic features, but their low BSEP
(Bile Salt Export Pump, or ABCB11, important transporter for bile
acid excretion, as well as major target for cholestatic agents)
activity and time-consuming differentiation procedure, limit their
use (Le Vee et al., 2013). More importantly, a lack of essential
anatomical structures limits their practical use for the
pharmaceutical industry. Alternatively, the described methods allow
for a simple, robust and high-throughput system to measure bile
transport activity by live fluorescent imaging in the presence of
testing compounds. The major advantages of the LoT assay include:
1. the cost effectiveness (S12.35 per 50 organoids; S94.85 per 384
well), 2. the assay throughput (measurable at single organoid) and
3. the multiplexed readouts for analyzing interplay between other
factors such as mitochondrial stress. Especially, as mentioned
above, retrospective studies revealed that multiple cell stress
potentials were associated with the incidence of DILI (Aleo et al.,
2014) and the LoT assay showed the comparable results with that
study, since cell viability was decreased dependent on dual
readouts; mitochondrial and cholestatic stress. Oxidative stress
plays an important role in cell death and has been linked to the
development of cholestatic liver injury (Serviddio et al., 2004).
Hydrophobic bile acids accumulate intracellularly during
cholestasis and interfere with normal mitochondrial electron
transport, inhibiting the activity of respiratory complexes I and
III and consequently reducing adenosine triphosphate synthesis
(Krahenbuhl et al., 1994), resulting in mitochondrial
dysfunction-induced apoptosis (Bernardi, 1996). In line with these
findings, Applicant's correlational analysis of these dual readouts
indicated cholestatic stress was a more dominating factor for liver
injury compared with mitochondria stress as was seen in FIG. 5.
Thus, the LoT system may be used as a model system for
investigating DILI mechanisms.
[0087] In addition, given the recent establishment of population of
iPSC panels, potential assessment for different susceptibility in
individuals is also promising (Inoue et al., 2014). Predicting SAE
in conventional in vitro assay system does not generally focus on
individual differences, however, SAE often occur in a small
SAE-prone patient subgroup (Stevens and Baker, 2009). Applying the
LoT system to diverse population iPSC panels will provide
previously inaccessible about different susceptibilities to SAE. In
light of the extremely rare nature of DILI, the use of patients'
cells with specific genomic or ethnic factors will aid in
elucidating the currently unknown idiosyncratic mechanism of DILI.
Thus, LoT might serve as a game changing strategy for
pharmaceutical industries by providing essential insight for
minimizing DILI potential (FIG. 7).
[0088] One limitation in this organoid model is the lack of
immunological reactions. The immunological effect resulting from a
hypersensitivity reaction is one possible mechanism for
idiosyncratic DILI. There are limited in vitro models to assess the
hypersensitivity by drugs, although the sensitivity by
Troglitazone-induced cytotoxicity was increased using in vitro
co-culture model using hepatic cell line, Huh? and THP-1 cells
(Edling et al., 2009). Thus, advancing the LoT platform by focusing
on immune lineages will be of interest to assess hepatocellular
inflammation. Nevertheless, the LoT testing platform seems superior
in generating reproducible and large data sets from an individual
organoid as the inhibition of bile efflux function by multiple FDA
approved drugs is reproducibly observed in this assay. Considering
cholestasis is induced by a broad spectrum of liver diseases
including drug-induced, lipotoxic, infectious, and congenital
conditions (Chatterjee et al., 2014), the organoid based LoT assay
is useful for analyzing intra-hepatic cholestasis in a variety of
contexts with the potential for mechanistic studies as well as drug
screening applications beyond DILI.
[0089] Studying Vulnerable Human Liver Condition with LoT Assay
[0090] Host factors such as obesity are known to significantly
influence the onset of DILI (Heidari et al., 2014), yet they are
often understudied in the clinical setting due to its complex
nature. The presence of obesity or fatty liver might make patients
vulnerable to hepatic injury induced by xenobiotics and non-toxic
chemicals (e.g., drugs) and these might become hepatotoxic in lower
doses in the presence of risk factors (Fromenty, 2013). Despite
this, the current clinical trial system is not designed for
stratifying volunteers under vulnerable liver conditions with
handful of biomarker (ALT, AST) levels. Since number of patients
with steatosis are subclinical with no detectable biomarkers before
dosing, it is critical to foresee the outcomes in this vulnerable
condition before entering the clinical phase.
[0091] With an effort to advance the LoT system to assess the
toxicity in these vulnerable conditions on early drug screening
stage such as lead compound generation/optimization, Applicant
applied lipotoxic stress to liver organoids and demonstrated the
exaggerated synergistic effect of Troglitazone, an antidiabetic
drug, on DILI. Indeed, the organoid system successfully reflects
this feature by showing massive hepatocyte death, promoted by
triglyceride accumulation into hepatocytes in an organoid. One of
the mechanisms of DILI in obesity could explain reduced levels of
glutathione (GSH) (Michaut et al., 2016). Drug-induced oxidative
stress could have several origins, in particular, through GSH
depletion and inhibition of the mitochondrial respiratory chain
(Begriche et al., 2011; Pessayre et al., 2010). The vulnerable
model might reflect the decrease in intracellular GSH levels and
worsened Troglitazone induced oxidative stress through
mitochondrial dysfunction, ameliorated by providing NAC.
Considering the dramatic rise in non-alcoholic steatohepatitis
(NASH) prevalence, it is noteworthy that there is still a minimal
list of drugs for either aggravating pre-existing NAFLD or inducing
more frequently an acute hepatitis. Additionally, an in vitro
reductionist system provides a previously unforeseen window to
study previously untested host factors as the isolated host factor
can effectively deployed into organoids.
[0092] LoT Based Precision Medicine
[0093] The selection of optimal drug therapy using LoT will be of
major interest in clinics from a personalized medicine perspective.
For instance, the strategy considered in the choice of
antipsychotic agent must take into account the hepatic tolerance
according to the non-negligible incidence of liver disorders among
psychiatric population; 16% of possible DILI agents are
neuropsychiatric drugs (Dumortier et al., 2002). Given that NASH is
often accompanied by psychological disorders such as depression,
safer combinatorial selection of anti-depressive drugs (tricyclic
agents or SSRI), mood stabilizing agents and neuroleptic drugs is
needed (Dumortier et al., 2002). Also, due to an age-associated
increase in chronic conditions, multiple medication use (i.e.,
polypharmacy) is a common consequence of providing health care to
older adults (Marcum and Gellad, 2012), making it extremely
difficult to identify causative drugs when DILI is suspected. As
patient derived iPSC-organoids provide an unlimited and
reproducible source, LoT may serve as a panel to stratify the
potential of DILI in patients and provide information to choose
safer medication from a personalization perspective.
[0094] LoT Based New Drug Discovery to DILI
[0095] Equally important is the potential use for anti-DILI
therapeutic compound screen using LoT system. A large number of
drugs have deleterious effects on liver and DILI and it is a major
clinical problem. Actually, acetaminophen accounts for
approximately half of cases of DILI in the United States (Russo et
al., 2004). In other regions of the world, for instance in
developing countries, other drugs such as anti-tuberculosis
medications might be the leading cause of DILI (Bell and Chalasani,
2009). However, there are only several symptomatic therapies
available. Here, as a proof-of-concept experiment, Applicant
established an organoid survival experiment to assess the
therapeutic effect of a compound to resist a toxic mechanism of
DILI as evidenced by Troglitazone. While NAC is the major treatment
choice for paracetamol overdose (Makin et al., 1995; Verma and
Kaplowitz, 2009), recently, the research focus has shifted to
investigating the use of NAC in non-paracetamol DILI (Chughlay et
al., 2016). LoT system is useful to assess the efficacy of NAC to
DILI by non-paracetamol drugs. Beyond, this higher throughput
approach will serve as a powerful tool for screening larger scale
compound libraries that restore DILI-like symptoms in vitro.
Combined, methods described here can be used to identify and study
cell-intrinsic and extrinsic factors associated with clinical DILI
phenotypes, and would facilitate lead compound optimization,
mechanistic study, and precision medicine, as well as anti-DILI
therapy screening applications.
[0096] Methods
[0097] Maintenance of PSCs
[0098] TkDA3 with CYP2C9*2 variant human iPSC clone used in this
study was kindly provided by K. Eto and H. Nakauchi. Other suitable
lines include human iPSC lines gifted from Kyoto University and
those purchased from Coriell Biorepository. were maintained as
described previously (Takahashi et al., 2007). Undifferentiated
hiPSCs were maintained on feeder-free conditions in mTeSR1 medium
(StemCell technologies, Vancouver, Canada). Other suitable media
include E8 from Lonza, or StemFit from Aijinomoto Co. Plates are
coated with Matrigel (Corning Inc., New York, N.Y., USA) of 1/30
dilution at 37.degree. C. in an incubator with 5% CO.sub.2/95% air.
hPSCs Maintenance. In place of Matrigel, Laminin511, Laminin411
from Mippi Co or Biolamina Co can be used.
[0099] Production of Liver Organoids (HLO)
[0100] Differentiation of hiPSCs into definitive endoderm was
induced using previously described methods with several
modifications (Spence et al., 2011). In brief, colonies of hiPSCs
were isolated in Accutase (Thermo Fisher Scientific Inc., Waltham,
Mass., USA) and 150000-300000 cells were plated on Matrigel or
laminin coated tissue culture 24 well plate (VWR Scientific
Products, West Chester, Pa.). When the cells become a high-density
(over 90% of the cells covering the well), medium was changed to
RPMI 1640 medium (Life Technologies, Carlsbad, Calif.) containing
100 ng/mL Activin A (R&D Systems, Minneapolis, Minn.) and 50
ng/mL bone morphogenetic protein 4 (BMP4; R&D Systems) at Day
1, 100 ng/mL Activin A and 0.2% fetal calf serum (FCS; Thermo
Fisher Scientific Inc.) at Day 2 and 100 ng/mL Activin A and 2% FCS
at Day 3. For Day 4-6, cells were cultured in Advanced DMEM/F12
(Thermo Fisher Scientific Inc.) with B27 (Life Technologies) and N2
(Gibco, Rockville, Md.) containing 500 ng/ml fibroblast growth
factor (FGF4; R&D Systems) and 3 .mu.M CHIR99021 (Stemgent,
Cambridge, Mass., USA). Cultures for cell differentiation were
maintained at 37.degree. C. in an atmosphere of 5% CO2/95% air and
the medium was replaced every day. Differentiated definitive
endoderm showed budding on the plate at Day 7. If the spheroids
were not enough to be embedded into Matrigel, Day4-6 media is added
again and incubated with at 37.degree. C. overnight.
[0101] Differentiation into Liver Organoids.
[0102] Three methods may be used to differentiate the DE into liver
organoids: The "Matrigel Drop Method," the "Matrigel Sandwich
Method," and the Matrigel-Free Method," each of which is described
below.
[0103] Matrigel Drop Method:
[0104] On Day 7-8, definitive endoderm organoids with plated cells
were gently pipetted to delaminate from dishes. Isolated spheroids
were centrifuged at 800 rpm for 3 minutes and, after removing
supernatant, embedded in 100% matrigel drop on the dishes. The
plates were placed at 37.degree. C. in an atmosphere of 5% CO2/95%
air for 5-15 min. After the Matrigel was solidified, Advanced
DMEM/F12 was added with B27, N2 and Retinoic acid (RA; Sigma, St.
Louis, Mo.) 2 .mu.M for 1-5 days. The media was replaced every
other day. After RA treatment, organoids embedded in Matrigel drop
were cultured in Hepatocyte culture medium (HCM Lonza,
Walkersville, Md.) with 10 ng/mL hepatocyte growth factor (HGF;
PeproTech, Rocky Hill, N.J.), 0.1 .mu.M Dexamethasone (Dex; Sigma)
and 20 ng/mL Oncostatin M (OSM; R&D Systems). Cultures for cell
differentiation were maintained at 37.degree. C. in an atmosphere
of 5% CO.sub.2/95% air and the medium was replaced every 3 days.
Around Day 20-30, organoids embedded in Matrigel drop were isolated
by scratching and gentle pipetting for any analyses.
[0105] Matrigel Sandwich Method:
[0106] On Day 7-8, definitive endoderm organoids with plated cells
were gently pipetted to delaminate from dishes. Isolated spheroids
were centrifuged at 800 rpm for 3 minutes, and after removing
supernatant, they were mixed with 100% Matrigel. At the same time,
hepatocyte culture medium with all supplements was mixed with the
same volume of 100% Matrigel. HCM and Matrigel mix was plated to
the bottom of dish to make a thick coating on the plate (0.3-0.5
cm), and placed at 37.degree. C. in an atmosphere of 5%
CO.sub.2/95% air for 15-30 min. After the Matrigel was solidified,
spheroids mixed with Matrigel was seeded on Matrigel thick coated
plated. The plate was placed at 37.degree. C. in an atmosphere of
5% CO.sub.2/95% air for 5 min. Advanced DMEM/F12 was added with
B27, N2 and Retinoic acid (RA; Sigma, St. Louis, Mo.) 2 .mu.M for
1-5 days. The media was replaced every other day. After RA
treatment, organoids embedded in Matrigel drop were cultured in
Hepatocyte culture medium (HCM Lonza, Walkersville, Md.) with 10
ng/mL hepatocyte growth factor (HGF; PeproTech, Rocky Hill, N.J.),
0.1 .mu.M Dexamethasone (Dex; Sigma) and 20 ng/mL Oncostatin M
(OSM; R&D Systems). Cultures for cell differentiation were
maintained at 37.degree. C. in an atmosphere of 5% CO.sub.2/95% air
and the medium was replaced every 3 days. Around Day 20-30,
organoids embedded in Matrigel drop were isolated by scratching and
gentle pipetting for any analyses.
[0107] Matrigel-Free Method:
[0108] On Day 7-8, definitive endoderm organoids with plated cells
were continued planar culture in Advanced DMEM/F12 (Thermo Fisher
Scientific Inc.) with B27 (Life Technologies) and N2 (Gibco,
Rockville, Md.) Retinoic acid (RA; Sigma, St. Louis, Mo.) 2 .mu.M
for 4 days. The media was replaced every other day. After the 4
days planar culture, the organoids begin to bud, whereas 2D cells
differentiate into hepatocytes. Both organoids and hepatocytes can
be maintained for over 60 days under Hepatocyte culture medium (HCM
Lonza, Walkersville, Md.) with 10 ng/mL hepatocyte growth factor
(HGF; PeproTech, Rocky Hill, N.J.), 0.1 .mu.M Dexamethasone (Dex;
Sigma) and 20 ng/mL Oncostatin M (OSM; R&D Systems) for 10
days. For organoid assays, floating organoids can be collected in
Ultra-Low attachment multiwell plates 6 well plate and used for
subsequent assays whenever appropriate. Cultures for cell
differentiation were maintained at 37.degree. C. in an atmosphere
of 5% CO.sub.2/95% air and the medium was replaced every 3
days.
[0109] H&E Staining and Immunohistochemistry
[0110] Liver organoids were collected from Matrigel, fixed in 4%
paraformaldehyde and embedded in paraffin. Sections were subjected
to H&E and immunohistochemical staining. The following primary
antibodies were used: anti-human albumin antibody (1:200 dilution
abcam, Cambridge, UK), anti-type IV collagen antibody (1:200
dilution eBioscience, San Diego, Calif., USA), anti-ZO-1 antibody
(1:200 dilution BD Transduction Laboratories (San Jose, Calif.,
USA) and anti-MRP2 antibody (1:200 dilution Novus Biologicals,
Littleton, Colo.). Dye-conjugated secondary antibodies, Alexa Fluor
568-conjugated donkey anti-rabbit immunoglobulin (IgG; 1:1000;
Invitrogen, A10042) was applied to the organoids at room
temperature for 2 h. Nuclei were stained with 10 .mu.g/mL Hoechst
33342 (Sigma) at room temperature for 10 min, after which organoids
were washed again three times with washing buffer. The specimens
were observed under a Fluorescent microscope or bright-field. For
whole mount immunohistochemical staining, after liver organoids
were fixed in 4% paraformaldehyde for 30 min and permeabilized with
2.5% Tween 20 (Sigma) at room temperature, organoids were incubated
overnight at 4.degree. C. with the following primary antibodies
diluted in PBS: polyclonal anti-BSEP antibody (1:200 Sigma).
Fluorescent dye-conjugated secondary antibodies, Alexa Fluor
568-conjugated donkey anti-rabbit immunoglobulin (IgG; 1:500;
Invitrogen, A10042) were applied to the organoids at room
temperature for 2 h. After the reaction, the cells were washed
three times with washing buffer (PBS containing 0.5% Triton-X 100
[Sigma] and 0.5% bovine serum albumin [BSA; Sigma]). Nuclei were
stained with 10 .mu.g/mL Hoechst 33342 (Sigma) at room temperature
for 10 min, after which organoids were washed again three times
with washing buffer. The specimens were observed under a confocal
imaging performed on a Nikon A1Rsi inverted confocal
microscope.
[0111] RNA Isolation, RT-qPCR
[0112] RNA was isolated using the RNeasy mini kit (Qiagen, Hilden,
Germany). Reverse transcription was carried out using the
SuperScriptlll First-Strand Sysnthesis Systen for RT-PCR
(Invitrogen, CA, USA) according to manufacturer's protocol. qPCR
was carried out using TaqMan gene expression master mix (Applied
Biosystmes) on a QuantStudio 3 Real-Time PCR System (Thermo). All
primers and probes information for each target gene were obtained
from the Universal ProbeLibrary Assay Design Center
(https://qper.probefinder.com/organism.jsp).
[0113] Principal Component Analysis of RNA-Seq Data
[0114] RNA isolation, cDNA synthesis, sequencing on Illumina HiSeq
2500 are described previously (Asai et al., 2017). The RNA-Seq
reads were aligned to the human genome (GRCh37/hg19) using TopHat
(version 2.0.13). The alignment data from Tophat were fed to an
assembler, Cufflinks (version 2.2.1), to assemble aligned RNA-Seq
reads into transcripts. Annotated transcripts were obtained from
the UCSC genome browser (http://genome.ucsc.edu) and the Ensembl
database. Transcript abundances were measured in Fragments Per
Kilobase of exon per Million fragments mapped (FPKM).
[0115] To compare the lineage of pHLO, Applicant combined in-house
RNA-seq data (pFG and organoids) with preprocessed public data as
follows: Transcript abundances of iPSC, DE, HS, HP, iDH and NHC
were obtained from GSE86007 (Jalan-Sakrikar et al., 2016); ones of
child liver tissue, adult liver tissue, adult right lobe tissue,
fetal liver tissue and primary hepatocyte were obtained from ENCODE
(ENCFF418BVF, ENCFF804QWF, ENCFF965IQH, ENCFF918SJO, ENCFF367FJJ,
ENCFF029IUF, ENCFF280YNO, ENCFF347TXW, ENCFF724CQI, ENCFF624LQL,
ENCFF962SOD, ENCFF170AEC) (Consortium, 2012; Sloan et al., 2016)
and GSE85223 (Asai et al., 2017). Genes were used if all dataset
have identical gene symbol after possible data preprocessing.
Applicant performed quantile-normalization of FPKM+1 and RPKM+1
data in log 2 space followed by selected genes within the top 10000
of median expression levels. Principal component analysis was
performed by using the scaled gene expression levels by using R
package FactoMineR (version 1.35) (Sebastien L , 2008).
[0116] Protein Secretion Analysis
[0117] For measuring albumin, fibrinogen and complement factors
secreted level of organoids, 200 .mu.L of culture supernatant of
organoids on Ultra-Low attachment 96 well plates (Corning) were
collected. The culture supernatants were collected and stored at
-80.degree. C. until use. The supernatant was assayed with Human
Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc., TX, USA)
and fibrinogen (Thermo Fisher Scientific) according to the
manufacturer's instructions. For analyzing complement factors,
supernatants were measured with Luminex System (Luminex
Corporation, Austin, Tex.) according to the manufacturer's
instructions. To calculate albumin production per the number of
cells, the linear regression equation of cell numbers by diameter
of an organoid was used. For measuring total bile acid secreted
level intra-luminal organoids, fluid inside organoids was absorbed
using microinjection Nanoject II (Drummond Scientific, Broomall,
Pa., USA). Fluid absorbed was dilute in PBS and assayed with Total
Bile Acid ELISA Kit (Antibodies-online, Inc., GA, USA). To
calculate the volume of total bile acid, the number of cells in
organoid was calculated using the linear regression equation in the
same manner for albumin production and the molecular weight of
cholic acid was used for calculation and comparing with volume in
previous report.
[0118] Transmission Electron Microscopy
[0119] For transmission electron microscopy, briefly, organoids
were fixed in 3% glutaraldehyde for overnight at 4.degree. C.,
washed in 0.1 M sodium cacodylate buffer, and incubated for 1 h in
4% osmium tetroxide. They were subsequently washed then dehydrated
in ethanol series, and finally embedded in propylene oxide/LX112.
Tissue was then sectioned and stained with 2% uranyl acetate
followed by lead citrate. Images were visualized on Hitachi
transmission electron microscope.
[0120] CGamF Assay
[0121] Briefly, organoids were pre-incubated with a transport
buffer (118 mM NaCl, 23.8 mM NaHCO.sub.3, 4.83 mM KCl, 0.96 mM
KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5 mM glucose, 1.53 mM CaCl2,
adjusted to pH 7.4) for 30 min. Next, organoids were treated by 10
.mu.M fluorescently labeled bile acid (CGamF; a kind gift from Dr
Hofmann) for 1h, after then, organoids were washed three times with
PBS. Images were captured on fluorescent microscopy BZ-X710
(Keyence, Osaka, Japan).
[0122] Evaluation of Bile Transport Inhibition
[0123] Fluorescein diacetate was used for evaluating bile transport
activity in organoids. Around Day 25, the organoids were rinsed
with PBS, and, fluorescein diacetate was treated to organoids in
medium. In addition, to investigate the direction of transport,
fluorescein diacetate was injected to organoids using by Nanoject
III (Drummond Scientific). After treatment or injection of
fluorescein diacetate, images were captured on fluorescent
microscopy BZ-X710 (Keyence). Next, to check the feasibility of
test system, 10 mg/mL fluorescein diacetate (Sigma) in HCM was
added with 20 .mu.M Cyclosporin A (CSA; Sigma) for 45 minutes and
images were captured sequentially using fluorescent microscopy
BZ-9000 (Keyence). For evaluation of bile transport inhibition, 10
mg/mL fluorescein diacetate in HCM was added after treatment of
dimethyl sulfoxide (DMSO; Sigma), Streptomycin (STP; Sigma) as a
negative control, Tolcapone (Tol; Sigma), Diclofenac (Diclo;
Sigma), Bosentan (BOS; Sigma), CSA, Troglitazone (Tro; Sigma),
Nefadozone (Nefa; Sigma), Entacapone (Enta; Sigma) and Pioglitazone
(PIO, Sigma). After 5 minutes incubation, the organoids were rinsed
three times with PBS and images were captured sequentially using
fluorescent microscopy BZ-X710. Analysis was performed by
calculating ratio between the intensities outside and inside
organoids using Imagej 1.48 k software (Wayne Rasband, NIHR, USA,
http://imagej nih gov/ij). Changes in brightness or contrast during
processing were applied equally across the entire image.
[0124] Mitochondria Toxicity Potential Evaluation
[0125] After being cultured in on Ultra-Low attachment multi-well
plates 6 well plate in each culture condition, organoids were
picked up and seeded in Microslide 8 Well Glass Bottom (Ibidi,
Wis., USA). For evaluation of mitochondria membrane potential
(MMP), 250 nM Tetramethylrhodamine, Methyl Ester, Perchlorate
(TMRM; Thermo Fisher Scientific) was added after treatment of
dimethyl sulfoxide (DMSO; Sigma), Streptomycin (STP; Sigma) as a
negative control, Tolcapone (Tol; Sigma), Diclofenac (Diclo;
Sigma), Bosentan (BOS; Sigma), Cyclosporin A (CSA; Sigma),
Troglitazone (Tro; Sigma), Nefadozone (Nefa; Sigma), Entacapone
(Enta; Sigma) and Pioglitazone (PIO, Sigma) for 24h. After 30
minutes incubation, the organoids were rinsed three times with PBS
and images were scanned on a Nikon A1 Inverted Confocal Microscope
(Japan) using 60.times. water immersion objectives. Arias and
intensity of TMRM were calculated as MMP by IMARIS8 (Bitplane AG,
Switzerland). For assessment of cholestatic and mitochondrial
stress, cell viability was measured by using the CellTiter-Glo.RTM.
luminescent cell viability assay (Promega, Mannheim, Germany) per
organoid at 24h after treatment of drugs and confirmed not to
decrease the viability in each dose for avoiding secondary change
due to cell damage toward death.
[0126] Analysis of Relationship of Cell Viability in Organoids with
Mitochondria and Cholestatic Stress
[0127] To demonstrate the relationship of cell viability with
mitochondrial and cholestatic stress, first, indexes were setup
using following formula; "Index=-(Sample value-Control
value).times.100" based on the value provided from mitochondrial
and cholestatic stress assays. For analyzing cell damage associated
with mitochondrial and cholestatic stress, at 72h after treatment
of drugs, the ATP content per organoid was determined using the
CellTiter-Glo.RTM. luminescent cell viability assay (Promega).
These data were shown as FIG. 4, panel B using Infogr.am
(http://infogr.am): a free, web-based tool.
[0128] Evaluation of Viability in Organoids on Vulnerable
Condition
[0129] The experiment was performed as shown in FIG. 5A. After
being excluded from Matrigel and washed, organoids were treated 800
.mu.M oleic acid on Ultra-Low attachment multiwell plates 6 well
plate (Corning) for 3 days. Next, 50 .mu.M of Troglitazone was
treated with or without 50 .mu.M NAC for 24h. Cell viability was
performed by using the CellTiter-Glo.RTM. luminescent cell
viability assay (Promega). Images were captured sequentially using
fluorescent microscopy BZ-9000.
[0130] Lipid Induced Mitochondria Stress Evaluation
[0131] After being cultured in on Ultra-Low attachment multiwell
plates 6 well plate in each culture condition, twenty organoids
were picked up and seeded in Microslide 8 Well Glass Bottom (Ibidi,
Wis., USA) and subjected to live-cell staining. The following
regents or kits were used: BODIPY.RTM. 493/503 for lipids (Thermo
Fisher Scientific), and SiR Actin Kit for cytoskeleton (USA
Scientific, FL, USA), CellROX.RTM. Green Reagent for ROS (Fisher
Scientific), TMRM (Thermo Fisher Scientific) for mitochondria.
Organoids were visualized and scanned on a Nikon A1 Inverted
Confocal Microscope (Japan) using 60.times. water immersion
objectives. ROS production, mitochondria size and number were
analyzed by IMARIS 8.
[0132] Statistics
[0133] Statistical significance was determined using unpaired
Student's t-test or one-way ANOVA with Dunnett's multiple
comparison post-hoc test. P<0.05 was considered significant.
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[0198] All percentages and ratios are calculated by weight unless
otherwise indicated.
[0199] All percentages and ratios are calculated based on the total
composition unless otherwise indicated.
[0200] It should be understood that every maximum numerical
limitation given throughout this specification includes every lower
numerical limitation, as if such lower numerical limitations were
expressly written herein. Every minimum numerical limitation given
throughout this specification will include every higher numerical
limitation, as if such higher numerical limitations were expressly
written herein. Every numerical range given throughout this
specification will include every narrower numerical range that
falls within such broader numerical range, as if such narrower
numerical ranges were all expressly written herein.
[0201] The dimensions and values disclosed herein are not to be
understood as being strictly limited to the exact numerical values
recited. Instead, unless otherwise specified, each such dimension
is intended to mean both the recited value and a functionally
equivalent range surrounding that value. For example, a dimension
disclosed as "20 mm" is intended to mean "about 20 mm"
[0202] Every document cited herein, including any cross referenced
or related patent or application, is hereby incorporated herein by
reference in its entirety unless expressly excluded or otherwise
limited. The citation of any document is not an admission that it
is prior art with respect to any invention disclosed or claimed
herein or that it alone, or in any combination with any other
reference or references, teaches, suggests or discloses any such
invention. Further, to the extent that any meaning or definition of
a term in this document conflicts with any meaning or definition of
the same term in a document incorporated by reference, the meaning
or definition assigned to that term in this document shall
govern.
[0203] While particular embodiments of the present invention have
been illustrated and described, it would be obvious to those
skilled in the art that various other changes and modifications can
be made without departing from the spirit and scope of the
invention. It is therefore intended to cover in the appended claims
all such changes and modifications that are within the scope of
this invention.
* * * * *
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