U.S. patent application number 16/346190 was filed with the patent office on 2020-02-20 for liver organoid disease models and methods of making and using same.
The applicant listed for this patent is Children's Hospital Medical Center, Japan Science and Technology Agency. Invention is credited to Masaki Kimura, Rie Ouchi, Takanori Takebe.
Application Number | 20200056157 16/346190 |
Document ID | / |
Family ID | 62076362 |
Filed Date | 2020-02-20 |
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United States Patent
Application |
20200056157 |
Kind Code |
A1 |
Takebe; Takanori ; et
al. |
February 20, 2020 |
LIVER ORGANOID DISEASE MODELS AND METHODS OF MAKING AND USING
SAME
Abstract
Disclosed herein are methods of making and using lipotoxic
organoid models. In certain aspects, the methods may comprise the
steps of contacting a liver organoid with a free fatty acid (FFA)
composition. In one aspect, the FFA composition may comprise oleic
acid, linoleic acid, palmitic acid, or combinations thereof.
Inventors: |
Takebe; Takanori;
(Cincinnati, OH) ; Ouchi; Rie; (Cincinnati,
OH) ; Kimura; Masaki; (Cincinnati, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Hospital Medical Center
Japan Science and Technology Agency |
Cincinnati
Kawaguchi-shi, Saitama |
OH |
US
JP |
|
|
Family ID: |
62076362 |
Appl. No.: |
16/346190 |
Filed: |
November 3, 2017 |
PCT Filed: |
November 3, 2017 |
PCT NO: |
PCT/US17/59860 |
371 Date: |
April 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62417371 |
Nov 4, 2016 |
|
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62517414 |
Jun 9, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0029 20130101;
C12N 5/0672 20130101; C12N 5/0696 20130101; A61K 35/407 20130101;
C12N 2500/36 20130101; A61P 1/16 20180101; C12N 2506/45 20130101;
C12N 2501/119 20130101; C12N 5/0697 20130101; G01N 33/5008
20130101; A61K 45/06 20130101; C12N 5/0671 20130101; A61K 31/575
20130101; C12N 15/01 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; G01N 33/50 20060101 G01N033/50 |
Claims
1. A method of making a lipotoxic organoid model, comprising the
steps of contacting a liver organoid with a free fatty acid (FFA)
composition, wherein said FFA composition comprises oleic acid,
linoleic acid, palmitic acid, or combinations thereof.
2. The method of claim 1, wherein said lipotoxic organoid model is
a model of fatty liver disease.
3. The method of claim 1, wherein said lipotoxic organoid model is
a model of steatohepatitis.
4. The method of claim 1, wherein said lipotoxic organoid model is
a model of cirrhosis.
5. The method of claim 1, wherein said lipotoxic organoid model is
a model of parenteral nutrition associated liver disease
(PNALD).
6. The method of claim 1, wherein said lipotoxic organoid model is
a model of NAFLD.
7. The method of claim 1, wherein said lipotoxic organoid model is
characterized by one or more features selected from cytoskeleton
filament disorganization, ROS increase, mitochondrial swelling,
trigycleride accumulation, fibrosis, hepatocyte ballooning, IL6
secretion, steatosis, inflammation, ballooning, Mallory's bodies,
tissue stiffening, cell-death, and combinations thereof.
8. A method of screening for a drug for treatment of a liver
disease, comprising the step of contacting a candidate drug with
the lipotoxic organoid model of claim 1.
9. (canceled)
10. A composition comprising a three-dimensional (3D) liver
organoid model of liver disease obtained by expansion of one or
more precursor cells.
11. The composition of claim 10, wherein said liver disease is drug
induced hepatotoxicity having one or both of inflammation and
fibrosis.
12. The composition of claim 10, wherein said liver disease is
parenteral nutrition associated liver disease (PNALD).
13. The composition of claim 10, wherein said three-dimensional
(3D) liver organoid model does not comprise inflammatory cells.
14. The composition of claim 10, wherein said three-dimensional
(3D) liver organoid model of is a model of fatty liver disease,
wherein said organoid is characterized by one or more
characteristics selected from steatosis, inflammation, ballooning,
Mallory's bodies, ROS accumulation, mitochondrial overload,
fibrosis, tissue stiffening, and cell death.
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] Irreversible epithelial organ remodeling is a major
contributing factor to worldwide death and disease, costing
healthcare systems billions of dollars every year (Hynds and
Giangreco, 2013). Diseases of epithelial remodeling include lung
and gastrointestinal cancers as well as chronic diseases such as
liver cirrhosis, chronic obstructive pulmonary disease (COPD), and
inflammatory bowel disease (Hynds and Giangreco, 2013). Sadly, most
epithelial organ research performed primarily on animals fails to
produce new therapies for these diseases and mortality rates remain
unacceptably high. This is partly due to a lack of predictive human
systems to test the efficacy of a vast growing number of compound
libraries in pharmaceutical industries, imposing a fundamental
challenge to develop a high-fidelity system for modeling
inflammation and fibrosis in humans towards clinically relevant
therapy development.
[0003] Non-alcoholic Fatty Liver Disease (NAFLD) is one of the
major challenges to be overcome in developed countries due to the
increased chance of developing lethal liver disorders, yet
effective treatment is lacking. Similarly, iatrogenic parenteral
nutrition associated liver disease (PNALD) is a disorder that
arises from parenteral nutrition and which currently has no
effective treatment. Models having steatohepatitis and/or clinical
histopathological features of liver disease such as lipid droplet
accumulation and cystoskeleton filament disorganization, steatosis,
and hepatocellular ballooning, and which can be used to address
these and other liver disorders or disease states, are needed.
Despite the promise of disease models using patients' stem cells,
current approaches are limited in their applications to
unicellular, monogenic and relatively simple pathologies, failing
to capture more prevailing and complex disease pathology such as
epithelial organ fibrosis.
[0004] The human liver is a vital organ that provides many
essential metabolic functions for life such as lipid metabolism,
ammonium and bile production, coagulation, as well as the
detoxification of exogenous compounds. Using induced pluripotent
stem cell (iPSC) technology, in vitro reconstitution of patients'
liver reaction is attractive to the pharmaceutical industry, due to
a number of promising applications including regenerative therapy,
drug discovery, and drug toxicity studies. To this aim, currently,
conventional in vitro approaches study two-dimensional (2-D) and
3-D differentiation platforms to generate liver cells. However,
most of the reported methods predominantly differentiate cells into
target epithelial cell type, completely lacking an essential
supporting component such as pro-fibrotic and/or inflammatory cell
types. Alternatively, Applicant and others have proposed co-culture
based approaches by mixing epithelial and supportive lineages,
however, these assays are heavily variable and often confounded by
a number of artefactual changes such as the difficulty in selecting
Epithelial Cell Medium (ECM) and medium in which they can be
co-maintained. Thus, there is a need for the establishment of a new
and robust assay system in which the supportive lineages co-develop
for disease modeling and further screening application.
BRIEF SUMMARY
[0005] Disclosed herein are methods of making and using lipotoxic
organoid models. In certain aspects, the methods may comprise the
steps of contacting a liver organoid with a free fatty acid (FFA)
composition. In one aspect, the FFA composition may comprise oleic
acid, linoleic acid, palmitic acid, or combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] 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.
[0007] 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.
[0008] FIG. 1. Co-differentiation of pre-fibrotic and inflammatory
lineages in human iPSC-liver organoid A. Schematic representation
of retinoic acid (RA)-based liver organoid differentiation method
and bright-field image of day 20 liver organoids. Scale bar, 100
.mu.m. B. Bright-field image of liver organoids in Matrigel at day
20 after the culture in the presence and absence of 4 days RA. C.
Organoid numbers were measured manually at day 20 after the culture
in the presence (black bar) and absence (gray bar) of 4 days RA. D.
Diameter of organoid was recognized and measured by Image J at day
20 after the culture in the presence (black bar) and absence (gray
bar) of 4 days RA. E. Albumin production was measured from the
culture supernatants collected in 24 hrs after the culture of day
22-25 liver organoids in Matrigel with (black bar) or without (gray
bar) 4 days RA. F. Organoids with internal luminal structure were
counted manually at day 25 after culturing in the presence and
absence of 4 days RA. Red and gray bars indicate the percent of
organoids with (HLO)/without (spheroid) internal luminal structure,
respectively. G. Bile transport activity was monitored by
fluorescein diacetate (FD), which turns into fluorescein (green)
after esterase hydrolysis in hepatocytes, in the culture media and
tracks the flow of the fluorescence. Results represent
mean.+-.s.d., n=3. Percentages of Epcam, CD166, and CD68 positive
population were determined by flow cytometry. Results represent
mean.+-.s.d., n=2-3. H. Single cell RNA sequencing!!FPKM (log 2)
values for hepatocyte, stellate cell, Kupffer cell, and
endothelial-associated genes expressed by human iPSC, human iPSC
derived definitive endoderm, foregut spheroid, HLO, human adult
liver tissue and human fetal liver tissue. I. Percentages of Epcam,
CD166, CD68, and F4/80 positive population were determined by flow
cytometry. Results represent mean.+-.s.d., n=3. J.
Immunofluorescent (IF) staining of day 25 HLO for albumin, CD68,
Vimentin, GFAP and Epcam. White arrow indicates the localization of
the cells positive for CD68, GFAP, and Vimentin. Results represent
mean.+-.s.d., n=3. K. Phagocyte activity was analyzed by monitoring
intracellular pH using pHrodo indicators that reflect phagocytosis
(red). The fluorescent expression was captured under confocal
microscopy. Results represent mean.+-.s.d., n=6.
[0009] FIG. 2. Generation of steatohepatitis organoids (sHLO) by
fatty acid treatment A. Schematic representing the method for
generating steatohepatitis HLO (sHLO) B. Live-cell imaging of lipid
droplets (green), membrane (red), and nuclear (blue). The image was
adopted from the overlay of 10-20 Z-stack images. Increase of lipid
droplet accumulation and enlargement of lipid droplets and cells
were observed in a dose depended manner. C. Representative total
lipid volume normalized by each organoid size. Bar shows the mean
of the total lipids volume. Lipid droplets were increased in a dose
depended manner 0 (black), 200 .mu.M OA (red), 400 .mu.M OA
(green), and 800 .mu.M OA (blue). D. Quantification of
triglycerides in HLO. HLOs were isolated from one Matrigel drop and
divided into HCM media in the presence (blue bar) or absence (black
bar) of oleic acid (800 .mu.M) and cultured for 3 days. E. ELISA
measurement of IL-6 with culture supernatants obtained from the
wells that contain 20-30 HLO cultured in the presence or absence of
oleic acid (800 .mu.M) and cultured for 3 days. The final values
were normalized by the number of organoids in each well. IL-6 was
2.2-fold released in 800 .mu.M OA treatment (blue bar), when
compared with non-treatment (black bar). F. Gene expression of
pro-inflammatory cytokines TNF-alpha and IL-8. They were normalized
by 18S. Both TNF-alpha and IL-8 gene expression were upregulated in
200 .mu.M OA (red bar) and 800 .mu.M OA (blue bar), compared with
untreated (black bar). G. 10-20 HLO were cultured in HCM media
including 0, 400, 800 uM OA for 3 days. The cultured supernatants
were collected from each well, and THP-1 migration was measured by
using transmembrane with those supernatants. Cells that had
migrated were counted and normalized by the exact number of
organoids in each well. H. Trichrome staining of day 25 HLO and
percentage of trichrome stained HLO on HLO (black bar), sHLO (red
bar) and cHLO (blue bar) populations. Results represent
mean.+-.s.d., n=8-20 organoids. I. IF staining of day 25 HLO for
Epcam and Vimentin and percentages of Epcam and Vimentin positive
HLO on HLO (black bar), sHLO (red bar) and cHLO (blue bar)
populations. Results represent mean.+-.s.d., n=8-20 organoids. J.
20-30 HLO were cultured in the presence or absence of oleic acid
(800 .mu.M) for 5 days. P3NP was measured by ELISA with those
supernatants. The final values were normalized by the exact number
of organoids in each well. P3NP was 2.8 fold increased in 800 .mu.M
OA treatment (blue bar), when compared with non-treatment (black
bar).
[0010] FIG. 3. Cirrhotic transition of steatohepatitis HLO measured
by AFM. A. Schematic representation for measuring the stiffness of
HLO by AFM. The top region of each single HLO (14.times.14 matrix
in a 25.times.25 .mu.m square) was scanned with an AFM cantilever,
which can provide a spatial mapping of topographical and mechanical
information of HLO. Scale bar, 100 .mu.m. B. The representative
histogram of calculated Young's modulus (stiffness of HLO; E, kPa)
of single HLO showed Gaussian-like distribution and its peak values
and width were increased in a dose depended manner 0 (black), 200
(red), 400 (green) and 800 (blue) .mu.M OA. C. Young's modulus
(stiffness of HLO) was determined from 7-12 organoids and
summarized by the dot plot with box-and-whisker plot. Increase of
the median value was observed in a dose dependent manner 0 (black),
200 (red), 400 (green) and 800 (blue) .mu.M OA.
[0011] FIG. 4. cHLO rigidity recapitulates clinical phenotype of
Wolman disease A. Bright-field image of HLO and cHLO established
from several iPSC lines including healthy person (317D6), NAFLD
patients (NAFLD150, NAFLD77, and NAFLD27), and the Wolman disease
patients (WD90, WD92, and WD91). B. Young's modulus (stiffness of
HLO: Pa) of average of single HLO and cLO derived from several iPSC
lines.
[0012] FIG. 5. Modeling human phenotypic variation of steatosis
using iPSC-sHLO A. Representative allelic functions of PNPLA3,
GCKR, and TM6SF2 responsible for hepatic TG content increase. B.
Pie chart showing percent distribution of publicly available 2504
cell lines based on total polygenic scoring of three risk allele:
0.6<x (green area), 0.3<x<=0.6 (yellow area), x<=0.3
(brown area). C. The table summarizes the donor characteristic
including polygenic score assigned in further study. D. Live-cell
imaging of lipid droplets (green) and nuclear (blue) on three group
of total polygenic scoring of three allele: 0.6<x,
0.3<x<=0.6, x<=0.3. The image was adopted from the overlay
of 10-20 Z-stack images. E. Representative total lipid volume
normalized by each organoid size on three group of total polygenic
scoring of three allele: 0.6<x, 0.3<x<=0.6, x<=0.3. Red
and navy bar indicates 800 NM OA and 200 .mu.M OA treated sHLO,
respectively. F. Representative gene expression of pro-inflammatory
cytokines on three group of total polygenic scoring of three
allele: 0.6<x (black bar), 0.3<x<=0.6 (red bar), x<=0.3
(blue bar). G. Young's modulus (stiffness of HLO: Pa) of average of
single cLO on three group of total polygenic scoring of three
allele: 0.6<x (black dots), 0.3<x<=0.6 (red dots),
x<=0.3 (blue dots). H. OCA response
[0013] FIG. 6. Phagocyte activity on THP-1
[0014] FIG. 7. Lipids accumulation comparison of 800 .mu.M of OA,
PA, LA, and SA.
[0015] FIG. 8. Actual migration cell number of THP-1 by 400 and 800
.mu.M of OA
[0016] FIG. 9. A. Photo of E-cad positive and negative sorted
reconstituted spheroids from E-cad-mruby organoids, HepG2, LX-2,
and THP-1. B. Gene expression of pro-inflammatory cytokines
TNF-alpha and IL-8. C. ELISA measurement of P3NP.
[0017] FIG. 10. No effectiveness of resveratrol on ROS production
of liver organoids
[0018] FIG. 11. Percentages of BODIPY positive cells (lipids) were
determined by flow cytometry. Results represent mean.+-.s.d.,
n=5.
[0019] FIG. 12. Lipids accumulation in the organoid in the presence
and absence of 400 .mu.M Intralipid
[0020] FIG. 13. Bright-field image of liver organoids in the
culture of non-treatment, 800 .mu.M of OA alone, and 800 .mu.M of
OA with 40 ng/ml FGF19.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Unless otherwise noted, terms are to be understood according
to conventional usage by those of ordinary skill in the relevant
art.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
Pluripotent Stem Cells Derived from Embryonic Cells.
[0028] 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.
[0029] 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); UC01 (HSF1); UC06 (HSF6); WA01 (H1); WA07 (H7); WA09
(H9); WA13 (H13); WA14 (H14).
[0030] 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.
Induced Pluripotent Stem Cells (iPSCs)
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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).
[0035] 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.
[0036] 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 may be derived from a morula. In
some embodiments, pluripotent stem cells may be stem cells. Stem
cells used in these methods can include, but are not limited to,
embryonic stem cells. Embryonic stem cells may 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.
[0037] Non-alcoholic Fatty Liver Disease (NAFLD) is one of the
major challenges to be overcome in developed countries to do the
increased chance of developing lethal liver disorders, yet
effective treatment is lacking. Similarly, iatrogenic parental
nutrition associated liver disease (PNALD) is a disorder that
currently has no effective treatment. Models having steatohepatitis
and/or clinical histopathological features of liver disease such as
lipid droplet accumulation and cystoskeleton filament
disorganization, steatosis, and hepatocellular ballooning are
needed. Despite the promise of disease models using patients' stem
cells, current approaches are limited in their applications to
unicellular, monogenic and relatively simple pathologies, failing
to capture more prevailing and complex disease pathology such as
epithelial organ fibrosis.
[0038] In one aspect, a method of making a lipotoxic organoid model
is disclosed. The method may comprise the steps of contacting a
liver organoid made according to the methods described herein, with
a free fatty acid (FFA) composition. The FFA composition may
comprise oleic acid, linoleic acid, palmitic acid, or combinations
thereof, preferably oleic acid. The amount of FFA may be determined
by one of ordinary skill in the art. In one aspect, the FFA,
preferably oleic acid, may be contacted with a liver organoid in an
amount of from about 10 .mu.M to about 10,000 .mu.M, or from about
20 .mu.M to about 5,000 .mu.M, or from about 30 .mu.M to about 2500
.mu.M, or from about 40 .mu.M to about 1250 .mu.M or from about 50
.mu.M to about 1000 .mu.M, or from about 75 .mu.M to about 900
.mu.M or from about 80 .mu.M to about 800 .mu.M, or from about 90
.mu.M to about 700 .mu.M, or about 100 .mu.M to about 500 .mu.M, or
from about 200 .mu.M to about 400 .mu.M. The FFA may be contacted
with the liver organoid for a period of time of from about 1 hour
to about 10 days, or from about 2 hours to about 9 days, or from
about 3 hours to about 8 days, or from about 4 hours to about 7
days, or from about 5 hours to about 6 days, or from about 6 hours
to about 5 days, or from about 7 hours to about 4 days, or from
about 8 hours to about 3 days, or from about 9 hours to about 2
days, or from about 10 hours to about 1 day. In one aspect, the
range is about 3 to about 5 days, +/-24 hours.
[0039] In one aspect, the lipotoxic organoid model is a model of
fatty liver disease.
[0040] In one aspect, the lipotoxic organoid model is a model of
steatohepatitis.
[0041] In one aspect, the lipotoxic organoid model is a model of
cirrhosis.
[0042] In one aspect, the lipotoxic organoid model is a model of
parenteral nutrition associated liver disease (PNALD).
[0043] In one aspect, the lipotoxic organoid model is a model of
NAFLD.
[0044] In one aspect, the lipotoxic organoid model may be
characterized by cytoskeleton filament disorganization, ROS
increase, mitochondrial swelling, trigycleride accumulation,
fibrosis, hepatocyte ballooning, IL6 secretion, steatosis,
inflammation, ballooning and Mallory's body-like, tissue
stiffening, cell-death, and combinations thereof.
[0045] In one aspect, a method of screening for a drug for
treatment of a liver disease, including NAFLD and/or cholestasis is
disclosed. The method may comprise the step of contacting a
candidate drug with a lipotoxic organoid model as disclosed
herein.
[0046] In one aspect, a method of assaying the effectiveness of a
nutritional supplement/TPN is disclosed. The method may comprise
the step of contacting the nutritional supplement/TPN with a
lipotoxic organoid model as disclosed herein.
[0047] In one aspect, the three-dimensional (3D) liver organoid
model of fatty liver disease is disclosed, wherein the organoid is
characterized by steatosis, inflammation, ballooning and Mallory's
bodies, ROS accumulation and mitochondrial overload; fibrosis and
tissue stiffening, and cell death.
[0048] In one aspect, the three-dimensional (3D) liver organoid
model is a model of drug induced hepatotoxicity and
inflammation/fibrosis.
[0049] In one aspect, the three-dimensional (3D) liver organoid
model is a model of parenteral nutrition associated liver disease
(PNALD)
[0050] In one aspect, the three-dimensional (3D) liver organoid
model does not comprise inflammatory cells, for example T-cells or
other inflammatory secreted proteins.
[0051] Also disclosed are methods of inducing formation of a liver
organoid from iPSC cells which may be used in the aforementioned
methods and/or to obtain the aforementioned compositions. The
method may comprise the steps of
[0052] 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.
[0053] 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.
[0054] In some embodiments, DE culture is treated with the one or
more molecules of a 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.
[0055] 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 GSK30. 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.
[0056] In one aspect, the stem cells may be mammalian, or human,
iPSCs.
[0057] 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.
[0058] 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.
[0059] In one aspect, the liver organoid may be characterized in
that the liver organoid has bile transport activity.
[0060] 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.
[0061] 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.
[0062] 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.
EXAMPLES
[0063] Human organoid systems that achieved the higher-order
function of 3D tissues closely resemble in vivo organ architecture
in health and disease, yet failed to capture more prevailing and
complex pathology such as inflammation and fibrosis. Here,
Applicant developed multi-cellular human liver organoid (HLO) model
that displays essential features of steatohepatitis. Cultural fatty
acid exposure enables persistent steatosis induction, followed by
progressive activation of pro-inflammatory and fibrotic lineages
that develops massive fibrosis in HLO. Interestingly, expression of
the steatohepatitis phenotype is strongly influenced by clinically
reported heritable factors. Atomic force microscopy measurement
revealed that overall organoid stiffening correlates the severity
of inflammation and fibrosis. The measurement fidelity to clinical
phenotype was confirmed using three monogenic
steatohepatitis-specific iPSC lines. Furthermore, Applicant
established an iatrogenic parental nutrition associated liver
disease (PNALD) model in organoids.
[0064] Retinoic acid (RA) signaling is a well-known important
specifier of thyroid, lung, and pancreas from foregut endoderm
during the early organ specification phase, whereas it is clearly
not essential for specification of the liver in model organisms
(Kelly and Drysdale, 2015) with the exception of zebrafish (Negishi
et al., 2010). Indeed, xenopus and chick are similar to mammals as
liver specification occurs in embryos lacking RA (Chen et al.,
2004; Stafford et al., 2004). Conversely, several animal studies
suggested that RA promotes hepatic stellate cell differentiation
from mesenchyme, which is controlled in part by the zinc finger
transcription factor WT1 expressed in the STM and stellate cells
(Ijpenberg et al., 2007; Wang et al., 2013). Additionally, several
studies suggest that the balanced RA regulation induces monocyte
fate specification from human stem cells (Purton et al., 2000; Ronn
et al., 2015). Also, later liver bud growth is promoted by RA
signaling via unknown mechanisms (Zorn and Wells, 2009). Given the
broader roles of RA for both parenchymal and non-parenchymal cell
specification, Applicant hypothesized that the timed RA exposure
might impact lineage diversification of hepatic stromal cells
including pro-inflammatory lineages, thereby facilitating creation
of human liver organoids for modeling inflammation and
fibrosis.
[0065] Recently, directed differentiation into intestinal organoids
via foregut spheroid generation from PSC has been reported
(McCracken et al., 2017; Spence et al., 2011). These organoids do
not merely generate epithelial cells but also co-develop
mesenchymal cell components. Here, by taking advantage of this
foregut generation method, Applicant tested the hypothesis that the
timely RA pulsing into liver organoids preferentially
co-differentiates supportive lineages after hepatic specification
from human iPSC. By fostering close interactions between epithelial
and supportive lineages in a 4-D condition wherein they can
co-develop, Applicant demonstrated the applicability of the liver
organoid to model inflammation and fibrosis. Applicant also
established a screen platform to determine the severity of fibrosis
by evaluating stiffness at single organoid level in the living
state. This method serves as an invaluable application platform for
the study of epithelial organ inflammation and fibrosis towards
drug development and personalized therapy.
Results
[0066] Generation of RA-Based Liver Organoid Model from Human
iPSCs
[0067] To elucidate whether RA signaling has an impact on
determining the lineage differentiation towards stromal cells,
Applicant established a liver organoid model from human iPSCs by
transient induction of RA. Applicant initially differentiated iPSCs
to foregut spheroids through definitive endoderm (DE) specification
as previously described (Spence et al., 2011). The foregut
spheroids were mixed with the attached cells grown in the same
well, and the mixture was embedded in Matrigel. Since RA is a known
specifier for diverse lineages through a context dependent process,
Applicant set the duration of initial RA signaling before the
hepatocyte maturation process which is cultured in hepatocyte
culture media (HCM) and characterized the established organoids at
day 20 as illustrated in FIG. 1, A. Since the highest concentration
of albumin in culture media was observed with 4 days of RA exposure
among the various duration of RA from 0 to 5 days, the further
characterization of the established organoids was compared between
two conditions of 0 and 4 days-RA exposure. The number of organoids
was increased 1.8-fold in RA treated wells compared to untreated
and the size was increased 1.5-fold after RA treatment (FIG. 1, B,
C, and D). Albumin secretion was increased two-fold in the RA
treated group (3.0-6.5 .mu.g/ml) when compared with untreated
(0.5-3.5 .mu.g/ml; FIG. 1, E). Of note, albumin secretion was
maintained more than 40 days after day 20 when albumin was first
detected (data not shown). Interestingly, the internal luminal
structure appeared in 95% of RA treated organoids, whereas it was
detected in only 12% of untreated organoids, indicating that the
lumenization of the organoid is dependent on RA signaling (FIG. 1,
F).
[0068] Applicant next tested the bile transport activity by adding
fluorescein diacetate, a marker of efflux transport in hepatocytes
which turns into fluorescein after estrase hydrolysis in
hepatocytes, into the culture media and tracked the flow of the
fluorescence. The fluorescent substance was absorbed into the cells
of organoids within minutes after fluorescein diacetate addition
and subsequently excreted from the cells into the internal lumen
(FIG. 1, G). By contrast, the fluorescence was not detected in
spheroids. Since bile transport activity is an important function
of the liver, human liver organoids, hereafter defined as HLO, may
be used as models that represent multiple human hepatocyte
functions including albumin secretion and bile transport
functions.
Co-Differentiation of Pre-Fibrotic and Inflammatory Lineages in
Human iPSC-Liver Organoid
[0069] Interestingly, single-cell RNA sequencing (scRNA-seq)
analysis of RA treated HLO showed the expression signatures
specific to stellate cells, liver resident macrophage Kupffer
cells, and endothelial cells as evidenced by stellate cell markers
(ACTA2, DES, PDGFRB), Kupffer cell markers (CD68, IRF7), and
endothelial cell markers (OIT3, DPP4, C1QTNF1; FIG. 1, H) (Bahar
Halpern et al., 2017; El Taghdouini et al., 2015; van de Garde et
al., 2016). To confirm the presence of stellate cells and Kupffer
cells, Applicant conducted a quantitative analysis by FACS with
epithelial cell marker EpCAM, stellate cell marker CD166/ALCAM, and
Kupffer cell marker CD68 and F4/80 (Yanagimachi et al., 2013).
While the frequency of EpCAM+ cells was 78.85.+-.7.35% of HLO,
EpCAM-CD166+, EpCAM-CD68+, and EpCAM-F4/80+ expressing cells was
32.4.+-.1.2%, 1.69.+-.0.3%, and 1.68.+-.0.2%, respectively (FIG. 1,
I). Upon immunohistochemistry, the expression of CD68 cells was
also detected in HLO, and CD68 expression was localized to the
cells of the internal lumen side (FIG. 1, J). Vimentin and GFAP,
novel markers for human hepatic stellate cells and reported to be
required for the process of stellate cell trans-differentiation in
rodents (Geerts et al., 2001; Kordes et al., 2014), were both
detected in HLO and were co-expressing in the same cells (FIG. 1,
J), indicating that stellate cells are resident in HLO.
[0070] To examine whether CD68 expressing cells are functionally
active in HLO similar to Kupffer cells, Applicant monitored the
phagocytic activity in HLO, which has been reported to be active in
liver resident Kupffer cells, with a pH-sensitive rhodamine-based
live-cell dye that undergoes a dramatic increase in fluorescence
(red) in response to an environmental shift from high to low pH
which occurs in phagocytosis as was observed in THP1 human
macrophage cells (FIG. 8). In HLO, the activity was detected and
localized similar to CD68 expressing cells, whereas it was rarely
detected in the spheroid (FIG. 1, K). These observations indicate
that RA-based liver organoid (HLO) is a unique liver model that
naturally develops multi-lineage liver stromal cells including
stellate cells and Kupffer cells from human iPSC.
Inflammation Response and Fibrosis Induction of HLO by Free Fatty
Acid
[0071] Currently, free fatty acids (FFA) are widely used as an
initiating factor to establish an in vitro lipotoxic hepatocyte
model; however, fat accumulation is the only phenotype (Kanuri and
Bergheim, 2013). Alternate co-culture models of hepatocyte and
Kupffer cells showed inflammatory responses such as the increased
expression of IL family cytokines, macrophage related cytokines,
and MMP related cytokines but not subsequent fibrosis (Hassan et
al., 2014). Given the presence of hepatocyte-, stellate- and
Kupffer-like cells with lipid metabolism function-associated genes
indicated by global transcriptome analysis, Applicant hypothesized
that FFA exposure naturally triggers an inflammation response and
resulting fibrotic response in HLO. To test this hypothesis,
Applicant treated HLO for 3 to 5 days with FFA. Applicant indicated
3 day and 5 days OA treated HLO as steatohepatitis (s)HLO and
cirrhosis (c)HLO (FIG. 4, A). sHLO is HLO treated with oleic acid
for three days and show lipid accumulation and inflammation. cHLO
is HLO treated with oleic acid for five days and shows fibrosis
(HLO is stiffened) in addition to lipid accumulation and
inflammation. To first compare the effect of multiple FFAs
including oleic acid (OA, 18:1 n9), linoleic acid (LA, 18:2),
palmitic acid (PA, 16:0), and stearic acid (SA, 18:0) on lipids
accumulation, live-cell imaging was performed with lipid dye BODIPY
at 3 days after the FFA exposure. As FIG. 7 shows, OA was the most
effective in inducing lipid accumulation in hepatocyte like cells,
and SA was the least effective. Considering that OA triggers
massive lipid accumulation, the concentration of OA was varied (0,
200, 400, and 800 .mu.M) to trigger inflammatory reaction in sHLO.
Live-cell imaging and subsequent quantitation displayed that lipid
accumulation was elevated in sHLO in a dose dependent fashion (up
to 18-fold; FIG. 4, B and C). In addition to increase in lipid
accumulation, the size of lipid droplets was enlarged (FIG. 4, B).
Hepatocyte ballooning (enlargement) was one of the pathological
grading indicators to determine the nonalcoholic steatohepatitis
(NASH) activity (NAS scoring), which was confirmed in 800 .mu.M OA
treated sHLO by live imaging of cellular membrane (FIG., 4B).
Triglycerides, a main constituent of lipids accumulated in the
liver, were also detected in 800 .mu.M treated sHLO, but not in
untreated sHLO (FIG. 4, D). More importantly, ELISA of culture
supernatant showed that IL-6 was secreted 2.2-fold in 800 .mu.M OA
treated sHLO culture media when compared to the untreated (FIG. 4,
E). IL-8 and TNF-alpha were also upregulated in a 200 .mu.M or 800
.mu.M OA treated condition (10- and 2-fold increase, respectively;
FIG. 4, F). Moreover, using the OA-treated sHLO conditioned media,
the migration of THP1 was assessed in 24 hrs after culturing in
transwells, and THP-1 migration cells were elevated in a OA treated
sHLO, suggesting FFA treatment naturally evoked an inflammatory
response in sHLO presumably by causing stress on hepatocyte like
cells (up to 2-fold: FIG. 4, G and FIG. 8.) To confirm whether
those inflammation and fibrosis features were specific to HLO,
Applicant isolated epithelial marker E-cad from E-cad mRuby
embryonic stem (ES) cells derived organoids, reconstructed the
spheroids from either E-cad positive or negative cells, and applied
for ELISA for IL6 and P3NP and RNA expression for IL8 and
TNF-alpha. As FIG. 9 indicates, neither E-cad positive nor negative
cells derived spheroids produced P3NP and IL-6 secreted production
and overexpressed the gene expression of inflammatory markers.
Applicant also tested same experiments using human hepatocyte cell
line HepG2, Macrophage cell line THP-1, and hepatic stellate cell
LX-2, and had a similar result of E-cad positive and negative
cells. The results demonstrated that liver organoids only response
to FFA treatment on inflammation and fibrosis response.
[0072] To further determine whether HLO progresses towards
fibrosis-like condition, Applicant performed Masson trichrome stain
on sHLO and cHLO. Trichrome positive HLO was not observed on sHLO
but significantly increased on cHLO (FIG. 2, H). Moreover, IF
staining of epithelial marker Epcam and fibrosis marker Vimentin
demonstrated increase of Epcam positive HLO and decrease of
vimentin positive HLO in cHLO (FIG. 2, I), suggesting that OA
treatment induced the selective increase in fibrotic population as
well as matrix deposition in cHLO. Moreover, ELISA of P3NP was also
measured at day 5 of OA exposure. P3NP secretion was increased
2.8-fold in the culture media of 800 .mu.M OA treated HLO when
compared with the untreated (FIG. 4, J). Collectively, these
observations indicated that FFA exposure not merely caused an
accumulation of triglycerides but also initiated an inflammatory
reaction and fibrosis in HLO.
cHLO Profile of Inflammation and Fibrosis by FFA Treatment at
Single Cell Level
High-Throughput Quantification of Fibrosis by AFM
[0073] Accumulating evidence has indicated that liver stiffness is
well correlated with the severity of liver fibrosis (Yoneda et al.,
2008) and thereby measuring the stiffness of HLO has potential to
evaluate the fibrosis severity of HLO. Applicant's preliminary
qualitative analysis of smooth muscle actin immunostaining
indicates dose dependent fibrosis progression by OA exposure (FIG.
2, H, I, and J). To gain more quantitative insights in a screenable
format, Applicant thus assessed in the living state whether HLO
fibrosis could increase the stiffness in a dose dependent fashion
using micro-indentation with Atomic Force Microscopy (AFM). As
shown in FIG. 4, A, the top region of each single HLO (14.times.14
matrix in a 25.times.25 .mu.m square) was scanned with an AFM
cantilever, which can provide a spatial mapping of topographical
and mechanical information of HLO. The representative histogram of
calculated Young's modulus (E, kPa) of single HLO clearly showed a
Gaussian-like distribution and its peak values and width were
increased in accordance with the OA concentration (FIG. 4, B; 0,
200, 400, 800 .mu.M). Young's moduli determined from HLOs were
summarized by dot plot with box-and-whisker plot (FIG. 4, C),
demonstrating that the rigidity of HLO was gradually elevated from
0 to 800 .mu.M OA i.e., gradual shift of median value and
enlargement of the stiffness range were observed according to the
elevation of OA concentration. The Emedian values were 1.2 kPa, 1.6
kPa, 2.4 kPa and 2.8 kPa for 0, 200, 400, and 800 .mu.M OA-treated
organoids, respectively. Furthermore, difference between 90th
percentile and 10th percentile (.DELTA.P90-10) was 3.2 kPa for
untreated organoids and 7.0 kPa for 800 .mu.M OA-treated organoids
was observed. As FIG. 3, F shows, TNF-alpha and IL-8 were also
upregulated by OA addition, known to be correlated with fibrosis
severity in NAFLD patients (Ajmera et al., 2017). These results
indicate that stiffness of HLO can be a quantifiable measurement
for the severity of fibrosis, and thus can be potentially applied
for a high-throughput screening of fibrosis.
cHLO Rigidity Recapitulates Clinical Phenotype of Wolman
Disease
[0074] The clinical relevance of a model relating to fibrosis and
cirrhosis from pluripotent stem cells is unclear due to the
undetermined nature of genetic influences in a patient. A
predisposition to fibrosis probably would not be captured in this
model. Applicant therefore has examined assay fidelity to clinical
phenotype by evaluating congenital steatohepatitis patient derived
iPSC including healthy people (317D6), NAFLD/NASH patients
(NAFLD150, 77, and 27) and Wolman disease patients (WD90, 91, and
92)(FIG. 5, A). Specifically, Applicant established three Wolman
disease patient-specific iPSC lines, which is a mono-genetic
disorder with lethal steatohepatitis, and confirmed the significant
rigidity increase relative to normal iPSC line-derived organoids,
notably with remarkable correlation to enzymatic activity at the
time of clinical diagnosis (FIG. 5, B).
Modeling Human Phenotypic Variation of Steatosis Using
iPSC-sHLO.
[0075] Second, Applicant extended this new organoid model to assess
the polygenic effects on steatohepatitis progression. Three protein
coding sequencing variants PNPLA3 (patatin-like phospholipase3)
p.I148M, TM6SF2 (Transmembrane 6 Superfamily Member 2) p.E167K, and
GCKR (Glucokinase regulatory protein) p.P446L which have been
repeatedly shown to be independent determinants of hepatic
triglyceride content (HTGC) by large-scale GWAS (Xu et al., 2015),
(Zain et al., 2015) (FIG. 6, A). Applicant therefore approached
large scale cell bank, and mapped out 2504 cell lines with the
polygenic score (FIG. 6, B) by a summation of reported effect size
.beta.(SNP) (Stender et al., 2017), per-allele change in
standardized HTGC multiplying weighted dosage as follow:
.SIGMA.{.beta. (SNP).times.(dosage of risk allele)}. After the
acquisition of iPSC lines with three different thresholds (FIG. 6,
B and C), Applicant has generated seven iPSC organoids, and induced
steatohepatitis to estimate polygenic impacts on the phenotype.
Strikingly, Applicant has found the remarkable correlation of
`steatosis` (by live imaging, FIG. 6, D and E) and `inflammation`
(qRT-PCR, FIG. 6, F), but NOT for `fibrosis` (AFM based stiffness
measurements, FIG. 6, G) to polygenic scores. Applicant also found
that OCA response differ among the cell line and response to OCA
are depended on SNP number (FIG. 6H)
DISCUSSION
[0076] Multi-Cellular Liver Organoid from Human PSC
[0077] A series of recent studies reported the successful
integration of supportive lineages into endoderm-derived organoids
by experimentally combining endothelial cells (Takebe et al.,
2013), mesenchymal cells (Takebe et al., 2015), and neural crest
cells (Workman et al., 2017). The RA-pulsation based method
naturally engages cells to diversify with a sustained cell
polarity, and is remarkably reproducible and scalable with
reasonable cost. Whether the precise nature of supportive lineages
is developmentally relevant or liver specific remains elusive, the
stromal populations are fully reactive to known fibrosis inducers
including LPS and fatty acids, paving a new way for modeling
multicellular and complex pathologies.
Promise of Mechanical Organoid Screening for Analyzing Inflammation
and Fibrosis
[0078] From a future screening standpoint, single organoid based
liver measurement is a highly attractive readout due to its
robustness, normalization and relative simplicity. For example,
based on live fluorescent imaging analysis of an organoid, a
functional swelling assay was established for cystic fibrosis
patients' gut organoids, demonstrating an effective drug selection
(Saini, 2016). Live rigidity assessment on a single liver organoid
from iPSC is an effective way to predict the severity of fibrosis.
A significant correlation between liver stiffness measurement and
fibrosis stage is clinically reported in fatty liver disease
patients by elastography (Yoneda et al., 2008). In addition, a
strong association between increased liver stiffness and presence
of diabetes mellitus (DM) and/or greater insulin resistance was
observed in a subgroup of subjects with ultrasonographically
defined NAFLD (Koehler et al., 2016). Interestingly, human liver
organoid (HLO) stiffness increased in proportion to both LPS and
FFAs, accompanied by inflammatory cytokine production and
fibroblast expansion. Considering a number of epithelial organ
fibrosis share a similar phenotype via diverse pathological
mechanisms, an organoid based rigidity detection assay could be
used to analyze fibrosis using lung, kidney, cardiac and gut
organoids.
[0079] In liver organoids, multiple measurements can be
intra-vitally evaluated with the use of high content imaging
system. In fact, increases in the number and size of lipid
droplets, bile transport activity, phagocytes, and the
deterioration of cellular morphology can be effectively imaged in
fat-treated liver organoids. Thus, organoid based assay platforms
may be used to better understand the human specific mechanisms of
fatty liver diseases and establishing high-content screening for
drug discovery for these diseases. Combined with compound libraries
and nutritional metabolites, organoid based mechanical screening
would be highly attractive model system for implicating effective
therapy in humans, otherwise not testable preclinically.
Nutritional Precision Medicine (Therapy Personalization)
[0080] Patient-specific iPSC-derived organoids might be used to
predict individualized drug efficacy and epithelial response. Human
iPSCs can be established from both healthy and diseased
individuals. In parallel to the establishment of a population iPSC
panel, the use of patients' iPSCs is expected to model
inter-individual differences of lipotoxicity, drug efficacy and
safety concerns (Warren et al., 2017a; Warren et al., 2017b). In
doing so, phenotypic screening using a liver organoid-based
platform will promote personalized selection of highly effective
interventions for the disorders. For instance, as the nutritional
supplementation might be altered depending on individuals, the
disclosed system may be a highly compatible assay for reflecting
nutrition associated conditions before administration.
Specifically, patient-specific iPSC-derived organoids can be used
to customize PN formulation to each patient with the aim of
minimizing the possible progression to hepatic steatosis and
fibrosis (PNALD). Indeed, in clinics, PN formulation is often
customized (Mercaldi et al., 2012) as commercial solutions do not
meet the caloric, amino acid, and electrolyte needs of critically
ill patients, who are often obese and require fluid restriction,
and display hepatic/renal dysfunction (Boullata et al., 2014).
Beyond nutritional needs, there is an urgent need to gain insights
for a reduction of possible adverse effects associated with PN
products. The liver organoid would thus be useful to evaluate
safety concerns especially for PNALD risk assessment, facilitating
the customization strategy of PN formulations for each patient.
[0081] Overall, Applicant has demonstrated that enabling crosstalk
between epithelial and stromal lineages in a 4-D organoid culture
is useful for modeling clinically relevant pathology associated
with steatosis and fibrosis of liver. This model will eventually
lead to analysis of more prevailing pathology such as NAFLD or
NASH, which is a growing concern with population aging. More
broadly, Applicant has established a viable strategy for modeling
humanistic complex pathology coupled with currently evolving
organoid technology (Lancaster and Knoblich, 2014), paving a new
way for the discovery of effective treatments against unrecoverable
diseases at single organoid level.
Methods
[0082] hPSCs Maintenance. The TkDA3 human iPSC clone used in this
study was kindly provided by K. Eto and H. Nakauchi. Human iPSC
lines were maintained as described previously (Takebe et al., 2015;
Takebe et al., 2014). Undifferentiated hiPSCs were maintained on
feeder-free conditions in mTeSR1 medium (StemCell technologies,
Vancouver, Canada) on plates coated with Matrigel (Corning Inc.,
NY, USA) at 1/30 dilution at 37.degree. C. in 5% CO.sub.2 with 95%
air.
[0083] Definitive endoderm induction. Human iPSCs into definitive
endoderm was differentiated using previously described methods with
slight modifications (Spence et al., 2011). In brief, colonies of
human iPSCs were isolated in Accutase (Thermo Fisher Scientific
Inc., MA, USA) and 150,000 cells/mL were plated on Matrigel coated
tissue culture plate (VWR Scientific Products, West Chester, Pa.).
Medium was changed to RPMI 1640 medium (Life Technologies)
containing 100 ng/mL Activin A (R&D Systems, MN, USA) 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. Day4-6 cells were cultured in Advanced DMEM/F12 (Thermo
Fisher Scientific Inc.) with B27 (Life Technologies) and N2 (Gibco,
CA, USA) containing 500 ng/ml fibroblast growth factor (FGF4;
R&D Systems) and 3 uM CHIR99021 (Stemgent, MA, USA). Cells were
maintained at 37.degree. C. in 5% CO.sub.2 with 95% air and the
medium was replaced every day. Spheroids appeared on the plate at
day 7 of differentiation.
[0084] HLO induction. At day 7, spheroids and attached cells are
gently pipetted to be delaminated from dishes. They were
centrifuged at 800 rpm for 3 minutes, embedded in a 100% Matrigel
drop on the dishes in Advanced DMEM/F12 with B27, N2 and 2 uM
retinoic acid (RA; Sigma, MO, USA) after removing supernatant, and
cultured for 4 days. After RA treatment, spheroids embedded in the
Matrigel drop were cultured in Hepatocytes Culture Medium (HCM;
Lonza, MD, USA) with 10 ng/mL hepatocyte growth factor (HGF;
PeproTech, NJ, USA), 0.1 uM Dexamethasone (Dex; Sigma) and 20 ng/mL
Oncostatin M (OSM; R&D Systems). Cultures for HLO induction
were maintained at 37.degree. C. in 5% CO.sub.2 with 95% air and
the medium was replaced every 2-3 days. To analyze HLO (day 20-30),
organoids were isolated from Matrigel by scratching and
pipetting.
[0085] Albumin, IL-6, and P3NP ELISA. To measure albumin secretion
level of HLO, 200 .mu.L of culture supernatant was collected from
HLO embedded in Matrigel. For IL-6 and P3NP, 20-30 organoids were
seeded and cultured on an ultra-low attachment multiwell plates 96
well plate (Corning). To define the exact number of organoids in
each well and lastly normalize the secreted level for IL-6 and P3NP
by the number, the organoids were captured on The KEYENCE BZ-X710
Fluorescence Microscope. The culture supernatants were collected at
24 hrs (for albumin), 96 hrs (for IL-6) and 120 hrs (P3NP) time
points after the culture and stored at -80.degree. C. until use.
The supernatant was centrifuged at 1,500 rpm for 3 min and to
pellet debris, and the resulting supernatant was assayed with Human
Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc., TX,
USA), Human IL-6 ELISA Kit (Biolegend, CA, USA), and Human
N-terminal procollagen III propeptide, PIIINP ELISA Kit (My
BioSource, CA, USA) according to the manufacturer's instructions.
Significance testing was conducted by Student's t-test.
[0086] Bile transport activity. Fluorescein diacetate was used for
evaluating bile transport activity in organoids. 10 mg/mL
fluorescein diacetate (Sigma) was added into HCM media cultured
with HLO and allowed to sit for 5 minutes and captured using
fluorescent microscopy BZ-X710 (Keyence, Osaka, Japan).
[0087] Phagocyte, lipids, ROS live-cell imaging. After being
cultured in an ultra-low attachment 6 multi-well plate, 5-10 HLO
were picked up and seeded in a Microslide 8 Well Glass Bottom plate
(Ibidi, WI, USA) and subjected to live-cell staining. The following
antibodies were used: pHrodo.RTM. Red S. aureus Bioparticles.RTM.
Conjugate for Phagocyte activity (Thermo Fisher Scientific Inc.),
BODIPY.RTM. 493/503 for lipids (Thermo Fisher Scientific Inc.),
Di-8-ANEPPS for membrane (Thermo Fisher Scientific Inc.), and
CellROX green reagent for ROS detection (Thermo Fisher Scientific
Inc.). Nuclear staining was marked by NucBlue Live ReadyProbes
Reagent (Thermo Fisher Scientific Inc.). hLOHLO was visualized and
scanned on a Nikon A1 Inverted Confocal Microscope (Japan) using
60.times. water immersion objectives. The final lipid droplet
volume was calculated by IMARIS8 and normalized by each organoid
size. Significance testing for lipid droplet volume and ROS
production (%) was conducted by Student's t-test.
[0088] HE staining and immunohistochemistry. HLO were isolated from
Matrigel and fixed in 4% paraformaldehyde and embedded in paraffin.
Sections were subjected to HE and immunohistochemical staining. The
following primary antibodies were used: anti-alpha smooth muscle
actin antibody (1:200 dilution; abcam, Cambridge, UK), Desmin
antibody (Pre-diluted; Roche, Basel, Switzerland), and CD68
antibody (1:200 dilution; abcam).
[0089] Flow cytometry. HLO were isolated from 10 Matrigel droplets
and washed by 1.times.PBS. HLO were dissociated to single cells by
the treatment of Trypsin-EDTA (0.05%), phenol red (Gibco) for 10
min. After PBS wash, the single cells were subjected to flow
cytometry with BV421-conjugated Epcam antibody (BioLegend),
PE-conjugated CD166 antibody (eBioscience, CA, USA), and
PE/Cy7-CD68 (eBioscience). DNA was measured by propidium iodide
staining.
[0090] LPS and FFA exposure and OCA and FGF19 treatment. 20-30 HLO,
which had been isolated from Matrigel and washed by 1.times.PBS,
were divided into each condition and cultured on an ultra-low
attachment 6 multi-well plates (Corning). HLO were cultured with
LPS (Sigma), OA (Sigma), LA (Sigma), SA (Sigma), or PA (Sigma) and
collected at day 1 and 3 (for LPS HLO) and at day 3 and 5 (for OA)
after the culture. To test the inhibitory effect of OCA (INT-747,
MedChem Express, NJ, USA) and human FGF19 recombinant (Sigma) on
HLO, 20-30 HLO were cultured in HCM media in the presence or
absence of oleic acid (800 .mu.M), and 1 .mu.M OCA and 40 ng/ml
FGF19 were added into 800 .mu.M OA condition. HLO were collected at
day 3 for lipids live-cell imaging and at day 5 for stiffness
measurement.
[0091] Whole mount immunofluorescence. HLO were fixed for 30 min in
4% paraformaldehyde and permeabilized for 15 min with 0.5% Nonidet
P-40. HLO were washed by 1.times.PBS three times and incubated with
blocking buffer for 1 h at room temperature. HLO were then
incubated with primary antibody; anti-alpha smooth muscle actin
antibody (1:50 dilution; abcam) overnight at 4.degree. C. HLO were
washed by 1.times.PBS and incubated in secondary antibody in
blocking buffer for 30 min at room temperature. HLO were washed and
mounted using Fluoroshield mounting medium with DAPI (abcam). The
stained HLO were visualized and scanned on a Nikon A1 Inverted
Confocal Microscope (Japan) using 60.times. water immersion
objectives.
[0092] RNA isolation, RT-qPCR. RNA was isolated using the RNeasy
mini kit (Qiagen, Hilden, Germany). Reverse transcription was
carried out using the SuperScriptIII First-Strand Synthesis System
for RT-PCR (Invitrogen, CA, USA) according to manufacturer's
protocol. qPCR was carried out using TaqMan gene expression master
mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR System
(Thermo Fisher Scientific Inc.). All primers and probe information
for each target gene was obtained from the Universal ProbeLibrary
Assay Design Center (https://qpcr.probefinder.com/organism.jsp).
Significance testing was conducted by Student's t-test.
[0093] HLO stiffness measurement by AFM. HLOs treated with 0,
50200, 1400, 2800 ng/mL.mu.M LPSOA were used for stiffness
measurement with a AFM (NanoWizard IV, JPK Instruments, Germany).
The AFM head with a silicon nitride cantilever (CSC37, k=0.3 N/m,
MikroMasch, Bulgaria) was mounted on a fluorescence stereo
microscope (M205 FA, Leica, Germany) coupled with a Z-axis piezo
stage (JPK CellHesion module, JPK Instruments, Germany), which
allows the indentation measurement up to the depth of .about.100
.mu.m. As a substrate for organoids, a fibronectin-coated dish was
used. The tissue culture dish (p=34 mm, TPP Techno Plastic
Products, Switzerland) was first incubated with a 1 .mu.g/mL
fibronectin solution (Sigma)) at 4.degree. C. for overnight. Then,
the tissue culture dish was washed twice by distilled water and
dried for 1 hour. Thereafter, HLOs incubated with OALPS for 51 days
were deposited to the fibronectin-coated dish and incubated for 1
hour at 37.degree. C. The sample dish was then placed onto the AFM
stage, and force-distance curves in a 14.times.14 matrix in a
25.times.25 .mu.m square were measured from each HLO. Finally,
Young's moduli (E, Pa) of HLO were determined by fitting the
obtained force-distance curves with the modified Hertz model
(Sneddon, 1965). Dunn-Holland-Wolfe test was performed for
significance testings.
[0094] THP-1 cell migration assay. THP-1 cell, which was gifted
from T. Suzuki, was maintained in Advanced DMEM/F12 (Thermo Fisher
Scientific Inc.) containing 10% FBS. THP-1 floating cells were
collected, and 200,000 cells were added with serum-free Advanced
DMEM/F12 to the membrane chamber of the CytoSelect.TM. 96-Well Cell
Migration Assay (5 .mu.m, Fluorometric Format; Cell Biolabs, CA,
USA). 10-20 HLO were cultured in HCM media including 0, 400, 800 uM
OA with an ultra-low attachment 96 multi-well plate (Corning) for
three days. To define the exact number of organoids in each well
and lastly normalize the final migrated cells by the number, the
organoids were captured on The KEYENCE BZ-X710 Fluorescence
Microscope. 150 .mu.L of culture supernatant of HLO was collected
and added to the feeder tray of the kit. The kit was incubated at
37.degree. C. for 24 h in a 5% CO.sub.2 cell culture incubator.
Cells that had migrated were counted using Countess II FL Automated
Cell Counter (Thermo Fisher Scientific Inc.). Significance testing
was conducted by Student's t-test.
[0095] Triglyceride assay. For quantitative determination of
triglycerides, HLO were isolated from one Matrigel drop and divided
into HCM media in the presence or absence of oleic acid (800
.mu.M). They were cultured on an ultra-Low attachment 6 multi-well
plate for three days. Quantitative estimation of hepatic
triglyceride accumulation was performed by an enzymatic assay of
triglyceride mass using the EnzyChrom Triglyceride assay kit
(Bioassay Systems, CA, USA).
[0096] HLO survival assay. HLO were collected from Matrigel and
washed by 1.times.PBS. 30-40 organoids were cultured on an
ultra-low attachment 6 multi-well plate (Corning). HLO were
captured on The KEYENCE BZ-X710 Fluorescence Microscope every day.
The surviving and dead organoids were counted manually from the
photo. HLO with a rounded configuration was counted as the
surviving while the organoids with out of shape is counted as dead.
To assess the survival rate of OA treated HLO at the same time
point, 3D cell titer glo assay was used (Promega, Wi, USA).
[0097] Statistics and reproducibility. Statistical analysis was
performed using unpaired two-tailed Student's t-test,
Dunn-Holland-Wolfe test, or Welch's t-test. Results were shown
mean.+-.s.e.m.; P values <0.05 were considered statistically
significant. N-value refers to biologically independent replicates,
unless noted otherwise.
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