U.S. patent application number 17/672528 was filed with the patent office on 2022-06-02 for methods and compositions for producing hepatocyte-like cells.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Gary Peltz, Dan Xu.
Application Number | 20220168359 17/672528 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-02 |
United States Patent
Application |
20220168359 |
Kind Code |
A1 |
Peltz; Gary ; et
al. |
June 2, 2022 |
METHODS AND COMPOSITIONS FOR PRODUCING HEPATOCYTE-LIKE CELLS
Abstract
Methods are provided for producing a population of
hepatocyte-like cells (iHeps) from a population of
adipocyte-derived stem cells (ASCs). Aspects of the methods include
placing a population of ASCs into a three dimensional culture
(e.g., hanging drop suspension culture, high density culture,
spinner flask culture, microcarrier culture, etc.), and contacting
the cells with a first and second culture medium. Also provided are
methods of treating an individual, which include producing a
population of iHeps from a population of ASCs, and administering an
effective number of iHeps into the individual. Kits for practicing
the methods are also described herein.
Inventors: |
Peltz; Gary; (Redwood City,
CA) ; Xu; Dan; (Fremont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Appl. No.: |
17/672528 |
Filed: |
February 15, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15974377 |
May 8, 2018 |
11291691 |
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17672528 |
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14917558 |
Mar 8, 2016 |
10004767 |
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PCT/US2014/056049 |
Sep 17, 2014 |
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15974377 |
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61880056 |
Sep 19, 2013 |
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International
Class: |
A61K 35/407 20060101
A61K035/407; C12N 5/071 20060101 C12N005/071; C12N 5/074 20060101
C12N005/074 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with Government support under
contract DK090992 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method of producing a population of hepatocyte-like cells from
a population of adipocyte-derived stem cells (ASCs), the method
comprising: (a) placing a population of ASCs into a three
dimensional culture for a period of time sufficient to produce an
ASC-derived cellular aggregate; (b) contacting cells of the
ASC-derived cellular aggregate with a first culture medium
comprising Activin A and a fibroblast growth factor (FGF) to
produce a precursor cell population; and (c) contacting cells of
the precursor cell population with a second culture medium
comprising hepatocyte growth factor (HGF) to produce an induced
cell population that comprises induced hepatocyte-like cells
(iHeps), wherein the total time elapsed from beginning step (a) to
the production of an induced cell population in step (c) is less
than 13 days.
2. The method of claim 1, wherein the three dimensional culture is
a spinner flask culture.
3. The method of claim 1, wherein the three dimensional culture is
a microcarrier culture.
4. The method of claim 1, wherein the three dimensional culture is
a hanging drop suspension culture.
5. The method of claim 1, wherein the total time elapsed from
beginning step (a) to the production of an induced cell population
in step (c) is 10 days or less.
6. The method of claim 1, wherein the period of time sufficient to
produce an ASC-derived cellular aggregate is 3 days or less.
7. The method of claim 1, wherein the cells of the ASC-derived
cellular aggregate are contacted with the first culture medium for
7 days or less.
8. The method of claim 1, wherein the cells of the ASC-derived
cellular aggregate are contacted with the first culture medium for
7 days or less.
9. The method of claim 1, wherein the FGF is FGF 4.
10. The method of claim 1, wherein the first culture medium further
comprises a Wnt signaling agonist.
11. The method of claim 3, wherein the Wnt signaling agonist is
Wnt3a.
12. The method of claim 1, wherein the second culture medium
further comprises a compound selected from the group consisting of:
oncostatinM (OSM), dexamethasone (Dex), Dimethyl sulfoxide (DMSO),
and a combination thereof.
13. The method of claim 1, wherein the second culture medium
further comprises oncostatinM (OSM), dexamethasone (Dex), and
Dimethyl sulfoxide (DMSO).
14. The method of claim 1, further comprising determining the
percentage of cells of the induced cell population that are
iHeps.
15. The method of claim 14, wherein determining the percentage of
cells comprises: contacting cells of the induced cell population
with a specific binding agent for a hepatocyte marker molecule; and
determining the percentage of cells positive for expression of the
hepatocyte marker molecule, wherein cells positive for expression
of the hepatocyte marker molecule are iHeps.
16. The method of claim 14, wherein 11% or more of the cells of the
induced cell population are iHeps.
17. The method of claim 16, wherein 25% or more of the cells of the
induced cell population are iHeps.
18. The method of claim 1, further comprising enriching the induced
cell population for iHeps.
19. The method of claim 18, wherein the enriching comprises
fluorescent activated cell sorting (FACS).
20. A method of treating an individual with reduced liver function,
the method comprising: (a) producing a population of
hepatocyte-like cells from a population of adipose-derived stem
cells (ASCs) by a method comprising: (i) placing a population of
ASCs into a three dimensional culture for a period of time
sufficient to produce an ASC-derived cellular aggregate, (ii)
contacting cells of the ASC-derived cellular aggregate with a first
culture medium comprising Activin A and a fibroblast growth factor
(FGF) to produce a precursor cell population, and (iii) contacting
cells of the precursor cell population with a second culture medium
comprising hepatocyte growth factor (HGF) to produce an induced
cell population that comprises induced hepatocyte-like cells
(iHeps), wherein the total time elapsed from beginning step (i) to
the production of an induced cell population in step (iii) is less
than 13 days; and (b) administering an effective number of iHeps
into the individual to improve liver function.
21. The method of claim 20, wherein the three dimensional culture
is a spinner flask culture.
22. The method of claim 20, wherein the three dimensional culture
is a microcarrier culture.
23. The method of claim 20, wherein the three dimensional culture
is a hanging drop suspension culture.
24. The method of claim 20, wherein the total time elapsed from
beginning step (a) to the production of an induced cell population
in step (c) is 10 days or less.
25. The method of claim 20, wherein the period of time sufficient
to produce an ASC-derived cellular aggregate is 3 days or less.
26. The method of claim 20, wherein the cells of the ASC-derived
cellular aggregate are contacted with the first culture medium for
7 days or less.
27. The method of claim 20, wherein the cells of the ASC-derived
cellular aggregate are contacted with the first culture medium for
7 days or less.
28. The method of claim 20, wherein the FGF is FGF 4.
29. The method of claim 20, wherein the first culture medium
further comprises a Wnt signaling agonist.
30. The method of claim 29, wherein the Wnt signaling agonist is
Wnt3a.
31. The method of claim 20, wherein the second culture medium
further comprises a compound selected from the group consisting of:
oncostatinM (OSM), dexamethasone (Dex), Dimethyl sulfoxide (DMSO),
and a combination thereof.
32. The method of claim 20, wherein the total time elapsed from
beginning step (i) to producing an induced cell population that
comprises induced hepatocyte-like cells (iHeps) in step (iii) is
less than 13 days.
33. The method of claim 20, further comprising determining the
percentage of cells of the induced cell population that are
iHeps.
34. The method of claim 33, wherein determining the percentage of
cells comprises: contacting cells of the induced cell population
with a specific binding agent for a hepatocyte marker molecule; and
determining the percentage of cells positive for expression of the
hepatocyte marker molecule, wherein cells positive for expression
of the hepatocyte marker molecule are iHeps.
35. The method of claim 20, wherein 15% or more of the cells of the
induced cell population are iHeps.
36. The method of claim 35, wherein 25% or more of the cells of the
induced cell population are iHeps.
37. The method of claim 20, further comprising, prior to step (b),
enriching the induced cell population for iHeps.
38. The method of claim 37, wherein enriching comprises fluorescent
activated cell sorting (FACS).
39. The method of claim 20, wherein at least 1.times.10.sup.4 iHeps
are administered.
40. The method of claim 20, wherein the iHeps are transplanted into
the liver.
41. The method of claim 40, wherein the iHeps are transplanted into
the liver using ultrasound guided injection.
42. The method of claim 20, wherein the individual is a mammal.
43. The method of claim 42, wherein the mammal is a human.
44. The method of claim 20, wherein the ASCs are ASCs isolated from
the individual.
45. The method of claim 44, further comprising, prior to step (a),
isolating the ASCs from the individual.
Description
CROSS REFERENCE
[0001] This application claims benefit and is a Continuation of
U.S. patent application Ser. No. 15/974,377 filed May 8, 2018,
which is a continuation of U.S. patent application Ser. No.
14/917,558 filed Mar. 8, 2016, which issued as U.S. Pat. No.
10,004,767 on Jun. 26, 2018, which is a national stage entry of PCT
Application No. PCT/US2014/056049, filed Sep. 17, 2014, which
claims benefit of U.S. Provisional Patent Application No.
61/880,056, filed Sep. 19, 2013, which applications are
incorporated herein by reference in their entirety.
BACKGROUND
[0003] A major goal for regenerative medicine is to facilitate
human tissue replacement through transplantation of stem cells that
can be harvested from readily accessible tissues. For example,
orthotopic liver transplantation is the only effective treatment
for end-stage liver disease or severe liver injury, but its utility
is severely limited by the lack of donor liver tissue and by the
requirement for lifelong immunosuppression.
[0004] A large number of adipocyte-derived stem cells (ASCs) can be
easily obtained using a commonly performed procedure, liposuction.
Additionally, methods for inducing ASC differentiation into
hepatocyte-like cells (iHeps) have been developed. Liver
regeneration via transplantation of iHeps (e.g., autologous iHeps)
is a highly attractive possibility for regenerative medicine. By
this method, ASC obtained by liposuction are induced to
differentiate into iHeps in vitro, and then iHeps are transplanted
into the donor's liver. The abundance and accessibility of adipose
tissue ensures that there is a source of readily available ASCs.
Moreover, liver regeneration using autologous iHeps would not
require immunosuppression.
[0005] However, since a patient can die rapidly after acute liver
failure (e.g., caused by acetaminophen toxicity), the prolonged
culture period associated with the currently known method to
produce iHeps from ASCs severely limits the clinical utility for
producing iHeps. Furthermore, the known method to produce iHeps
from ASCs is also characterized by low efficiency and low
yield.
[0006] The production of hepatocyte-like cells from
adipocyte-derived stem cells for use in therapy and/or research
will benefit from reduced cost, reduced culture time, increased
efficiency, and increased yield.
PUBLICATIONS
[0007] Amos et al., Tissue Eng Part A. 2010 May; 16(5):1595-606;
Macisaac et al., Exp Cell Res. 2012 Feb. 15; 318(4): 416-423; Kapur
et al, Biofabrication. 2012 June; 4(2):025004; Gutierrez et al.,
Exp Hematol. 2005 October; 33(10):1083-91; Banerjee et al,
Cytotechnology. 2006 May; 51(1):1-5.-2006; Banas et al.,
Hepatology. 2007 July; 46(1):219-28; Banas et al., J Gastroenterol
Hepatol. 2009 January; 24(1):70-7; Green et al., 2011. Nat
Biotechnol 29:267-272; So et al., Biol Open. 2013 Jan. 15;
2(1):30-6; Payne et al., Vitam Horm. 2011; 85:207-16; Hay et al.,
Proc Natl Acad Sci USA. 2008 Aug. 26; 105(34):12301-6; Lue et al,
Liver Int. 2010 July; 30(6):913-22; Hasegawa et al, Biochem Biophys
Res Commun. 2011 Feb. 18; 405(3):405-10; Haraguchi et al, J Tissue
Eng Regen Med. 2013 Jun. 3; Cameron et al, Biotechnol Bioeng. 2006
Aug. 5; 94(5):938-48; WO2007089798; US20100098739; and
US20100112031.
SUMMARY
[0008] Methods are provided for producing a population of
hepatocyte-like cells (iHeps) from a population of
adipocyte-derived stem cells (ASCs). Aspects of the methods include
placing a population of ASCs into a three dimensional culture
(e.g., hanging drop suspension culture, high density culture,
spinner flask culture, microcarrier culture, etc.) and contacting
the cells with a first and second culture medium. Also provided are
methods of treating an individual, which include producing a
population of iHeps from a population of ASCs, and administering an
effective number of iHeps into the individual.
[0009] The presence of iHeps may be confirmed by various methods,
including for example, contacting the induced cell population with
an antibody specific for a hepatocyte marker protein, and
determining the percentage of cells positive for expression. In
some embodiments, 15% or more of cells of the induced cell
population are hepatocyte-like cells. In some cases, 37% or more of
the cells of the induced cell population are hepatocyte-like cells.
In some embodiments, the elapsed time to generate iHeps from ASCs
is less than 13 days (e.g., less than 10 days). The high efficiency
of iHep production and the short time frame over which iHeps can be
produced by the subject methods facilitate treatment methods
because liver damage (e.g., liver failure, e.g., due to
acetaminophen toxicity) can rapidly cause death (e.g., in 2 weeks
or less). Kits for practicing the methods of the disclosure are
also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. 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. It is
emphasized that, according to common practice, the various features
of the drawings are not to-scale. On the contrary, the dimensions
of the various features are arbitrarily expanded or reduced for
clarity. Included in the drawings are the following figures.
[0011] FIGS. 1A-1E provide a comparison of SCi-Heps to Chi-Heps,
and a comparison of the methods of generating SCi-Heps and
Chi-Heps. (FIG. 1A) The two different methods for inducing the
differentiation of ASC into iHeps are shown. The top panel shows
the method for chemically differentiating ASC into Chi-Heps. The
cells isolated from lipo-aspirates are cultured for 3-15 days ASC
cells. These cells are then differentiated from mesoderm into
endodermal cells over a 3-day period (stage 1), and then further
differentiated into Chi-Heps (stage 2) using defined media
containing growth factors. The bottom panel shows one embodiment of
a subject method for producing SCi-Heps. (FIG. 1B) Bright field
images (100.times.) showing the change in the morphology of the
spindle-shaped ASC as they differentiate into Chi-Heps (via
chemical differentiation) or SCi-Heps using a subject method for
producing SCi-Heps. Relative to Chi-Heps, SCi-Heps have a greater
cellular density and colony-like morphology that more closely
resembles hepatocytes. (FIG. 1C) The percentage of ASC, Chi-Heps,
SCi-Heps and iPS-Heps expressing the CK8/18 marker found on mature
hepatocytes. Unstained ASC were used to characterize the background
level of staining (control), and 100% of human hepatocytes express
this marker. (FIG. 1D) Immunofluorescence staining images (at
100.times.) of control ASC, Chi-Heps, SCi-Heps or hepatocytes for
human CK8/18 expression, LDL uptake or PAS staining. Chi-Heps and
SCi-Heps, but not ASC, can endocytose LDL and can synthesize
glycogen (PAS stain). (FIG. 1E) The amount of human albumin or urea
secreted into the supernatant by Chi-Heps, SCi-Heps and ASC
cultured for the indicated time period. SCi-Heps produced albumin
and urea well before Chi-Heps produced detectable amounts of these
analytes, while ASC did not produce albumin or urea. In panels C
and E, each bar represents the average (+SEM) of 4 biologically
independent samples analyzed.
[0012] FIGS. 2A-2C depict gene expression in Chi-Heps, SCi-Heps and
adipocytes. (FIG. 2A) iHeps have increased levels of
hepatocyte-specific mRNAs and decreased levels of
adipocyte-specific mRNAs. RT-PCR analysis was used to measure the
level of hepatocyte-specific (FoxA2, AFP, ALB, AAT, TDO2) or
adipocyte-specific (CD37) mRNA expression in Chi-Heps, SCi-Heps,
iPS-Heps or ASCs. For each gene, the mRNA levels in Chi-Heps (after
15 days of differentiation), SCi-Heps (after 9 days of
differentiation) and iPS-Heps (after 30 days of differentiation)
are normalized relative to that in ASCs. (FIG. 2B) RT-PCR analysis
of the gene expression in ASCs, and in SCi-Heps after 3, 6 and 12
days of induced differentiation. The level of CD105 mRNA expression
(which is an ASC marker) was decreased by 35-fold (p=0.001); while
the level of hepatocyte (FoxA2 and ALB) mRNA expression was
increased by 25- and 51-fold, respectively (p=0.0007 and
9.times.10.sup.-5, respectively), in SCi-Heps after 12 days of
hepatocyte differentiation. The mRNA levels in SCi-Heps were
normalized relative to that in ASCs. Each data point in panels A-B
represents the average.+-.SEM for 3 independent determinations. As
shown in FIG. 11, there were significant changes in the level of
expression of each gene in SCi-Heps after 3, 6 and 12 days of
differentiation. (FIG. 2C) An illustration of the spatial
relationship between the gene expression profiles obtained from
ASCs, Chi-Heps, SCi-Heps, iPS-Heps and hepatocytes.
Microarray-based global gene expression data was analyzed as
described in the methods section. The lengths of each connecting
edge (and the number shown) indicate the distance between the
expression profiles for the cell types at each of the corresponding
vertices. This distance is determined by summing the squares of the
differences in the level of expression for each gene on the array
for the two cell types at the vertices of each line. This diagram
indicates that the gene expression pattern in Chi- and SCi-Heps is
closer to that of hepatocytes than is that of iPS-Heps. Also, the
deviation of the iPS-Heps pattern from the ASC-hepatocyte axis is
larger than that of iHeps, which indicates that iPS-Heps have a
larger number of gene expression changes that are external to both
ASCs and hepatocytes than are found in Chi- or SCi-Heps.
[0013] FIGS. 3A-3D demonstrate the implantation (via injection) of
SCi-Heps into the mouse liver. (FIG. 3A) An ultrasound generated
image (in transverse view) of SCi-Heps being injected through a 30
G needle directly into the right lobe of the liver of a TK-NOG
mouse. (FIG. 3B) Chimeric mice produced human albumin after SCi-Hep
transplantation. The amount of human albumin in plasma was serially
measured over an 8-week period after transplantation of SCi-Heps
into 4 ganciclovir-conditioned mice. Each dashed line shows the
amount of human albumin measured in a chimeric mouse at the
indicated time. In all 4 chimeric mice, human serum albumin was
detectable 4 weeks after transplantation, and the amount increased
with time after transplantation. In contrast, albumin was not
produced by any of the 4 mice that were recipients of control,
undifferentiated ASCs (red triangles and solid line). (FIG. 3C)
Immunofluorescent staining of liver sections (at 100.times.
magnification) obtained from TK-NOG mice 4 weeks after implantation
of SCi-Heps, (a-c) or undifferentiated ASCs (d-f). The liver
sections were stained with human specific anti-albumin (a, d),
anti-CK8/18 (b, e), or anti ASGR1 antibodies, and counter-strained
with DAPI to show the cell nuclei. (FIG. 3D) Immunofluorescent
staining of liver sections (200.times. magnification) obtained from
TK-NOG mice 4 weeks after transplantation with SCi-Heps or
undifferentiated ASCs. The liver sections were stained with
anti-CK8/18, anti-Ki67 or anti-ZO-1 antibodies; and were then
counter-strained with DAPI to show the cell nuclei.
[0014] FIGS. 4A-4B demonstrate that SCi-Heps do not form tumors in
immunocompromised mice, but iPS-Heps do. (FIG. 4A) As early as
three weeks after 5.times.10.sup.4 iPS-Heps were implanted under
the kidney capsule of NOG mice, palpable tumors were formed in the
area of implantation. In contrast, no tumors were detected 2-months
after the same number of Chi- or SCi-Heps were implanted (top row).
The tumors were visible in tissue sections obtained from the area
of iPS-Hep implantation, while only normal tissue was present in
the area of Chi-Hep implantation (middle row). The magnification of
images in the top and middle rows are 10.times.. The images in the
bottom rows were obtained from the boxed regions of the
corresponding image and are shown at 200.times. magnification.
(FIG. 4B) Tumor formation after iPS-Heps implantation. Top row:
Images of the tumors formed 3 weeks after iPS-Hep cells were
implanted under the kidney capsule. The 300.times. and 100.times.
images were obtained from the indicated region (dashed box) of the
corresponding 40.times. and 100.times. images. Bottom row: Two
months after the iPS-iHep cells were implanted, the tumors
contained cells from all three germ cell layers. These images are
at 200.times. magnification. The scale bars shown are 50 .mu.m.
[0015] FIGS. 5A-5C provide FACS analysis performed using an
anti-Sox 17 antibody, which recognizes to a marker for endodermal
cells. ASC were isolated from three different donors and induced to
differentiate using the standard (Chi-Hep) or spherical culture
(SCi-Heps) methods. After 4 days of differentiation, FACS was
performed using an anti-Sox 17 antibody, which recognizes to a
marker for endodermal cells. The percentage of ASC differentiating
into endodermal cells after spherical culture (61.2.+-.2.4) more
than doubled that obtained using the standard method
(25.7.+-.1.3).
[0016] FIGS. 6A-6B provides FACS analysis performed using an
anti-CD105 antibody, which binds to a marker for ASC. ASC were
isolated from three different donors and induced to differentiate
into SCi-Heps using the spherical culture method. After 9 days of
differentiation, FACS was performed using an anti-CD105 antibody,
which binds to a marker for ASC. While 98.6.+-.0.5% of ASC
expressed CD105, the percentage of SCi-Heps expressing this ASC
marker was only 9.0.+-.0.5%.
[0017] FIG. 7 demonstrates that iPS-Heps can synthesize glycogen
(a,b) and endocytose low-density lipoproteins (LDL) (c,d). The LDL
immunofluorescence and PAS stained images are shown at 100.times.
(a, c) and 400.times. magnification (b, d). In contrast, ASC could
not endocytose LDL (e, 100.times.), nor did they stain with PAS (f,
100.times.).
[0018] FIGS. 8A-8D demonstrate that iPS-Heps have hepatocyte
properties. The amount of human albumin (FIG. 8A) or urea nitrogen
(FIG. 8B) secreted into the supernatant by Chi-Heps, iPS-Heps and
ASCs are shown at the indicated time points. Chi-Heps produced
albumin and urea well before iPS-Heps produced detectable amounts
of these analytes, while control ASCs did not produce albumin or
urea. (FIG. 8C) Chi-Heps (after 15 days) and iPS-Heps (after 30
days of differentiation), but not ASCs, can mediate a
CYP3A4-dependent drug biotransformation reaction. (FIG. 8D)
iPS-Heps have increased levels of hepatocyte-specific and decreased
levels of adipocyte-specific mRNAs. RT-PCR analysis was used to
measure the level of hepatocyte-specific (FoxA2, AFP, ALB, AAT,
A1AT, TDO2) or adipocyte-specific (CD37, CD29) mRNA expression in
s, iPS-Heps or ASCs. For each gene, the mRNA levels in in Chi-Heps
(after 15 days of differentiation) and iPS-Heps (after 30 days of
differentiation) are normalized relative to that of the
corresponding mRNA level in ASCs. Each bar or data point in panels
A-D represents the average (.+-.SEM) of 4 biologically independent
samples that were analyzed.
[0019] FIG. 9 provides principal component analysis for the gene
expression data obtained from ASC, Chi-Hep, SCi-Hep, iPS-Hep and
hepatocytes. The first (x-axis) and the second (y-axis) principle
components (PC) explained 46.4% and 26.5%, respectively, of the
total variance in the expression profile. Of note, iPS-Heps had a
large amount of deviation along PC2 relative to ASCs and
hepatocytes, while Chi-Heps and SCi-heps were right in between the
ASCs and hepatocytes. Consistent with other analyses (including the
analysis shown in FIG. 2C), this graph indicates that iPS-Heps had
a large number of gene expression changes that were not present in
hepatocytes (relative to ASCs).
[0020] FIG. 10 demonstrates that SCi-Heps do not express ASGR-1 in
vitro. Permeabilized human hepatocytes (a-c) or SCi-Heps (d-f),
which were analyzed after 9 days of in vitro differentiation, were
stained with an anti-human ASGR-1 antibody and counter-stained with
DAPI. The immunofluorescence images (200.times. magnification) show
that human hepatocytes, but not SCi-Heps expressed ASGR-1. The
scale bars shown are 50 .mu.m.
[0021] FIG. 11 presents a table depicting the numbers of
differentially expressed genes in hepatocytes, Chi-Heps, SCi-Heps,
and iPS-heps relative to ASCs. From analysis of microarray data,
the indicated number of genes whose expression was significantly
increased, decreased or unchanged in hepatocytes relative to ASCs
were selected. (A gene's expression was considered as altered in
hepatocytes if the absolute value of the fold change was >5 and
the adjusted p value was <0.01.) Each of the 3 panels indicates
the number (and percentage) of the selected genes whose expression
was increased, decreased or not significantly changed (NS) when the
expression profiles in Chi-Heps, SCi-Heps or iPS-Heps (relative to
ASCs). For each of these comparisons, the expression of a gene was
considered altered in iHeps if the absolute value of the fold
change was >2 and the adjusted p value was <0.01. It is
noteworthy that 18% of the genes whose expression was not altered
in hepatocytes had an altered level of expression in iPS-Heps.
[0022] FIG. 12 presents a table depicting changes in the level of
expression of genes (Foxa2, Albumin) expressed in hepatocytes and
in adipocyte-specific (CD105) mRNAs during induced SCi-Hep
differentiation. RT-PCR was used to measure the level of expression
of 3 genes (Foxa2, Albumin and CD105) in ASC, and in SCi-Heps after
3, 6 and 12 days of induced differentiation. The measured average
expression levels for each gene in SCi-Heps on the indicated day
(relative to its level of expression in ASC) and the corresponding
p-values for the expression differences are shown. Each value is
the average fold-change for 3 independent measurements; a positive
value indicates that its expression was increased in SCi-Heps
relative to ASC, while a negative value indicates that it was
decreased. A one-sample t-test was applied using the
log-transformed expression level to assess the statistical
significance (P-value) of the measured expression differences.
[0023] FIGS. 13A-13B demonstrate that ASCs cultured by stirred
suspension culture (spinner flask culture in this case) form a high
density of cellular aggregates that resemble the spheres formed
during hanging drop suspension culture. Images shown are at
10.times. magnification. (FIG. 13A) The morphology of ASC cellular
aggregates formed after spinner flask culture of ASCs for 24 hours.
The morphology of the aggregates is very similar to that of the
`spheres` formed after culturing ASCs by the hanging drop method.
The increased cell density (hence the term "high density culture")
in the spinner flask culture is readily apparent when comparing
panel (FIG. 13A) to panel (FIG. 13B). (FIG. 13B) `spheres` formed
after culturing ASCs by the hanging drop method for 48 hours.
[0024] FIGS. 14A-14B demonstrate that ASCs cultured by stirred
suspension culture (spinner flask culture in this case) form a
greater percentage (FIG. 14A) of CD34+ cells (adipose stem cells)
and a roughly equal percentage (FIG. 14B) of SOX17+ cells
(endodermal precursor cells) compared to ASCs cultured by the
hanging-drop method. X-axis is the intensity of signal
detected.
[0025] FIGS. 15A-15B demonstrate that iHeps produced using spinner
flask culture (SS-Hep) had the specific cytochrome CYP enzymes
CYP3A4 and CYP1A1, and the activity of CYP3A4 was strongly induced
by dexamethasone (Dex) treatment. YJM is a human hepatocyte cell
line. CYP activity was normalized to cell viability. Results are
presented with (+) and without (-) Dex induction.
[0026] FIG. 16 demonstrates that iHeps produced using spinner flask
culture (SS-Hep) secreted an increased level of human albumin
(hAlb) compared to Chi-Heps and SCi-Heps (approximately 16-fold
relative to SCi-Heps and approximately 96-fold relative to
Chi-Heps).
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0027] Methods are provided for producing a population of
hepatocyte-like cells (iHeps) from a population of
adipocyte-derived stem cells (ASCs). Aspects of the methods include
placing a population of ASCs into a three dimensional culture
(e.g., hanging drop suspension culture, high density culture,
spinner flask culture, microcarrier culture, etc.), and contacting
the cells with a first and second culture medium. Also provided are
methods of treating an individual, which include producing a
population of iHeps from a population of ASCs, and administering an
effective number of iHeps into the individual. Kits for practicing
the methods are also described herein.
[0028] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to
particular method or composition described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0029] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0030] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supercedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0031] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0032] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the peptide" includes reference to one or more
peptides and equivalents thereof, e.g. polypeptides, known to those
skilled in the art, and so forth.
[0033] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed
Definitions
[0034] The terms "specific binding," "specifically binds," and the
like, refer to non-covalent or covalent preferential binding to a
molecule relative to other molecules or moieties in a solution or
reaction mixture (e.g., an antibody specifically binds to a
particular polypeptide or epitope relative to other available
polypeptides). In some embodiments, the affinity of one molecule
for another molecule to which it specifically binds is
characterized by a K.sub.D (dissociation constant) of 10.sup.-5 M
or less (e.g., 10.sup.-6 M or less, 10.sup.-7 M or less, 10.sup.-8
M or less, 10.sup.-9 M or less, 10.sup.-10 M or less, 10.sup.-11 M
or less, 10.sup.-12 M or less, 10.sup.-13 M or less, 10.sup.-14 M
or less, 10.sup.-1 M or less, or 10.sup.-16 M or less). "Affinity"
refers to the strength of binding, increased binding affinity being
correlated with a lower K.sub.D.
[0035] The term "specific binding member" as used herein refers to
a member of a specific binding pair (i.e., two molecules, usually
two different molecules, where one of the molecules, e.g., a first
specific binding member, through non-covalent means specifically
binds to the other molecule, e.g., a second specific binding
member).
[0036] The term "specific binding agent" as used herein refers to
any agent that specifically binds a biomolecule (e.g., a marker
such as a nucleic acid marker molecule, a protein marker molecule,
etc.). In some cases, a "specific binding agent" for a marker
molecule (e.g., a hepatocyte marker molecule) is used. Specific
binding agents can be any type of molecule. In some cases, a
specific binding agent is an antibody or a fragment thereof. In
some cases, a specific binding agent is nucleic acid probe (e.g.,
an RNA probe; a DNA probe; an RNA/DNA probe; a modified nucleic
acid probe, e.g., a locked nucleic acid (LNA) probe, a morpholino
probe, etc.; and the like)
[0037] As used herein, a "marker molecule" does not have to be
definitive (i.e., the marker does not have to definitely mark the
cell as being of a particular type. For example, the expression of
a marker molecule by a cell can be indicative (i.e., suggestive)
that the cell is of a particular cell type. For example, if 3 cell
types (type A, type B, and type C) express a particular marker
molecule (e.g., a particular mRNA, a particular protein, etc.),
expression of that marker molecule by a cell cannot necessarily be
used by itself to definitively determine that the cell is a type A
cell. However, expression of such a marker can suggest that the
cell is a type A cell. In some cases, expression of such a marker,
combined with other evidence, can definitively show that the cell
is a type A cell. As another illustrative example, if a particular
cell type is known to express two or more particular marker
molecules (e.g., mRNAs, proteins, a combination thereof, etc.) then
the expression by a cell of one of the two or more particular
marker molecules can be suggestive, but not definitive, that the
cell is of the particular type in question. In such a case, the
marker is still considered a marker molecule.
[0038] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired biological activity. "Antibodies"
(Abs) and "immunoglobulins" (Igs) are glycoproteins having the same
structural characteristics. While antibodies exhibit binding
specificity to a specific antigen, immunoglobulins include both
antibodies and other antibody-like molecules which lack antigen
specificity. Polypeptides of the latter kind are, for example,
produced at low levels by the lymph system and at increased levels
by myelomas.
[0039] "Antibody fragment", and all grammatical variants thereof,
as used herein are defined as a portion of an intact antibody
comprising the antigen binding site or variable region of the
intact antibody, wherein the portion is free of the constant heavy
chain domains (i.e. CH2, CH3, and CH4, depending on antibody
isotype) of the Fc region of the intact antibody. Examples of
antibody fragments include Fab, Fab', Fab'-SH, F(ab').sub.2, and Fv
fragments; diabodies; any antibody fragment that is a polypeptide
having a primary structure consisting of one uninterrupted sequence
of contiguous amino acid residues (referred to herein as a
"single-chain antibody fragment" or "single chain polypeptide"),
including without limitation (1) single-chain Fv (scFv) molecules
(2) single chain polypeptides containing only one light chain
variable domain, or a fragment thereof that contains the three CDRs
of the light chain variable domain, without an associated heavy
chain moiety (3) single chain polypeptides containing only one
heavy chain variable region, or a fragment thereof containing the
three CDRs of the heavy chain variable region, without an
associated light chain moiety and (4) nanobodies comprising single
Ig domains from non-human species or other specific single-domain
binding modules; and multispecific or multivalent structures formed
from antibody fragments. In an antibody fragment comprising one or
more heavy chains, the heavy chain(s) can contain any constant
domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fc
region of an intact antibody, and/or can contain any hinge region
sequence found in an intact antibody, and/or can contain a leucine
zipper sequence fused to or situated in the hinge region sequence
or the constant domain sequence of the heavy chain(s).
[0040] As used in this disclosure, the term "epitope" means any
antigenic determinant on an antigen to which the paratope of an
antibody binds. Epitopic determinants usually consist of chemically
active surface groupings of molecules such as amino acids or sugar
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
[0041] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms also apply to amino acid polymers in which one
or more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0042] A "TK-NOG mouse", as used herein refers to a mouse in which
a herpes simplex virus type 1 thymidine kinase (HSVtk) transgene is
expressed within the liver of a highly immunodeficient NOG mouse.
Mouse liver cells expressing the HSVtk transgene can be ablated
after a brief exposure to a non-toxic dose of ganciclovir (GCV). In
some embodiments, an individual receiving treatment can be a mouse.
For example, a TK-NOG mouse with ablated liver cells can be
considered an individual with reduced liver function (i.e., an
individual with liver damage) and the mouse can therefore be
considered to be an individual receiving treatment when subject
iHEPS (describe in detail below) are transplanted into the mouse.
Transplanted human liver cells can be stably maintained within the
liver of TK-NOG mice, and TK-NOG mice with transplanted human liver
cells are referred herein as humanized TK-NOG mice. The
reconstituted liver of humanized TK-NOG mice can be a mature and
functioning human organ, and can generate a human-specific profile
of drug metabolism. The `humanized liver` can be stably maintained
in humanized TK-NOG mice with a high level of function for a
prolonged period (e.g., at least 8 months). For more information
about TK-NOG mice, refer to: (i) Hasegawa et al, Biochem Biophys
Res Commun. 2011 Feb. 18; 405(3):405-10; (ii) Yamazaki et al, Chem
Res Toxicol. 2012 Feb. 20; 25(2):274-6; (iii) Hu et al.,
Pharmacogenet Genomics. 2013 February; 23(2):78-83; and (iv)
Yamazaki et al, Chem Res Toxicol. 2013 Mar. 18; 26(3):486-9; which
are hereby incorporated by reference in their entirety.
[0043] The term "stem cell" is used herein to refer to a mammalian
cell that has the ability both to self-renew and to generate a
differentiated cell type (see Morrison et al. (1997) Cell
88:287-298). In the context of cell ontogeny, the adjective
"differentiated", or "differentiating" is a relative term. A
"differentiated cell" is a cell that has progressed further down
the developmental pathway than the cell it is being compared with.
Thus, pluripotent stem cells can differentiate into
lineage-restricted progenitor cells (e.g., adipocyte-derived stem
cells), which in turn can differentiate into end-stage cells (e.g.,
adipocytes, osteoblasts, chondrocytes, etc.), which play a
characteristic role in a certain tissue type, and may or may not
retain the capacity to proliferate further. Stem cells (and
differentiated progeny) may be characterized by both the presence
of specific markers (e.g., proteins, RNAs, etc.) and the absence of
specific markers. Stem cells may also be identified by functional
assays both in vitro and in vivo, particularly assays relating to
the ability of stem cells to give rise to multiple differentiated
progeny.
[0044] The stem cells of interest are mammalian, where the term
refers to cells isolated from any animal classified as a mammal,
including humans, domestic and farm animals, and zoo, laboratory,
sports, or pet animals, such as dogs, horses, cats, cows, mice,
rats, rabbits, etc. In some embodiments, the mammal is a human and
the mammalian cells (e.g., a population of adipocyte-derived stem
cells) are therefore human cells (e.g., a population of human
adipocyte-derived stem cells).
[0045] The terms "passaging" or "passage" (i.e., splitting or
split) in the context of cell culture are known in the art and
refer to the transferring of a small number of cells into a new
vessel. Cells can be cultured if they are split regularly because
it avoids the senescence associated with high cell density. For
adherent cells, cells are detached from the growth surface as part
of the passaging protocol. Detachment is commonly performed with
the enzyme trypsin and/or other commercially available reagents
(e.g., TrypLE, EDTA (Ethylenediaminetetraacetic acid), a policemen
(e.g., a rubber policemen) for physically scrapping the cells from
the surface, etc.). A small number of detached cells (e.g., as few
as one cell) can then be used to seed a new cell population, e.g.,
after dilution with additional media. Therefore, to passage a cell
population means to dissociate at least a portion of the cells of
the cell population, dilute the dissociated cells, and to plate the
diluted dissociated cells (i.e., to seed a new cell
population).
[0046] The terms "media" and "medium" are herein used
interchangeably. Cell culture media is the liquid mixture that
baths cells during in vitro culture.
[0047] The term "population", e.g., "cell population" or
"population of cells", as used herein means a grouping (i.e., a
population) of two or more cells that are separated (i.e.,
isolated) from other cells and/or cell groupings. For example, a
6-well culture dish can contain 6 cell populations, each population
residing in an individual well. The cells of a cell population can
be, but need not be, clonal derivatives of one another. A cell
population can be derived from one individual cell. For example, if
individual cells are each placed in a single well of a 6-well
culture dish and each cell divides one time, then the dish will
contain 6 cell populations. A cell population can be any desired
size and contain any number of cells greater than one cell. For
example, a cell population can be 2 or more, 10 or more, 100 or
more, 1,000 or more, 5,000 or more, 10.sup.4 or more, 10.sup.5 or
more, 10.sup.6 or more, 10.sup.1 or more, 10.sup.8 or more,
10.sup.9 or more, 10.sup.10 or more, 10.sup.11 or more, 10.sup.12
or more, 10.sup.13 or more, 10.sup.14 or more, 10.sup.15 or more,
10.sup.16 or more, 10.sup.17 or more, 10.sup.18 or more, 10.sup.19
or more, or 10.sup.20 or more cells.
Methods
[0048] Aspects of the disclosure include methods of producing a
population of hepatocyte-like cells from a population of
adipocyte-derived stem cells (ASCs). The methods generally involve
placing a population of ASCs into a three dimensional culture
(e.g., hanging drop suspension culture, high density culture,
spinner flask culture, microcarrier culture, etc.) to produce an
ASC-derived cellular aggregate (e.g., sphere); contacting cells of
the ASC-derived cellular aggregate with a first culture medium
comprising Activin A and a fibroblast growth factor (FGF) to
produce a precursor cell population; and contacting cells of the
precursor cell population with a second culture medium comprising
hepatocyte growth factor (HGF) to produce an induced cell
population that comprises induced hepatocyte-like cells
(iHeps).
[0049] Adipose derived stem cells (ASCs). The term "adipose-derived
stem cell" refers to a population of adipose cells found in
post-natal mammals that are pluripotent and have the potential to
differentiate into a variety of cell types including but not
limited to cells of osteogenic, adipogenic, and chondrogenic
lineages. ASCs can also differentiate into hepatocyte-like cells.
ASCs have been referred to in the literature as adipose-derived
adult stem (ADAS) cells, adipose-derived adult stromal cells,
adipose-derived stromal cells (ADSCs), adipose stromal cells
(ASCs), adipose mesenchymal stem cells (AdMSCs), lipoblast,
pericyte, preadipocyte, and processed lipoaspirate (PLA) cells. The
International Fat Applied Technology Society (IFATS) reached a
consensus to adopt the term "adipose-derived stem cells" (ASCs) to
identify the isolated, plastic-adherent, multipotent cell
population. Thus, the term "Adipose-derived stem cell" (ASC) is
used herein in accordance with the IFATS consensus, which will be
known to one of ordinary skill in the art. In contrast to cell
lines, ASCs have not undergone immortalization.
[0050] A number of scientific publications have described the
underlying biology of ASCs, preclinical studies for the use of ASCs
in regenerative medicine in various fields have been performed, and
the efficacy of ASCs has been determined in several clinical
trials.
[0051] ASCs for use in the subject methods can be isolated by any
convenient methods, which will be known to one of ordinary skill in
the art. Subcutaneous adipose tissue samples can generally be
obtained under local anesthesia. Current methods used for isolating
ASCs generally include collagenase digestion followed by
centrifugal separation to isolate the Stromal Vascular Fraction
from primary adipocytes.
[0052] As a non-limiting example, to isolate ASCs, adipose tissue
obtained as excised surgical specimens (e.g., a biopsy) or as
lipoaspirates can be digested with a collagenase enzyme (e.g., a
bacterially-derived collagenase) in the presence of calcium to
release the individual cell components. Subsequently, mature
adipocytes can be separated (e.g., via differential
centrifugation), from the remaining cells, which form a Stromal
Vascular Fraction (SVF) pellet. The SVF cell population includes
endothelial cells, fibroblasts, Band T-lymphocytes, macrophages,
myeloid cells, pericytes, pre-adipocytes, smooth muscle cells, and
the culture adherent ASCs. After culture (e.g., 4 to 6 days) with
medium containing about 10% fetal bovine serum (any convenient
culture medium can be used), a single milliliter of human
lipoaspirate will yield between 0.25 to 0.375.times.10.sup.6 ASCs
capable of differentiating along the adipogenic (adipocyte),
chondrogenic (chondrocyte), and osteogenic (osteoblast) lineages in
vitro. ASCs display a fibroblast-like morphology and lack the
intercellular lipid droplets seen in adipocytes. Isolated ASCs are
typically expanded in monolayer culture on standard tissue culture
plastics with a basal medium containing 10% fetal bovine serum.
Since liposuction from a single patient often results in >1 L of
tissue, it is feasible to generate hundreds of millions of ASCs
from a single donor within a single in vitro cell culture passage
In contrast to the SVF cells, ASCs are relatively homogeneous based
on their expression profile of surface antigens.
[0053] In general, the isolation of ASCs from a lipoaspirate can
include: (1) wash the lipoaspriate in buffered saline solution; (2)
subject the lipoaspirate to collagenase digestion; (3) centrifuge
and isolate the stromal vascular fraction (SVF) pellet; (4) culture
the heterogeneous SVF cells on an adherent surface; and (5) isolate
adherent ASCs. Culture of SVF cells under standard conditions
eventually (within the first few passages) results in the
appearance of ASCs, which is a relatively homogeneous population of
mesodermal or mesenchymal cells. ASCs can be verified, for example,
by demonstrating that the cells can differentiate into multiple
different lineages (e.g., adipocytes can be identified using, for
example, Oil Red O stain; osteoblasts can be identified using, for
example, Alizarin Red stain; and chondrocytes can be identified
using, for example, Alcian Blue stain). Protein and nucleic acid
markers can also be used to verify differentiation into multiple
lineages.
[0054] The International Society for Cellular Therapy (ISCT) and
IFATS have established minimal criteria defining SVF cells and ASC
based on functional and quantitative criteria. The four criteria
used herein are: (1) ASCs are plastic-adherent when maintained
under standard culture conditions; (2) ASCs have the capacity for
osteogenic, adipogenic, and chondrogenic differentiation; (3) ASCs
express the markers (i.e., molecular markers) CD29, CD34, CD36,
CD49f, CD73, CD90 (Thy-1), CD105, CD133, c-kit, and c-met; and (4)
ASCs are negative for CD45, CD106, and CD31. The adipocytic,
chondroblastic and osteoblastic differentiation assays (e.g., Oil
Red O stain, Alcian blue stain, and Alizarin red stain,
respectively) can be used to assess potency and differentiation
capacity, and can be used in conjunction with a quantitative
evaluation of differentiation either biochemically or by reverse
transcription polymerase chain reaction. The colony-forming
unit--fibroblast (CFU-F) assay is recommended by the IFATS to
calculate population doublings capacity of ASCs.
[0055] For more information regarding the nature of ASCs, including
the isolation and culture of ASCs, see (i) Gimble et al., Circ Res.
2007 May 11; 100(9):1249-60: "Adipose-derived stem cells for
regenerative medicine"; (ii) Gimble et al., Organogenesis. 2013
Jan. 1; 9(1): "Adipose-derived stromal/stem cells: A primer"; (iii)
Bourin et al, Cytotherapy. 2013 June; 15(6):641-8: "Stromal cells
from the adipose tissue-derived stromal vascular fraction and
culture expanded adipose tissue-derived stromal/stem cells: a joint
statement of the International Federation for Adipose Therapeutics
and Science (IFATS) and the International Society for Cellular
Therapy (ISCT)"; (iv) Gentile et al, Stem Cells Transl Med. 2012
March; 1(3):230-6: "Concise review: adipose-derived stromal
vascular fraction cells and platelet-rich plasma: basic and
clinical implications for tissue engineering therapies in
regenerative surgery"; and (v) Mizuno et al., Stem Cells. 2012 May;
30(5):804-10: "Concise review: Adipose-derived stem cells as a
novel tool for future regenerative medicine"; all of which are
hereby incorporated by reference in their entirety.
[0056] As used herein, the term "adipose tissue" refers to fat
including the connective tissue that stores fat. Adipose tissue
contains multiple regenerative cell types, including ASCs and
endothelial progenitor and precursor cells.
[0057] Three dimensional culture. Methods of the disclosure include
placing a population of ASCs into a three dimensional culture to
produce an ASC-derived cellular aggregate (e.g., sphere). The term
"three dimensional culture" as used herein refers to the culture of
cells in a way that includes low shear force (and consequently low
turbulence), and high mass transfer of nutrients. One non-limiting
example of "three dimensional culture" is the culture of cells as
aggregates (e.g., spheres, i.e., spheroid formation). Placing a
population of cells (e.g., ASCs) into a three dimensional culture
can cause the cells to form cellular aggregates (e.g., cellular
spheres). Thus, subject cells (e.g., a population of ASCs) can be
placed into a three dimensional culture to produce an ASC-derived
cellular aggregate (e.g, sphere). Cellular aggregates (e.g,
spheres) can be produced by a number of three dimensional culture
techniques, including but not limited to hanging drop suspension
culture, high density cell culture (e.g., stirred suspension
culture such as spinner flask culture, e.g., with or without
microcarriers; bioreactor culture, e.g., with or without
microcarriers; etc.), and the like (see examples section).
[0058] In some embodiments, cells can be placed into a three
dimensional culture at any convenient chosen density (e.g., via
dilution or concentration). For example, in some cases, cells are
placed into a three dimensional culture at a cell density in a
range of from 1.times.10.sup.2 cells/ml to 1.times.10.sup.7
cells/ml (e.g., from 5.times.10.sup.2 cells/ml to 5.times.10.sup.6
cells/ml, from 1.times.10.sup.3 cells/ml to 5.times.10.sup.6
cells/ml, from 5.times.10.sup.3 cells/ml to 5.times.10.sup.6
cells/ml, from 5.times.10.sup.3 cells/ml to 1.times.10.sup.5
cells/ml, from 5.times.10.sup.3 cells/ml to 5.times.10.sup.4
cells/ml, from 7.times.10.sup.3 cells/ml to 3.times.10.sup.4
cells/ml, from 8.times.10.sup.3 cells/ml to 3.times.10.sup.4
cells/ml, 1.times.10.sup.4 cells/ml, from 1.times.10.sup.4 cells/ml
to 1.times.10.sup.6 cells/ml, from 5.times.10.sup.4 cells/ml to
1.times.10.sup.6 cells/ml, from 7.times.10.sup.4 cells/ml to
7.times.10.sup.5, from 1.times.10.sup.5 cells/ml to
7.times.10.sup.5, from 3.times.10.sup.5 cells/ml to
7.times.10.sup.5, from 4.times.10.sup.5 cells/ml to
6.times.10.sup.5, or 5.times.10.sup.5).
[0059] In practicing the methods of the disclosure, ASCs can be
cultured in three dimensional culture for a period of time
sufficient for the formation of cellular aggregates (e.g., spheres,
which can resemble embryoid bodies). In some embodiments, ASCs are
cultured using three dimensional culture (i.e., ASCs are placed
into a three dimensional culture) for 3 days or less (e.g., 2.5
days or less, 2 days or less, 1.5 days or less, 1 day or less, 12
hours or less, 8 hours or less, 6 hours or less, or 4 hours or
less). In some embodiments, ASCs are cultured using three
dimensional culture for a period of time in a range of from 2 hours
to 3 days (e.g., from 2 hours to 3 days, from 2 hours to 2.5 days,
from 2 hours to 2 days, from 2 hours to 1.5 days, from 2 hours to 1
day, from 6 hours to 3 days, from 6 hours to 2.5 days, from 6 hours
to 2 days, from 6 hours to 1.5 days, from 6 hours to 1 day, from 6
hours to 12 hours, from 8 hours to 3 days, from 8 hours to 2.5
days, from 8 hours to 2 days, from 8 hours to 1.5 days, from 8
hours to 1 day, from 8 hours to 12 hours, from 12 hours to 2.5
days, from 12 hours to 2 days, from 12 hours to 1.5 days, from 12
hours to 1 day, from 1 day to 3 days, from 1 day to 2.5 days, from
1 day to 2 days, from 1 day to 1.5 days, from 1.5 days to 3 days,
from 1.5 days to 2.5 days, from 1.5 days to 2 days, from 2 days to
3 days, from 1 day, 1.5 days, from 2 days, from 2.5 days, or 3
days).
[0060] Hanging drop suspension culture. In some embodiments, the
three dimensional culture is a hanging drop suspension culture.
Thus, in some embodiments, aggregates of subject cells (e.g., ASCs)
are formed by placing the cells in a hanging drop suspension
culture. Hanging drop suspension culture is a technique often used
to form embryoid bodies from embryonic stem cells (ESCs). Cells in
fluid are placed in droplets (usually in the range of 5-50 .mu.l in
size) onto a surface (e.g., surface of a Petri dish, a coverslip,
glass, plastic etc.) and the surface is inverted such that the
cells are suspended from the surface. The cells are thus cultured
under the surface in a suspended droplet instead of being cultured
on top of the surface. The suspended droplets are sometimes
referred to as hanging drops. Some cells form aggregates, (referred
to as `spheres` and/or `spheroids`) when cultured using the hanging
drop suspension culture.
[0061] In some embodiments, cells can be placed into hanging drop
suspension culture at a chosen density (e.g., via dilution or
concentration). For example, in some cases, cells are placed into
hanging drop suspension culture at a cell density in a range of
from 1.times.10.sup.2 cells/ml to 1.times.10.sup.7 cells/ml (e.g.,
from 5.times.10.sup.2 cells/ml to 5.times.10.sup.6 cells/ml, from
1.times.10.sup.3 cells/ml to 5.times.10.sup.6 cells/ml, from
5.times.10.sup.3 cells/ml to 5.times.10.sup.6 cells/ml, from
5.times.10.sup.3 cells/ml to 1.times.10.sup.5 cells/ml, from
5.times.10.sup.3 cells/ml to 5.times.10.sup.4 cells/ml, from
7.times.10.sup.3 cells/ml to 3.times.10.sup.4 cells/ml, from
8.times.10.sup.3 cells/ml to 3.times.10.sup.4 cells/ml,
1.times.10.sup.4 cells/ml, from 1.times.10.sup.4 cells/ml to
1.times.10.sup.6 cells/ml, from 5.times.10.sup.4 cells/ml to
1.times.10.sup.6 cells/ml, from 7.times.10.sup.4 cells/ml to
7.times.10.sup.5, from 1.times.10.sup.5 cells/ml to
7.times.10.sup.5, from 3.times.10.sup.5 cells/ml to
7.times.10.sup.5, from 4.times.10.sup.5 cells/ml to
6.times.10.sup.5, or 5.times.10.sup.5).
[0062] In practicing the methods of the disclosure, ASCs can be
cultured by the hanging drop method (i.e., cultured using hanging
drop suspension culture) for a period of time sufficient for the
formation of cellular aggregates (e.g., spheres, which can resemble
embryoid bodies). In some embodiments, ASCs are cultured using
hanging drop suspension culture (i.e., ASCs are placed into a
hanging drop suspension culture) for 3 days or less (e.g., 2.5 days
or less, 2 days or less, 1.5 days or less, 1 day or less, 12 hours
or less, 8 hours or less, 6 hours or less, or 4 hours or less). In
some embodiments, ASCs are cultured using hanging drop suspension
culture for a period of time in a range of from 2 hours to 3 days
(e.g., from 2 hours to 3 days, from 2 hours to 2.5 days, from 2
hours to 2 days, from 2 hours to 1.5 days, from 2 hours to 1 day,
from 6 hours to 3 days, from 6 hours to 2.5 days, from 6 hours to 2
days, from 6 hours to 1.5 days, from 6 hours to 1 day, from 6 hours
to 12 hours, from 8 hours to 3 days, from 8 hours to 2.5 days, from
8 hours to 2 days, from 8 hours to 1.5 days, from 8 hours to 1 day,
from 8 hours to 12 hours, from 12 hours to 2.5 days, from 12 hours
to 2 days, from 12 hours to 1.5 days, from 12 hours to 1 day, from
1 day to 3 days, from 1 day to 2.5 days, from 1 day to 2 days, from
1 day to 1.5 days, from 1.5 days to 3 days, from 1.5 days to 2.5
days, from 1.5 days to 2 days, from 2 days to 3 days, from 1 day,
1.5 days, from 2 days, from 2.5 days, or 3 days).
[0063] In some embodiments, an ASC-derived cellular aggregate
(e.g., sphere) is removed from three dimensional culture (e.g.,
hanging drop suspension culture, high density culture, spinner
flask culture, microcarrier culture, etc.). For example, a cellular
aggregate (e.g., sphere) can be suspended in fluid and plated onto
a two-dimensional surfaced that is not considered a three
dimensional culture. In some cases, aggregates (e.g., spheres) are
collected (e.g., by centrifugation) and suspended. In some cases,
the aggregates (e.g., spheres) are collected and suspended at a
density in a range of from 15 aggregates/ml to 45 aggregates/ml
(e.g., from 20 aggregates/ml to 40 aggregates/ml, from 25
aggregates/ml to 35 aggregates/ml, or 30 aggregates/ml). The
aggregates can be suspended in any convenient media (e.g., stage 1
media, described below). In some cases, the collected (suspended)
aggregates (e.g., spheres) are seeded (i.e., plated, e.g., onto
matrigel-coated dishes). In some embodiments, an ASC-derived
cellular aggregate (e.g., sphere) is removed from three dimensional
culture (e.g., hanging drop suspension culture, high density
culture, spinner flask culture, microcarrier culture, etc.)
simultaneous with, or prior to, contacting cells of the ASC-derived
cellular aggregate (e.g., sphere) with a stage 1 medium. Thus, in
some cases, cells are cultured (e.g., contacted with) a stage 1
medium after being removed from three dimensional culture. In some
embodiments, cells of the ASC-derived cellular aggregate (e.g.,
sphere) are contacted with a stage 1 medium while still in three
dimensional culture (e.g., in order to transfer the cells to
non-inverted culture conditions).
[0064] High density culture--microcarrier culture. In some
embodiments, the three dimensional culture is a high density
culture. Thus, aggregates (e.g., spheres) of subject cells (e.g.,
ASCs) are formed by placing the cells in a high density culture. In
some embodiments, a high density culture is a microcarrier culture.
In some embodiments, the three dimensional culture is a
microcarrier culture. Thus, aggregates (e.g., spheres) of subject
cells (e.g., ASCs) are formed by placing the cells in a
microcarrier culture. The term "microcarrier culture" is used
herein to refer to the culture of cells on a support matrix (e.g.,
a spherical support matrix). In this system, cells are propagated
on the surface of small solid particles suspended in the growth
medium by slow agitation. The cells attach and grow to confluence
on the surface of the microcarriers.
[0065] Microcarriers can be produced in various shapes and sizes,
spherical being the most common, and their density allows them to
be maintained in suspension with gentle stirring. Each microcarrier
should have dimensions that can facilitate cell growth for several
doublings. In this way at the end of the cell growth, each
microcarrier will support several hundred cells on its surface.
Spherical microcarriers usually have diameters of 100-250 .mu.m. In
some embodiments, microcarriers are spheres with a diameter in a
range of from 100 .mu.m to 250 .mu.m. The size distribution of the
spherical microcarriers should be low (e.g., .+-.25 .mu.m) in order
to prevent uneven distribution of cells on the microcarriers.
Density of the microcarriers should be slightly above 1 (e.g.,
1.02-1.05 g/ml) in order to maintain the microcarriers in
suspension at minimal agitation speed.
[0066] Microcarriers can be made from a number of different
materials including diethylaminoethanol (DEAE)-dextran, dextran,
glass, polystyrene plastic, acrylamide (e.g., polyacrylamide),
collagen, etc. In some cases, biodegradable microcarriers can be
used as the scaffold for in vivo transplantation of cells. The MC
surface is available for cell growth while the mobility of MCs in
the medium generates a homogeneity that is similar to the
suspension environment used in traditional mammalian and microbial
submerged cultures.
[0067] The surface of a microcarrier can be derivatized with
functional groups such as recombinant proteins, positively charged
tertiary quaternary or primary amines, gelatin, collagen, other
extracellular matrix (ECM) proteins and peptides (e.g. RGD
peptide). The positively charged MCs attract the cells (which are
negatively charged), by electrostatic forces. The optimal amount of
positive charge is generally found to be between 1 and 2
milliequivalents/g dry materials (for cross-linked dextran or
polyacrylamide beads derivatized with tertiary amines). At this
level, cells attach to the microcarriers efficiently (about 90%
within 1 h) without negative effect on cell growth. Coating with
collagen or ECM protein results in lower cell attachment but
usually supports better growth of cells at low inoculation
levels.
[0068] Several types of microcarriers are available commercially
including dextran-based (Cytodex, GE Healthcare), collagen-based
(Cultispher, Percell), and polystyrene-based (SoloHill Engineering)
microcarriers. They differ in their porosity, specific gravity,
optical properties, presence of animal components, and surface
chemistries. It is possible to categorize the microcarriers into
six groups:
[0069] Group 1: Non-porous smooth (e.g. polystyrene microcarriers)
or microporous microcarriers (e.g. Cytodex 1) with positive
charges. These microcarriers are suitable for culturing adherent
cells that form a continuous monolayer of cells on the surface of
the microcarriers in stirred cultures. This group includes also
Whatman's anion exchange celluloses (DE-53), cylindrical shaped
microcarriers that have been used successfully in culturing cell
lines (BHK and MDCK).
[0070] Group 2: Collagen coated microcarriers (e.g. Cytodex 3 and
FACT 102-L). These microcarriers are chemically coupled with
collagen and are suitable for culturing sensitive cells with low
plating efficiency. The collagen coating is also designed to
facilitate cell harvesting.
[0071] Group 3: ECM coated microcarriers (Pro-F 102-L). Pro-F 102-L
is coated with recombinant fibronectin which is designed for
culturing of sensitive cells in serum free conditions.
[0072] Group 4: Non-charged microcarriers (e.g. Glass beads and
tissue culture Polystyrene MC P 102-L). These microcarriers have
similar surface properties as classical 2D tissue culture
surfaces.
[0073] Group 5: Macroporous microcarriers (e.g. Cytopore and
Cultispher). Macroporous microcarriers with pore sizes in the range
of 10-70 .mu.m on the surface. They provide higher cell surface
areas for growth and offer better mechanical protection to the
cells from shear stress generated by stirrers, spargers or spin
filters.
[0074] Group 6: Weighted microcarriers (Cytoline). These
microcarriers are designed for use in fluidized bed perfusion
cultures. These commercial microcarriers have been designed
according to the needs for propagating anchorage dependent cell
lines used in production of vaccine and biopharmaceuticals.
[0075] In practicing the methods of the disclosure, ASCs can be
cultured by microcarrier culture (i.e., cultured using microcarrier
culture) for a period of time sufficient for the formation of
cellular aggregates (e.g., spheres, which can resemble embryoid
bodies). In some embodiments, ASCs are cultured using microcarrier
culture (i.e., ASCs are placed into a microcarrier culture) for 3
days or less (e.g., 2.5 days or less, 2 days or less, 1.5 days or
less, 1 day or less, 12 hours or less, 8 hours or less, 6 hours or
less, or 4 hours or less). In some embodiments, ASCs are cultured
using microcarrier culture for a period of time in a range of from
2 hours to 3 days (e.g., from 2 hours to 3 days, from 2 hours to
2.5 days, from 2 hours to 2 days, from 2 hours to 1.5 days, from 2
hours to 1 day, from 6 hours to 3 days, from 6 hours to 2.5 days,
from 6 hours to 2 days, from 6 hours to 1.5 days, from 6 hours to 1
day, from 6 hours to 12 hours, from 8 hours to 3 days, from 8 hours
to 2.5 days, from 8 hours to 2 days, from 8 hours to 1.5 days, from
8 hours to 1 day, from 8 hours to 12 hours, from 12 hours to 2.5
days, from 12 hours to 2 days, from 12 hours to 1.5 days, from 12
hours to 1 day, from 1 day to 3 days, from 1 day to 2.5 days, from
1 day to 2 days, from 1 day to 1.5 days, from 1.5 days to 3 days,
from 1.5 days to 2.5 days, from 1.5 days to 2 days, from 2 days to
3 days, from 1 day, 1.5 days, from 2 days, from 2.5 days, or 3
days).
[0076] For examples of the use of microcarriers (e.g., using
spinner flasks, bioreactors, etc.) for various purposes and with
various types of cells, refer to scientific literature such as: (i)
Chen et al, Biotechnol Adv. 2013 Mar. 24. pii:
S0734-9750(13)00065-7: Application of human mesenchymal and
pluripotent stem cell microcarrier cultures in cellular therapy:
Achievements and future direction; (ii) Pacak et al, PLoS One.
2013; 8(1):e55187: Microcarrier-based expansion of adult murine
side population stem cells; (iii) Torgan et al, Med Biol Eng
Comput. 2000 September; 38(5):583-90: Differentiation of mammalian
skeletal muscle cells cultured on microcarrier beads in a rotating
cell culture system; and (iv) Park et al, Tissue Eng Part B Rev.
2013 April; 19(2):172-90: Microcarriers designed for cell culture
and tissue engineering of bone; as well as patent literature such
as U.S. Pat. Nos. 8,524,492, 8,426,176, 7,947,471, 7,670,839,
7,361,493, 6,214,618, and 5,153,133, 4,910,142, and 4,824,946; all
of which are hereby incorporated by reference in their
entirety.
[0077] Microcarrier culture generally requires agitation of the
microcarriers (e.g., the cell-loaded microcarriers). Agitation can
include simple agitation or shaking, agitation using a spinner
flask, and/or agitation using a bioreactor (also known as a
rotating cell culture system (RCCS), a rotary cell culture system
(RCCS), or a rotating chamber system).
[0078] High density culture--Spinner flask culture. In some
embodiments, the three dimensional culture is a high density
culture. Thus, aggregates (e.g., spheres) of subject cells (e.g.,
ASCs) are formed by placing the cells in a high density culture. In
some embodiments, a high density culture is a spinner flask
culture. Spinner flask culture is an example of a stirred
suspension culture. Stirred suspension culture (i.e., mass
suspension culture)(e.g., spinner flask culture, bioreactor
culture, etc.) can be used to culture cells in the absence of
microcarriers or in the presence of microcarriers. Thus, in some
embodiments, a microcarrier culture is agitated using a spinner
flask. In some embodiments, a three dimensional culture (e.g., a
high density culture) is a spinner flask culture without (i.e., in
the absence of) microcarriers. In the absence of microcarriers,
ASCs placed in stirred suspension culture (e.g., spinner flask
culture, bioreactor culture) still form cellular aggregates (e.g.,
spheres)(FIG. 13). Thus, in some embodiments, a three dimensional
culture (e.g., a high density culture) is a spinner flask culture
without (i.e., in the absence of) microcarriers.
[0079] Spinner flasks (i.e., stirrer bottles) are generally
designed to facilitate the culture of high volumes of cells by
providing homogenous circulation of cells (e.g., cells in the
absence of microcarriers, cells on microcarriers, etc.) and medium
(i.e., nutrients), and superior gas exchange (e.g., oxygenation).
Spinner flasks can be made of any convenient material (e.g.,
borosilicate glass, tissue culture grade plastics, etc.) and are
usually a flat-bottom flask with a stirring device (e.g., usually a
vertical impeller or a hanging stir bar) that can be controlled
using a magnetic stir plate. A spinner flask can also include two
angled side arms to allow for the introduction of pipettes. Any
convenient spinner flask that provides for high density culture can
be used in practicing the subject methods.
[0080] In some embodiments, cells can be placed into a spinner
flask culture at any convenient chosen density (e.g., via dilution
or concentration). For example, in some cases, cells are placed
into a spinner flask culture at a cell density in a range of from
1.times.10.sup.2 cells/ml to 1.times.10.sup.7 cells/ml (e.g., from
5.times.10.sup.2 cells/ml to 5.times.10.sup.6 cells/ml, from
1.times.10.sup.3 cells/ml to 5.times.10.sup.6 cells/ml, from
5.times.10.sup.3 cells/ml to 5.times.10.sup.6 cells/ml, from
5.times.10.sup.3 cells/ml to 1.times.10.sup.5 cells/ml, from
5.times.10.sup.3 cells/ml to 5.times.10.sup.4 cells/ml, from
7.times.10.sup.3 cells/ml to 3.times.10.sup.4 cells/ml, from
8.times.10.sup.3 cells/ml to 3.times.10.sup.4 cells/ml,
1.times.10.sup.4 cells/ml, from 1.times.10.sup.4 cells/ml to
1.times.10.sup.6 cells/ml, from 5.times.10.sup.4 cells/ml to
1.times.10.sup.6 cells/ml, from 7.times.10.sup.4 cells/ml to
7.times.10.sup.5, from 1.times.10.sup.5 cells/ml to
7.times.10.sup.5, from 3.times.10.sup.5 cells/ml to
7.times.10.sup.5, from 4.times.10.sup.5 cells/ml to
6.times.10.sup.5, or 5.times.10.sup.5).
[0081] In practicing the methods of the disclosure, ASCs can be
cultured by spinner flask culture (i.e., cultured using spinner
flask culture) for a period of time sufficient for the formation of
cellular aggregates (e.g., spheres, which can resemble embryoid
bodies). In some embodiments, ASCs are cultured using spinner flask
culture (i.e., ASCs are placed into a spinner flask culture) for 3
days or less (e.g., 2.5 days or less, 2 days or less, 1.5 days or
less, 1 day or less, 12 hours or less, 8 hours or less, 6 hours or
less, or 4 hours or less). In some embodiments, ASCs are cultured
using spinner flask culture for a period of time in a range of from
2 hours to 3 days (e.g., from 2 hours to 3 days, from 2 hours to
2.5 days, from 2 hours to 2 days, from 2 hours to 1.5 days, from 2
hours to 1 day, from 6 hours to 3 days, from 6 hours to 2.5 days,
from 6 hours to 2 days, from 6 hours to 1.5 days, from 6 hours to 1
day, from 6 hours to 12 hours, from 8 hours to 3 days, from 8 hours
to 2.5 days, from 8 hours to 2 days, from 8 hours to 1.5 days, from
8 hours to 1 day, from 8 hours to 12 hours, from 12 hours to 2.5
days, from 12 hours to 2 days, from 12 hours to 1.5 days, from 12
hours to 1 day, from 1 day to 3 days, from 1 day to 2.5 days, from
1 day to 2 days, from 1 day to 1.5 days, from 1.5 days to 3 days,
from 1.5 days to 2.5 days, from 1.5 days to 2 days, from 2 days to
3 days, from 1 day, 1.5 days, from 2 days, from 2.5 days, or 3
days).
[0082] High density culture--Bioreactor culture. In some
embodiments, the three dimensional culture is a high density
culture. Thus, aggregates (e.g., spheres) of subject cells (e.g.,
ASCs) are formed by placing the cells in a high density culture. In
some embodiments, a high density culture is a bioreactor culture.
Bioreactor culture is an example of a stirred suspension culture.
Stirred suspension culture (i.e., mass suspension culture)(e.g.,
spinner flask culture, bioreactor culture, etc.) can be used to
culture cells in the absence of microcarriers or in the presence of
microcarriers. Thus, in some embodiments, a microcarrier culture is
agitated using a bioreactor. In some embodiments, a three
dimensional culture (e.g., a high density culture) is a bioreactor
culture without (i.e., in the absence of) microcarriers. In the
absence of microcarriers, ASCs placed in stirred suspension culture
(e.g., spinner flask culture, bioreactor culture) still form
cellular aggregates (e.g., spheres). Thus, in some embodiments, a
three dimensional culture (e.g., a high density culture) is a
bioreactor culture without (i.e., in the absence of)
microcarriers.
[0083] Bioreactors (also known as a rotating cell culture system
(RCCS), a rotary cell culture system (RCCS), or a rotating chamber
system) can control environmental conditions such as gas (e.g.,
air, oxygen, nitrogen, carbon dioxide) flow rates, temperature, pH,
dissolved oxygen levels, agitation speed/circulation rate, and the
like. Like spinner flasks, bioreactors are designed to facilitate
the culture of high volumes of cells by providing homogenous
circulation of cells (e.g., cells in the absence of microcarriers,
cells on microcarriers, etc.) and medium (i.e., nutrients), and
superior gas exchange (e.g., oxygenation). Any convenient
bioreactor that provides for high density cell culture can be used
with the disclosed methods and various suitable bioreactors are
known in the art. Examples of suitable biorecactors include, but
are not limited to: rotating wall microgravity bioreactors,
rotating wall vessel bioreactors (RWVB), stirred tank bioreactors,
perfused bioreactors, fluidized bed bioreactors. Bioreactors can be
made from a large variety of different materials, including, but
not limited to: stainless steel, polytetraflouroethylene (PTFE),
glass, and the like.
[0084] Bioreactors can allow for the scaling up of culture in, for
example, conventional stainless steel or disposable bioreactors
that are used for propagation of suspended mammalian cells.
Bioreactors range in size. In some cases a bioreactor culture is a
culture in the range of from 5 liters to 105 liters (e.g., from 5
liters to 15 liters, from 8 liters to 12 liters, from 15 liters to
25 liters, from 25 liters to 40 liters, from 40 liters to 50
liters, from 45 liters to 55 liters, from 55 liters to 65 liters,
from 65 liters to 75 liters, from 75 liters to 85 liters, from 85
liters to 95 liters, or from 95 liters to 105 liters).
[0085] In some embodiments, cells can be placed into a bioreactor
culture at any convenient chosen density (e.g., via dilution or
concentration). For example, in some cases, cells are placed into a
bioreactor culture at a cell density in a range of from
1.times.10.sup.2 cells/ml to 1.times.10.sup.7 cells/ml (e.g., from
5.times.10.sup.2 cells/ml to 5.times.10.sup.6 cells/ml, from
1.times.10.sup.3 cells/ml to 5.times.10.sup.6 cells/ml, from
5.times.10.sup.3 cells/ml to 5.times.10.sup.6 cells/ml, from
5.times.10.sup.3 cells/ml to 1.times.10.sup.5 cells/ml, from
5.times.10.sup.3 cells/ml to 5.times.10.sup.4 cells/ml, from
7.times.10.sup.3 cells/ml to 3.times.10.sup.4 cells/ml, from
8.times.10.sup.3 cells/ml to 3.times.10.sup.4 cells/ml,
1.times.10.sup.4 cells/ml, from 1.times.10.sup.4 cells/ml to
1.times.10.sup.6 cells/ml, from 5.times.10.sup.4 cells/ml to
1.times.10.sup.6 cells/ml, from 7.times.10.sup.4 cells/ml to
7.times.10.sup.5, from 1.times.10.sup.5 cells/ml to
7.times.10.sup.5, from 3.times.10.sup.5 cells/ml to
7.times.10.sup.5, from 4.times.10.sup.5 cells/ml to
6.times.10.sup.5, or 5.times.10.sup.5).
[0086] In practicing the methods of the disclosure, ASCs can be
cultured in bioreactor culture for a period of time sufficient for
the formation of cellular aggregates (e.g., spheres, which can
resemble embryoid bodies). In some embodiments, ASCs are cultured
using bioreactor culture (i.e., ASCs are placed into a bioreactor
culture) for 3 days or less (e.g., 2.5 days or less, 2 days or
less, 1.5 days or less, 1 day or less, 12 hours or less, 8 hours or
less, 6 hours or less, or 4 hours or less). In some embodiments,
ASCs are cultured using bioreactor culture for a period of time in
a range of from 2 hours to 3 days (e.g., from 2 hours to 3 days,
from 2 hours to 2.5 days, from 2 hours to 2 days, from 2 hours to
1.5 days, from 2 hours to 1 day, from 6 hours to 3 days, from 6
hours to 2.5 days, from 6 hours to 2 days, from 6 hours to 1.5
days, from 6 hours to 1 day, from 6 hours to 12 hours, from 8 hours
to 3 days, from 8 hours to 2.5 days, from 8 hours to 2 days, from 8
hours to 1.5 days, from 8 hours to 1 day, from 8 hours to 12 hours,
from 12 hours to 2.5 days, from 12 hours to 2 days, from 12 hours
to 1.5 days, from 12 hours to 1 day, from 1 day to 3 days, from 1
day to 2.5 days, from 1 day to 2 days, from 1 day to 1.5 days, from
1.5 days to 3 days, from 1.5 days to 2.5 days, from 1.5 days to 2
days, from 2 days to 3 days, from 1 day, 1.5 days, from 2 days,
from 2.5 days, or 3 days).
[0087] Culturing cells in differentiation media. The term
"differentiation media" as used herein refers to media can be used
to induce cells to differentiate. For example, stage 1 and stage 2
media (described below) are two types of differentiation media used
to induce ASCs to differentiate into hepatocyte-like cells. In some
embodiments, the methods include removing the ASC-derived cellular
aggregate (e.g., sphere) from three dimensional culture (e.g.,
hanging drop suspension culture, high density culture, spinner
flask culture, bioreactor culture, stirred suspension culture,
microcarrier culture, etc.) and contacting cells of the ASC-derived
cellular aggregate (e.g., for 3 days or less) with a first culture
medium (e.g., stage 1 media) comprising Activin A and a fibroblast
growth factor (FGF) to produce a precursor cell population. In some
embodiments, cells of the precursor cell population are contacted
with a second culture medium (e.g., stage 2 media) comprising
hepatocyte growth factor (HGF) (e.g., for 7 days or less) to
produce an induced cell population that comprises induced
hepatocyte-like cells (iHeps). All proteins listed below that can
be included in a suitable differentiation media, can be provided
from any convenient source (e.g., purified from tissue,
recombinant, etc.).
[0088] In some embodiments, cells of an ASC-derived cellular
aggregate (e.g., sphere) are contacted with stage 1 media (as
defined below, e.g., a culture medium having Activin A and an FGF)
for 3 days or less (e.g., 2.5 days or less, 2 days or less, 1.5
days or less, 1 day or less, or 12 hours or less) to produce a
precursor cell population. In some embodiments, cells of an
ASC-derived cellular aggregate (e.g., sphere) are contacted with
stage 1 media for a period of time in a range of from 12 hours to 3
days (e.g., 12 hours to 2.5 days, 12 hours to 2 days, 12 hours to
1.5 days, 12 hours to 1 day, 1 day to 3 days, 1 day to 2.5 days, 1
day to 2 days, 1 day to 1.5 days, 1.5 days to 3 days, 1.5 days to
2.5 days, 1.5 days to 2 days, 2 days to 3 days, 1 day, 1.5 days, 2
days, 2.5 days, or 3 days) to produce a precursor cell
population.
[0089] In some embodiments, cells of the precursor cell population
are contacted with stage 2 media (as defined below, e.g., a culture
medium having hepatocyte growth factor (HGF)) for 8 days or less
(e.g., 7.5 days or less, 7 days or less, 6.5 days or less, 6 days
or less, 5.5 days or less, 5 days or less, 4.5 days or less, 4 days
or less, 3.5 days or less, 3 days or less, 2.5 days or less, 2 days
or less, 1.5 days or less, 1 day or less, or 12 hours or less) to
produce an induced cell population that comprises induced
hepatocyte-like cells. In some embodiments, cells of the precursor
cell population are contacted with stage 2 media for a period of
time in a range of from 12 hours to 8 days (e.g., 12 hours to 8
days, 1 day to 8 days, 2 days to 8 days, 3 days to 8 days, 4 days
to 8 days, 5 days to 8 days, 6 days to 8 days, 2 days to 7 days, 3
days to 7 days, 4 days to 7 days, 5 days to 7 days, 6 days to 7
days, 5.5 days to 8 days, 5.5 days to 7.5 days, 5.5 days to 6.5
days, 6 days to 7.5 days, 6.5 days to 7.5 days, 1 day, 2 days, 3
days, or 3 days) to produce an induced cell population that
comprises induced hepatocyte-like cells.
[0090] In some embodiments, the total time elapsed from (i) placing
a population of ASCs into a three dimensional culture, to (ii)
producing an induced cell population that comprises induced
hepatocyte-like cells (iHeps), is less than 13 days (e.g., less
than 12.5 days, less than 12 days, less than 11.5 days, less than
11 days, less than 10.5 days, less than 10 days, less than 9.5
days, less than 9 days, less than 8.5 days, less than 8 days, less
than 7.5 days, less than 7 days, less than 6.5 days, less than 6
days, less than 5.5 days, less than 5 days, less than 4.5 days, or
less than 4 days).
[0091] In some embodiments, the total time elapsed from (i) placing
a population of ASCs into a three dimensional culture, to (ii)
producing an induced cell population that comprises induced
hepatocyte-like cells (iHeps), is 13 days or less (e.g., 12.5 days
or less, 12 days or less, 11.5 days or less, 11 days or less, 10.5
days or less, 10 days or less, 9.5 days or less, 9 days or less,
8.5 days or less, 8 days or less, 7.5 days or less, 7 days or less,
6.5 days or less, 6 days or less, 5.5 days or less, 5 days or less,
4.5 days or less, or 4 days or less).
[0092] In some embodiments, the total time elapsed from (i) placing
a population of ASCs into a three dimensional culture, to (ii)
producing an induced cell population that comprises induced
hepatocyte-like cells (iHeps), is in a range of from 12 hours to 13
days (e.g., from 1 day to 13 days, from 2 days to 13 days, from 3
days to 13 days, from 4 days to 13 days, from 4.5 days to 13 days,
from 5 days to 13 days, from 5.5 days to 13 days, from 6 days to 13
days, from 6.5 days to 13 days, from 7 days to 13 days, from 7.5
days to 13 days, from 8 days to 13 days, from 8.5 days to 13 days,
from 9 days to 13 days, from 9.5 days to 13 days, from 10 days to
13 days, from 10.5 days to 13 days, from 11 days to 13 days, from
11.5 days to 13 days, from 12 hours to 12.5 days, from 1 day to
12.5 days, from 2 days to 12.5 days, from 3 days to 12.5 days, from
4 days to 12.5 days, from 4.5 days to 12.5 days, from 5 days to
12.5 days, from 5.5 days to 12.5 days, from 6 days to 12.5 days,
from 6.5 days to 12.5 days, from 7 days to 12.5 days, from 7.5 days
to 12.5 days, from 8 days to 12.5 days, from 8.5 days to 12.5 days,
from 9 days to 12.5 days, from 9.5 days to 12.5 days, from 10 days
to 12.5 days, from 10.5 days to 12.5 days, from 11 days to 12.5
days, from 11.5 days to 12.5 days, from 12 hours to 12 days, from 1
day to 12 days, from 2 days to 12 days, from 3 days to 12 days,
from 4 days to 12 days, from 4.5 days to 12 days, from 5 days to 12
days, from 5.5 days to 12 days, from 6 days to 12 days, from 6.5
days to 12 days, from 7 days to 12 days, from 7.5 days to 12 days,
from 8 days to 12 days, from 8.5 days to 12 days, from 9 days to 12
days, from 9.5 days to 12 days, from 10 days to 12 days, from 10.5
days to 12 days, from 11 days to 12 days, from 3 days to 11 days,
from 3 days to 10 days, from 3 days to 9.5 days, from 3 days to 9
days, from 6 days 10 days, or from 4 days to 10 days).
[0093] Basal cell culture media. Subject cells (e.g., ASCs, iHeps,
differentiating ASCs, etc.) are cultured in an appropriate liquid
nutrient medium, referred to herein as "basal cell culture media.".
The stage 1 and stage 2 media are basal culture media supplemented
with at least one additional component, as described below. Various
basal media formulations are available (e.g., Dulbecco's Modified
Eagle Medium (DMEM), RPMI, Iscove's medium, hepatocyte culture
medium (HCM) (Lonza, Cat: cc-3198) etc.).
[0094] Any convenient culture media can serve as a basal culture
medium. For example, a suitable tissue culture medium can contain
components such as a vitamin; an amino acid (e.g., an essential
amino acid, a non-essential amino acid, etc.); a pH buffering
agent; a salt; an antimicrobial agent (e.g., an antibacterial
agent, an antimycotic agent, etc.); serum (e.g., fetal bovine
serum, human serum, calf serum, horse serum, goat serum etc.); an
energy source (e.g., a sugar); a nucleoside; a lipid; trace metals;
a cytokine; a growth factor; a stimulatory factor; additives (e.g.,
pyruvate (0.1-5 mM), glutamine (0.5-5 mM), etc.) and the like. Any
convenient cell culture media can be used, and as is known in the
art, various cell types grow better in particular media
preparations. For example, in some cases, particular media
formulations have been optimized to culture specific types of cells
(e.g., ASCs, hepatocytes (hepatocyte culture medium (HCM) (Lonza,
Cat: cc-3198)), neural progenitors, embryonic stem cells, etc.).
Accordingly, any convenient cell culture media can be used as a
basal culture media and may be tailored to the culture of ASCs
and/or their differentiating progeny.
[0095] One exemplary, non-limiting, suitable basal medium is RPMI
(Roswell Park Memorial Institute) 1640, which is well known in the
art as a basal medium that allows the culture of mammalian cells
with serum (Fetal Bovine Serum (FBS)) supplementation. Another
suitable basal culture media is Advanced RPMI 1640, which is
similar to classic RPMI 1640, but allows for serum supplementation
to be reduced by 50-90% with no change in growth rate or
morphology. Advanced RPMI 1640 can contain, for example, glucose,
non-essential amino acids, sodium pyruvate, and phenol red. The
complete formulation is readily available (e.g., online) to one of
ordinary skill in the art for RPMI 1640, for Advanced RPMI 1640,
and for many suitable commercially available basal culture media.
Another suitable basal culture media is CLONETICS.TM. hepatocyte
culture medium (HCM.TM.) (available, for example, from
Lonza--Catalog number 3198).
[0096] Stage 1 medium. "Stage 1 medium" is also referred to herein
as a "first culture medium." A suitable stage 1 medium is a basal
culture medium (e.g., advanced RPMI 1640, RPMI 1640, etc.) suitable
for the culture of cells (e.g., ASCs, differentiating ASCs, etc.)
supplemented with at least activin A (e.g., 100 ng/ml) and a
fibroblast growth factor (e.g., FGF4, e.g., 20 ng/ml). In some
cases, stage 1 medium includes a Wnt signaling agonist (e.g.,
Wnt3a, e.g., 50 ng/ml). In some cases, stage 1 medium includes a
supplement such as B27 (e.g., 20%), which is known in the art. In
some cases, a stage 1 medium includes a Wnt signaling agonist and a
supplement (e.g., B27). In some cases, the basal culture media used
for stage 1 media is advanced RPMI 1640 and it is supplemented with
activin A (e.g., 100 ng/ml), FGF4 (e.g., 20 ng/ml), Wnt3a (e.g., 50
ng/ml), and 20% B27. In general, Stage 1 medium drives ASC
differentiation toward the endoderm lineage.
[0097] Activins are dimeric proteins consisting of .beta. subunits,
which are connected by disulfide bonds. There are three different
forms of activin: (i) homodimeric activin A (2 .beta..sub.A
subunits); (ii) homodimeric activin B (2 .beta..sub.B subunits);
and (iii) heterodimeric activin AB (1 .beta..sub.A and 1
.beta..sub.B subunit). The term "Activin A" as used herein refers
to a homodimer of .beta..sub.A subunits. A .beta..sub.A subunit is
the polypeptide also referred to as "Inhibin beta A chain" or
"INHBA." The amino acid sequence of Inhibin beta A chain is:
TABLE-US-00001 (SEQ ID NO: 1)
MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALAALPKDVPNS
QPEMVEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGY
VEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVV
ERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGE
RSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQES
GASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQS
EDHPHRRRRRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYC
EGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMS
MLYYDDGQNIIKKDIQNMIVEECGCS
In some embodiments, a subject stage 1 medium comprises Activin A
at a concentration in a range of from about 50 ng/ml to about 150
ng/ml (e.g., from about 60 ng/ml to about 140 ng/ml, from about 70
ng/ml to about 130 ng/ml, from about 80 ng/ml to about 120 ng/ml,
from about 85 ng/ml to about 115 ng/ml, from about 90 ng/ml to
about 110 ng/ml, from about 95 ng/ml to about 105 ng/ml, from about
97.5 ng/ml to about 102.5 ng/ml, or about 100 ng/ml).
[0098] Fibroblast growth factors (FGFs) are a well described family
of proteins (related by sequence, structure, and function), with 18
mammalian members. The FGFs are grouped into 6 subfamilies based on
differences in sequence homology and phylogeny: FGFs 1 and 2; FGFs
3, 7, 10, and 22; FGFs 4-6; FGFs 8, 17, and 18; FGFs 9, 16 and 20;
and FGFs 19, 21 and 23. In some cases, a suitable FGF is FGF 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 16, 17, 18, 20, and/or 22. In some cases,
a suitable FGF is FGF4, FGF5, and/or FGF6. In some cases, a
suitable FGF is FGF4.
The amino acid sequences of exemplary human FGFs are:
TABLE-US-00002 FGF1: (SEQ ID NO: 2)
MAEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQ
LSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEK
NWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD FGF2: (SEQ ID NO: 3)
MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAA
GSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAA
PAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDG
RVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERL
ESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS FGF3: (SEQ ID NO:
4) MGLIWLLLLSLLEPGWPAAGPGARLRRDAGGRGGVYEHLGGAPRRRKLYCATKYHLQLHP
SGRVNGSLENSAYSILEITAVEVGIVAIRGLFSGRYLAMNKRGRLYASEHYSAECEFVER
IHELGYNTYASRLYRTVSSTPGARRQPSAERLWYVSVNGKGRPRRGFKTRRTQKSSLFLP
RVLDHRDHEMVRQLQSGLPRPPGKGVQPRRRRQKQSPDNLEPSHVQASRLGSQLEASAH FGF4:
(SEQ ID NO: 5)
MSGPGTAAVALLPAVLLALLAPWAGRGGAAAPTAPNGTLEAELERRWESLVALSLARLPV
AAQPKEAAVQSGAGDYLLGIKRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSLLELSP
VERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGMFIA
LSKNGKTKKGNRVSPTMKVTHFLPRL FGF5: (SEQ ID NO: 6)
MSLSFLLLLFFSHLILSAWAHGEKRLAPKGQPGPAATDRNPRGSSSRQSSSSAMSSSSAS
SSPAASLGSQGSGLEQSSFQWSPSGRRTGSLYCRVGIGFHLQIYPDGKVNGSHEANMLSV
LEIFAVSQGIVGIRGVFSNKFLAMSKKGKLHASAKFTDDCKFRERFQENSYNTYASAIHR
TEKTGREWYVALNKRGKAKRGCSPRVKPQHISTHFLPRFKQSEQPELSFTVTVPEKKKPP
SPIKPKIPLSAPRKNTNSVKYRLKFRFG FGF6: (SEQ ID NO: 7)
MALGQKLFITMSRGAGRLQGTLWALVFLGILVGMVVPSPAGTRANNTLLDSRGWGTLLSR
SRAGLAGEIAGVNWESGYLVGIKRQRRLYCNVGIGFHLQVLPDGRISGTHEENPYSLLEI
STVERGVVSLFGVRSALFVAMNSKGRLYATPSFQEECKFRETLLPNNYNAYESDLYQGTY
IALSKYGRVKRGSKVSPIMTVTHFLPRI FGF7: (SEQ ID NO: 8)
MHKWILTWILPTLLYRSCFHIICLVGTISLACNDMTPEQMATNVNCSSPERHTRSYDYME
GGDIRVRRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGIVAIKGVESEFYLA
MNKEGKLYAKKECNEDCNFKELILENHYNTYASAKWTHNGGEMFVALNQKGIPVRGKKTK
KEQKTAHFLPMAIT FGF8: (SEQ ID NO: 9)
MGSPRSALSCLLLHLLVLCLQAQEGPGRGPALGRELASLFRAGREPQGVSQQHVREQSLV
TDQLSRRLIRTYQLYSRTSGKHVQVLANKRINAMAEDGDPFAKLIVETDTFGSRVRVRGA
ETGLYICMNKKGKLIAKSNGKGKDCVFTEIVLENNYTALQNAKYEGWYMAFTRKGRPRKG
SKTRQHQREVHFMKRLPRGHHTTEQSLRFEFLNYPPFTRSLRGSQRTWAPEPR FGF9: (SEQ ID
NO: 10)
MAPLGEVGNYFGVQDAVPFGNVPVLPVDSPVLLSDHLGQSEAGGLPRGPAVTDLDHLKGI
LRRRQLYCRTGFHLEIFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGVDSGLYLGMNE
KGELYGSEKLTQECVFREQFEENWYNTYSSNLYKHVDTGRRYYVALNKDGTPREGTRTKR
HQKFTHFLPRPVDPDKVPELYKDILSQS FGF10: (SEQ ID NO: 11)
MWKWILTHCASAFPHLPGCCCCCFLLLFLVSSVPVTCQALGQDMVSPEATNSSSSSFSSP
SSAGRHVRSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGV
VAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVAL
NGKGAPRRGQKTRRKNTSAHFLPMVVHS FGF16: (SEQ ID NO: 12)
MAEVGGVFASLDWDLHGFSSSLGNVPLADSPGFLNERLGQIEGKLQRGSPTDFAHLKGIL
RRRQLYCRTGFHLEIFPNGTVHGTRHDHSRFGILEFISLAVGLISIRGVDSGLYLGMNER
GELYGSKKLTRECVFREQFEENWYNTYASTLYKHSDSERQYYVALNKDGSPREGYRTKRH
QKFTHFLPRPVDPSKLPSMSRDLFHYR FGF17: (SEQ ID NO: 13)
MGAARLLPNLTLCLQLLILCCQTQGENHPSPNFNQYVRDQGAMTDQLSRRQIREYQLYSR
TSGKHVQVTGRRISATAEDGNKFAKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKP
SGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQ
GQLPFPNHAEKQKQFEFVGSAPTRRTKRTRRPQPLT FGF18: (SEQ ID NO: 14)
MYSAPSACTCLCLHFLLLCFQVQVLVAEENVDFRIHVENQTRARDDVSRKQLRLYQLYSR
TSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKP
DGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPK
GQPELQKPFKYTTVTKRSRRIRPTHPA FGF20: (SEQ ID NO: 15)
MAPLAEVGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQLAHL
HGILRRRQLYCRTGFHLQILPDGSVQGTRQDHSLFGILEFISVAVGLVSIRGVDSGLYLG
MNDKGELYGSEKLTSECIFREQFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTPRDGAR
SKRHQKFTHFLPRPVDPERVPELYKDLLMYT FGF22: (SEQ ID NO: 16)
MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDVRWRRLFSSTHFFLRVDPGGR
VQGTRWRHGQDSILEIRSVHVGVVVIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFRERIE
ENGHNTYASQRWRRRGQPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS
In some embodiments, a subject stage 1 medium comprises an FGF
(e.g., FGF4, FGF5, FGF6, etc.) at a concentration in a range of
from 5 ng/ml to 50 ng/ml (e.g., from 5 ng/ml to 40 ng/ml, from 10
ng/ml to 40 ng/ml, from 10 ng/ml to 30 ng/ml, from 15 ng/ml to 25
ng/ml, from 17.5 ng/ml to 22.5 ng/ml, 15 ng/ml, 17.5 ng/ml, 20
ng/ml, 22.5 ng/ml, 25 ng/ml or 30 ng/ml).
[0099] Wnt signaling agonist--In some embodiments, a suitable
"Stage 1 medium" includes a Wnt signaling agonist. A Wnt signaling
agonist triggers the nuclear localization of .beta.-Catenin and
results in the activation of canonical Wnt signaling. There are two
main branches of the Wnt signaling pathway: (1) the canonical
.beta.-Catenin dependent Wnt signaling pathway and (2) the
non-canonical .beta.-Catenin independent pathways, which include
planar cell polarity (PCP) signaling as well as Calcium signaling
(Gao, et. al, Cell Signal. 2010 May; 22(5):717-27. Epub 2009 Dec.
13). As used herein, the terms "Wnt signaling" and
"Wnt/.beta.-Catenin signaling" are used interchangeably to refer to
the canonical .beta.-Catenin dependent Wnt signaling pathway. As
such, a "Wnt signaling agonist" increases output from the
.beta.-Catenin dependent Wnt signaling pathway. Activation of the
Wnt pathway culminates when the protein .beta.-Catenin enters the
cell nucleus (for recent review of the canonical .beta.-Catenin
dependent Wnt signaling pathway see Clevers et. al., Cell. 2012
Jun. 8; 149(6):1192-205: Wnt/.beta.-catenin signaling and disease).
However, in the absence of Wnt signaling, free cytosolic
.beta.-Catenin is incorporated into a complex, known in the art as
the .beta.-Catenin destruction complex, which includes the proteins
Axin, Adenomatous Polyposis Coli (APC), and glycogen synthase
kinase (GSK-3.beta.). Phosphorylation of .beta.-Catenin by
GSK-3.beta. designates .beta.-Catenin for the ubiquitin pathway and
degradation by proteasomes (via .beta.TRCP).
[0100] The binding of a canonical Wnt to its receptor (a Frizzled
protein) leads to the activation of Dishevelled (Dvl) proteins,
which inhibit glycogen synthase kinase-3.beta. (GSK-3.beta.)
activity (i.e., phosphorylation of .beta.-Catenin), leading to the
cytosolic stabilization of .beta.-Catenin. Stabilized
.beta.-Catenin then enters the nucleus and associates with the
TCF/LEF (T Cell-specific transcription Factor/Lymphoid Enhancer
Factor) family of transcription factors to induce transcription of
important downstream target genes. Thus, in the absence of Wnt
signaling, cytosolic (and therefore nuclear) levels of
.beta.-Catenin are kept low by negative regulatory components of
the pathway while in the presence of Wnt signaling, cytosolic (and
therefore nuclear) levels of .beta.-Catenin are stabilized by
positive regulatory components of the pathway.
[0101] By "negative regulatory components" of the Wnt pathway, it
is meant proteins that function by antagonizing the Wnt pathway,
thus resulting in decreased pathway output (i.e., decreased target
gene expression). Examples of known negative regulatory components
of the Wnt pathway include, but are in no way limited to: WIF,
sFRP, Dkk, APCDD1, Notum, SOST, Axin, APC, GSK-3.beta., CK1.gamma.,
WTX, and .beta.TrCP.
[0102] By "positive regulatory components" of the Wnt pathway, it
is meant proteins that function by enhancing the Wnt pathway, thus
resulting in increased pathway output (i.e., increased target gene
expression). Examples of known positive regulatory components of
the Wnt pathway include, but are in no way limited to: Wnt (e.g.,
Wnt1, Wnt3, Wnt3a, Wnt7a, and/or Wnt8), Norrin, R-spondin, PORCN,
Wls, Frizzled, LRP5 and LRP6, Tspan12, Lgr4, Lgr5, Lgr6, Dvl,
.beta.-Catenin, and TCF/LEF. In some embodiments, a subject Wnt
signaling agonist is a positive regulatory component of the
canonical Wnt signaling pathway (e.g., Wnt1, Wnt3, Wnt3a, Wnt7a,
Wnt8, etc.).
[0103] In some embodiments, a subject Wnt signaling agonist is
"specific for" or "specifically binds to" a component of the Wnt
signaling pathway such that binding results in increased pathway
output (i.e., transcription of target genes). The binding of an
agonist can be mediated by covalent or non-covalent interactions or
a combination of covalent and non-covalent interactions. When the
interaction of the agonist with the component to which it
specifically binds produces a non-covalently bound complex, the
binding which occurs is typically electrostatic, hydrogen-bonding,
or the result of lipophilic interactions. In particular, specific
binding is characterized by the preferential binding of one member
of a pair to a particular species relative to other species within
the family of compounds to which the corresponding member of the
binding member belongs. Thus, for example, a Wnt signaling agonist
that is specific for the negative regulatory component GSK-3
preferably binds to GSK-3 relative to other proteins in the
cell.
[0104] A subject Wnt signaling agonist is any molecule (e.g., a
chemical compound; a non-coding nucleic acid, e.g., a non-coding
RNA; a polypeptide; a nucleic acid encoding a polypeptide, etc.)
that results in increased output (i.e., increased target gene
expression) from the Wnt signaling pathway. For example, a Wnt
signaling agonist can function by stabilizing, enhancing the
expression of, or enhancing the function of a positive regulatory
component of the pathway or by destabilizing, decreasing the
expression of, or inhibiting the function of a negative regulatory
component of the pathway. Thus, a Wnt signaling agonist can be a
polypeptide positive regulatory component (e.g., Wnt3, wnt3a, Wnt1,
and the like), and/or a nucleic acid encoding one or more positive
regulatory components of the pathway. A Wnt signaling agonist can
also be a small molecule or nucleic acid that stabilizes a positive
regulatory component of the pathway either at the level of mRNA or
protein.
[0105] In some embodiments, a Wnt signaling agonist functions by
stabilizing .beta.-Catenin, thus allowing nuclear levels of
.beta.-Catenin to rise. .beta.-Catenin can be stabilized in
multiple different ways. As multiple different negative regulatory
components of the Wnt signaling pathway function by facilitating
the degradation of .beta.-Catenin, a subject Wnt signaling agonist
can be a small molecule or nucleic acid inhibitor (e.g., microRNA,
shRNA, etc.)(functioning at the level of mRNA or protein) of a
negative regulatory component of the pathway. For example, in some
embodiments, the Wnt signaling agonist is an inhibitor of GSK-3p.
In some such embodiments, the inhibitor of GSK-3p is a small
molecule chemical compound (e.g., TWS119, BIO, CHIR-99021, SB
216763, SB 415286, CHIR-98014 and the like). [0106] TWS119:
3-(6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy)phenol
(Ding et. al, Proc Natl Acad Sci USA. 2003 Jun. 24; 100(13):7632-7.
Epub 2003 Jun. 6) [0107] BIO:
6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3-dihy-
dro-(3Z)-2H-indol-2-one or (2'Z,3'E)-6-Bromoindirubin-3'-oxime
(Meijer et. al, Chem Biol. 2003 December; 10(12):1255-66) [0108]
CHIR-99021:
6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidiny-
l]amino]ethyl]amino]-3-pyridinecarbonitrile (Bennett et al., J Biol
Chem. 2002 Aug. 23; 277(34):30998-1004. Epub 2002 Jun. 7) [0109] SB
216763:
3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione
(Cross et al., J Neurochem. 2001 April; 77(1):94-102) [0110] SB
415286:
3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione
(Cross et al., J Neurochem. 2001 April; 77(1):94-102) [0111]
CHIR-98014:
N2-(2-(4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-ylamino)eth-
yl)-5 nitropyridine-2,6-diamine [0112] (Ring et al., Diabetes. 2003
March; 52(3):588-95)
[0113] Any convenient Wnt agonist may be used. In some embodiments,
the Wnt agonist is Wnt3a. The amino acid sequence of human Wnt3a
is:
TABLE-US-00003 (SEQ ID NO: 17)
MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVP
KQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGP
VLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGW
KWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQAIASHMH
LKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESR
GWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVS
SHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHT CK.
In some cases, a subject stage 1 medium comprises Wnt3a at a
concentration in a range of from about 20 ng/ml to about 80 ng/ml
(e.g., from about 20 ng/ml to about 80 ng/ml, from about 25 ng/ml
to about 75 ng/ml, from about 30 ng/ml to about 70 ng/ml, from
about 35 ng/ml to about 65 ng/ml, from about 40 ng/ml to about 60
ng/ml, from about 45 ng/ml to about 55 ng/ml, from about 47.5 ng/ml
to about 52.5 ng/ml, or about 50 ng/ml).
[0114] Stage 2 medium. "Stage 2 medium" is also referred to herein
as a "second culture medium." A suitable stage 2 medium is a basal
culture medium (e.g., hepatocyte culture medium (HCM.TM.), advanced
RPMI 1640, RPMI 1640, etc.) suitable for the culture of cells
(e.g., differentiating ASCs, hepatocytes, etc.) supplemented with
at least hepatocyte growth factor (HGF). In some cases, stage 2
medium includes a fibroblast growth factor (e.g., FGF4, e.g., 25
ng/ml).
[0115] In some embodiments, a stage 2 media further includes a
component selected from: an FGF (e.g., FGF4, e.g., 25 ng/ml),
oncostatinM (OSM, e.g., 30 ng/ml), dexamethasone (Dex, e.g.,
2.times.10.sup.-5 M), dimethyl sulfoxide (DMSO, e.g., 0.1%), and a
combination thereof. In some cases, the basal culture media used
for stage 2 media is CLONETICS.TM. (available, for example, from
Lonza--Catalog number 3198) and it is supplemented with HGF (e.g.,
150 ng/ml), an FGF (e.g., FGF4, e.g., 25 ng/ml), oncostatinM (e.g.,
30 ng/ml), dexamethasone (e.g., 2.times.10.sup.-5 M), and DMSO
(e.g., 0.1%).
[0116] Hepatocyte growth factor (HGF) is a protein that functions
as a potent mitogen, hepatotrophic factor, and/or a growth factor.
The amino acid sequence of human HGF is:
TABLE-US-00004 (SEQ ID NO: 18)
MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTL
IKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFP
FNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQ
PWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEV
CDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERY
PDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPL
ETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLR
ENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYM
GNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGP
WCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIP
TRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGI
HDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP
NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKV
TLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIP
NRPGIFVRVAYYAKWIHKIILTYKVPQS.
In some embodiments, a subject stage 2 medium includes HGF at a
concentration in a range of from 75 ng/ml to 250 ng/ml (e.g., from
100 ng/ml to 200 ng/ml, from 120 ng/ml to 180 ng/ml, from 125 ng/ml
to 175 ng/ml, from 130 ng/ml to 170 ng/ml, from 135 ng/ml to 165
ng/ml, from 140 ng/ml to 160 ng/ml, from 145 ng/ml to 155 ng/ml,
120 ng/ml, 125 ng/ml, 130 ng/ml, 135 ng/ml, 140 ng/ml, 145 ng/ml,
150 ng/ml, 155 ng/ml, 160 ng/ml or 175 ng/ml).
[0117] In some embodiments, a subject stage 2 medium includes an
FGF (e.g., FGF4) at a concentration in a range of from 5 ng/ml to
55 ng/ml (e.g., from 5 ng/ml to 45 ng/ml, from 10 ng/ml to 45
ng/ml, from 10 ng/ml to 35 ng/ml, from 15 ng/ml to 35 ng/ml, from
20 ng/ml to 30 ng/ml, from 22.5 ng/ml to 27.5 ng/ml, 15 ng/ml, 17.5
ng/ml, 20 ng/ml, 22.5 ng/ml, 25 ng/ml, 27.5 ng/ml, or 30
ng/ml).
[0118] Oncostatin M (OSM) is a polypeptide that functions as a
cytokine and belongs to the interleukin 6 group of cytokines. The
amino acid sequence of human OSM is:
TABLE-US-00005 (SEQ ID NO: 19)
MGVLLTQRTLLSLVLALLFPSMASMAAIGSCSKEYRVLLGQLQKQTDLMQ
DTSRLLDPYIRIQGLDVPKLREHCRERPGAFPSEETLRGLGRRGFLQTLN
ATLGCVLHRLADLEQRLPKAQDLERSGLNIEDLEKLQMARPNILGLRNNI
YCMAQLLDNSDTAEPTKAGRGASQPPTPTPASDAFQRKLEGCRFLHGYHR
FMHSVGRVFSKWGESPNRSRRHSPHQALRKGVRRTRPSRKGKRLMTRGQL PR
In some embodiments, a subject stage 2 medium includes oncostatinM
(OSM) at a concentration in a range of from 10 ng/ml to 50 ng/ml
(e.g., from 15 ng/ml to 45 ng/ml, from 20 ng/ml to 40 ng/ml, from
22.5 ng/ml to 37.5 ng/ml, from 25 ng/ml to 35 ng/ml, from 27.5
ng/ml to 32.5 ng/ml, 25 ng/ml, 27.5 ng/ml, 30 ng/ml, 32.5 ng/ml, or
35 ng/ml).
[0119] Dexamethasone (Dex) is a synthetic member of the
glucocorticoid class of steroid drugs that has anti-inflammatory
and immunosuppressant properties. Dex is also known as
(11.beta.,16.alpha.)-9-Fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-die-
ne-3,20-dione,
9.alpha.-Fluoro-16.alpha.-methyl-11.beta.,17.alpha.,21-trihydroxy-1,4-pre-
gnadiene-3,20-dione, 9.alpha.-Fluoro-16.alpha.-methylprednisolone,
and Prednisolone F. Dex has the CAS number 50-02-2 and the chemical
structure:
##STR00001##
In some embodiments, a subject stage 2 medium comprises
dexamethasone (Dex) at a concentration in a range of from
1.times.10.sup.-6 M to 1.times.10.sup.-4 M (e.g., from
5.times.10.sup.-6 to 5.times.10.sup.-5 M, from 1.times.10.sup.-5 M
to 3.times.10.sup.-5 M, from 1.5.times.10.sup.-5 M to
2.5.times.10.sup.-5 M, or 2.times.10.sup.-5 M).
[0120] Dimethylsulfoxide (DMSO) is an organosulfur compound with
the formula (CH.sub.3).sub.2SO that is a colorless liquid and is a
polar aprotic solvent that dissolves both polar and nonpolar
compounds. DMSO is also known as Methyl sulfoxide, has the CAS
number 67-68-5, and has the chemical structure:
##STR00002##
In some embodiments, a subject stage 2 medium comprises DMSO at a
concentration in a range of from 0.01% to 1% (e.g., from 0.025% to
0.5%, from 0.05% to 0.2%, or 0.1%).
[0121] Hepatocyte-like cells. The term "hepatocyte-like cell" as
used herein refers to a cell (e.g., of a cell population, e.g., a
cell that is induced according to the subject methods) that is
positive for (i.e., exhibits) one or more hepatocyte characters.
Hepatocyte characters (i.e., characteristics of hepatocyte-like
cells) include, but are not limited to:
(i) expression of (i.e., positive for) one or more hepatocyte
markers (e.g., glucose-6-phosphatase, albumin (ALB),
alpha-1-antitrypsin (AAT, also known as SERPINA1), cytokeratin 8
(CK8), cytokeratin 18 (CK18), cytokeratin 8/18 (CK8/18),
asialoglycoprotein receptor 1 (ASGR1), alcohol dehydrogenase 1,
arginase Type I, cytochrome p450 3A4 (CYP3A4), liver-specific
organic anion transporter (LST-1), forkhead box protein A2 (FoxA2),
alpha-fetoprotein (AFP), tryptophan 2,3-dioxygenase (TDO2), or a
combination thereof); (ii) activity of liver enzymes such as
glucose-6-phosphatase, CYP3A4, and/or CYP1A1; (iii) production
and/or secretion of liver products (e.g., as measured in bodily
fluids such as blood, serum, plasma, etc.)(e.g., bile, urea, and/or
albumin); (iv) exhibition of a hepatocyte metabolic properties
(e.g., ability to detoxify xenobiotics; endocytosis of LDL,
synthesis of glycogen, cytochrome P450 1A2 detoxification activity,
and the like); (v) exhibition of hepatocyte morphological features;
(vi) ability to engraft into the liver of an immunodeficient
individual (e.g., a mouse, a human, etc.); and (vii) lack of
expression of (negative for) one or more non-hepatocyte markers
(e.g., adipocyte markers, e.g., CD37, CD29, etc.; ASC markers,
e.g., CD105; and the like).
[0122] A subject hepatocyte-like cell does not have to be positive
for all tested hepatocyte characters in order for the cell to be
considered a hepatocyte-like cell. For example, in some cases, a
subject hepatocyte-like cell is positive for all tested hepatocyte
characters. However, in some cases, a subject hepatocyte-like cell
is positive for one or more hepatocyte character; and is also
negative for one or more hepatocyte characters. In some cases
(described below in more detail), a hepatocyte-like cell is
transplanted into an individual. In such cases, the hepatocyte-like
cell can be (i) positive for all tested hepatocyte characters; or
(ii) positive for one or more hepatocyte character(s); and negative
for one or more hepatocyte character(s).
[0123] Thus, in some cases, hepatocyte-like cells do not express
all known hepatocyte markers and/or test positive for all
hepatocyte functional tests. For example, in some cases,
hepatocyte-like cells that are induced according to the subject
methods do not express ASGR1. However, as demonstrated in the
examples section below (e.g., FIG. 3C), in some cases,
hepatocyte-like cells exhibit a hepatocyte character (e.g., express
a hepatocyte marker such as ASGR1) after transplantation (e.g.,
into an individual such as a mouse and/or a human) that they did
not exhibit prior to transplantation. For example, in some cases,
hepatocyte-like cells that are induced according to the subject
methods do not express the hepatocyte marker ASGR1 prior to
transplantation into an individual, but do express ASGR1 after
transplantation. Thus, in some cases, it is not required that
hepatocyte-like cells induced according to the subject methods
exhibit all known hepatocyte characters (e.g., production of a
liver product, expression of a hepatocyte marker, etc.) or even
exhibit all tested hepatocyte characters prior to the use of the
cells for transplantation. For example, in some cases,
hepatocyte-like cells that are induced according to the subject
methods are positive for one or more hepatocyte characters, and are
negative for one or more hepatocyte characters, but are still
considered hepatocyte-like cells and can still be transplanted into
an individual.
[0124] In some embodiments, the subject methods include the step of
verifying the presence of induced hepatocyte-like cells (iHeps) in
the induced cell population. Verifying can rely on cellular
phenotypes (e.g., gene or protein expression, drug metabolism
profile, responsiveness to particular drugs, etc.) known in the art
to be characteristic of hepatocytes. For example, verifying can be
performed by testing for any one or more of the hepatocyte
characters listed above (e.g., expression of a hepatocyte marker,
lack of a expression of a non-hepatocyte marker, production and/or
secretion of a liver product, activity of a liver enzyme,
exhibition of ta hepatocyte metabolic property, etc.). In some
cases, verifying comprises determining the percentage of cells of
the induced cell population that are iHeps.
[0125] In some embodiments, verifying includes contacting cells of
the induced cell population with a specific binding agent (e.g., a
nucleic acid probe, an antibody, etc.) specific for a hepatocyte
marker (e.g., mRNA, protein, etc.), and determining the percentage
of cells positive for expression, wherein cells positive for
expression are iHeps. Suitable markers are listed above. In some
embodiments, verifying includes contacting the induced cell
population with a binding agent (e.g., a nucleic acid probe, an
antibody, etc.) specific for a non-hepatocyte marker (e.g., mRNA,
protein, etc.), and determining the percentage of cells negative
for expression, wherein cells negative for expression are iHeps.
Suitable markers are listed above. In some cases, 10% or more of
the cells of the induced cell population are determined to be iHeps
(e.g., 10.5% or more, 11% or more, 12.5% or more, 15% or more,
17.5% or more, 20% or more, 22.5% or more, 25% or more, 27.5% or
more, 30% or more, 32.5% or more, 35% or more, 37% or more, 40% or
more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or
more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or
more, 95% or more, 98% or more, 99% or more, or 100%). In some
embodiments, the percent of cells of the induced cell population
that are determined to be iHeps is in a range of from 10% to 100%
(e.g., from 10% to 90%, from 10% to 80%, from 10% to 70%, from 10%
to 60%, from 10% to 50%, from 10% to 45%, from 10% to 100%, from
40% to 100%, from 10% to 37.5%, from 10% to 37%, from 15% to 90%,
from 15% to 80%, from 15% to 70%, from 15% to 60%, from 15% to 50%,
from 15% to 45%, from 15% to 100%, from 40% to 100%, from 15% to
37.5%, from 15% to 37%, from 20% to 90%, from 20% to 80%, from 20%
to 70%, from 20% to 60%, from 20% to 50%, from 20% to 45%, from 20%
to 100%, from 40% to 100%, from 20% to 37.5%, from 20% to 37%, from
25% to 90%, from 25% to 80%, from 25% to 70%, from 25% to 60%, from
25% to 50%, from 25% to 45%, from 25% to 100%, from 40% to 100%,
from 25% to 37.5%, from 25% to 37%, from 30% to 90%, from 30% to
80%, from 30% to 70%, from 30% to 60%, from 30% to 50%, from 30% to
45%, from 30% to 100%, from 40% to 100%, from 30% to 37.5%, or from
30% to 37%).
[0126] It will be understood by those of skill in the art that
expression levels reflect detectable amounts of the marker (e.g.,
nucleic acid or protein) on and/or in the cell. A cell that is
negative for staining (e.g., the level of binding of a marker
specific reagent is not detectably different from a matched
control) may still express minor amounts of the marker. And while
it is commonplace in the art to refer to cells as "positive" or
"negative" for a particular marker, actual expression levels are
quantitative traits. The number of detected molecules can vary by
several logs, yet still be characterized as "positive".
[0127] When a protein marker is used, the staining intensity (e.g.,
of a marker-specific antibody) can be monitored by flow cytometry,
where lasers detect the quantitative levels of fluorochrome (which
is proportional to the amount of cell marker bound by specific
reagents, e.g. antibodies). Flow cytometry, or FACS, can also be
used to separate cell populations based on the intensity of binding
to a specific reagent, as well as other parameters such as cell
size and light scatter. Although the absolute level of staining may
differ with a particular fluorochrome and reagent preparation, the
data can be normalized to a control.
[0128] In order to normalize the distribution to a control, each
cell is recorded as a data point having a particular intensity of
staining. These data points may be displayed according to a log
scale, where the unit of measure is arbitrary staining intensity.
In one example, the brightest stained cells in a sample can be as
much as 4 logs more intense than unstained cells. When displayed in
this manner, it is clear that the cells falling in the highest log
of staining intensity are bright, while those in the lowest
intensity are negative. The "low" positively stained cells have a
level of staining brighter than that of an isotype matched control,
but is not as intense as the most brightly staining cells normally
found in the population. An alternative control may utilize a
substrate having a defined density of marker on its surface, for
example a fabricated bead or cell line, which provides the positive
control for intensity.
[0129] Enrichment of hepatocyte-like cells. To increase the
fraction of obtained cells that are iHeps, it is sometimes
advantageous to enrich for (i.e., purify) the produced iHeps. In
particular aspects, the hepatocyte-like cells provided herein may
be selected or enriched by using a screenable or selectable
reporter expression cassette comprising a mature
hepatocyte-specific transcriptional regulatory element operably
linked to a reporter gene, or by cell sorting (e.g., magnetic cell
sorting, Fluorescence Activated Cell Sorting (FACS), and the like)
using an antibody against a hepatocyte-specific marker (e.g., a
cell surface antigen such as ASGR1).
[0130] To aid selection or enrichment, the ASCs, may comprise a
selectable or screenable reporter expression cassette comprising a
reporter gene. The reporter expression cassette may comprise a
hepatocyte-specific transcriptional regulatory element operably
linked to a reporter gene (e.g., any of the fluorescent proteins,
e.g., blue, yellow, red, green, etc.). Non-limiting examples of
hepatocyte-specific transcriptional regulatory element include a
promoter of glucose-6-phosphatase, albumin (ALB),
alpha-1-antitrypsin (AAT, also known as SERPINA1), cytokeratin 8
(CK8), cytokeratin 18 (CK18), asialoglycoprotein receptor 1
(ASGR1), alcohol dehydrogenase 1, arginase Type I, cytochrome p450
3A4 (CYP3A4), liver-specific organic anion transporter (LST-1),
forkhead box protein A2 (FoxA2), alpha-fetoprotein (AFP), and
tryptophan 2,3-dioxygenase (TDO2).
[0131] Enrichment using antibodies (e.g., magnetic cell sorting,
FACS, and the like) specific for cell surface markers of ASCs
(e.g., CD29, CD34, CD36, CD49f, CD73, CD90 (Thy-1), CD105, CD133,
c-kit, c-met, and the like), endodermal precursor cells (e.g.,
SOX17), hepatocyte precursor/progenitor cells (e.g., N-cadherin,
E-cadherin, EpCAM, and NCAM), and/or hepatocytes (e.g., ASGR1) have
the advantage of not requiring genetic modification of the cells to
be enriched. Magnetic cell sorting and FACS have the ability to
analyze multiple surface markers simultaneously, and they can be
used to sort ASCs, endodermal precursor cells, hepatocyte
precursor/progenitor cells, and/or hepatocytes (e.g., iHeps) based
on the expression of a cell surface marker. In some cases, iHeps do
not express ASGR1. However, in some cases, e.g, if they are
cultured longer and/or are further differentiated, iHeps do express
ASGR1. In cases where cells that (i) do not express ASGR1; and/or
(ii) express markers of hepatocyte precursor cells and/or
endodermal precursor cells, are administered to an individual, the
cells can further differentiate after administration into the
individual (as demonstrated in the examples section below).
[0132] In some cases, cells that are produced by the subject
methods can be enriched (e.g., sorted) by using an expressed cell
surface marker. For example, cells of the precursor cell population
(e.g., ASCs that have been contacted by a stage 1 medium) and/or
cells of the induced cell population (e.g., cells of the precursor
cell population that have been contacted with stage 2 medium) can
be enriched using any marker that is expressed by hepatocyte
precursor/progenitor cells (e.g., N-cadherin, E-cadherin, EpCAM,
and NCAM) and/or endodermal precursor cells (e.g., SOX17). Cells of
the induced cell population (e.g., cells of the precursor cell
population that have been contacted with stage 2 medium) can be
enriched using any marker that is expressed by hepatocyte
precursor/progenitor cells (e.g., N-cadherin, E-cadherin, EpCAM,
NCAM, etc.), endodermal precursor cells (e.g., SOX17), and/or
hepatocytes (e.g., ASGR1) prior to further culture (e.g., to
facilitate for further differentiation) and/or prior to
administration to an individual. A cell population that includes
ASCs (e.g., a population of cells from liposuction) can be enriched
for ASCs using ASC cell surface markers (e.g., CD29, CD34, CD36,
CD49f, CD73, CD90 (Thy-1), CD105, CD133, c-kit, c-met, and the
like).
[0133] Cryopreservation. In some embodiments, subject cells (e.g.,
iHeps) are preserved for future use. Specifically, iHeps can be
cryopreserved by performing the following steps: (i) Detach cells
from growth cell surface, (ii) rinse cells (e.g., wash with PBS via
centrifugation), (iii) resuspend cells (e.g., cell pellet) in media
with 10% DMSO, and (vi) freeze cells and store in liquid nitrogen
using standard tissue culture techniques commonly used in the art
to preserve cells. Cells can be frozen at 1-50 million cells per
vial. When ready for use, iHeps should be thawed in a manner as
commonly known in the art for thawing frozen cultured cells.
Methods of Treatment
[0134] Aspects of the disclosure include methods of treating an
individual. The subject methods of treatment generally include
producing a population of hepatocyte-like cells from a population
of adipose-derived stem cells (e.g., using the methods described
above), and administering (e.g., injecting, transplanting, etc.) an
effective number of hepatocyte-like cells into an individual. The
ASCs can be from any source and can be derived by any convenient
method. Exemplary methods of isolating/extracting/obtaining ASCs
are described above. In some cases the ASCs are autologous (i.e.,
from the same individual into which the iHeps will be administered)
(e.g., to reduce the possibility and/or severity of an immune
response). In some cases, the ASCs are from a related individual
(e.g., to reduce the possibility and/or severity of an immune
response). In some cases, the ASCs are from an unrelated
individual. In some cases, the ASCs are from an individual of
another species (e.g., human ASCs administered to a mouse).
[0135] The terms "treatment", "treating", "treat" and the like are
used herein to generally refer to obtaining a desired pharmacologic
and/or physiologic effect. The effect can be prophylactic in terms
of completely or partially preventing a disease or symptom(s)
thereof and/or may be therapeutic in terms of a partial or complete
stabilization or cure for a disease and/or adverse effect
attributable to the disease. The term "treatment" encompasses any
treatment of a disease in a mammal, particularly a human, and
includes: (a) preventing the disease and/or symptom(s) from
occurring in a subject who may be predisposed to the disease or
symptom but has not yet been diagnosed as having it; (b) inhibiting
the disease and/or symptom(s), i.e., arresting development of a
disease and/or the associated symptoms; or (c) relieving the
disease and the associated symptom(s), i.e., causing regression of
the disease and/or symptom(s). Those in need of treatment can
include those already inflicted (e.g., those with liver
malfunction, liver disease, liver damage, etc.) as well as those in
which prevention is desired.
[0136] The terms "recipient", "individual", "subject", "host", and
"patient", are used interchangeably herein and refer to any
mammalian subject for whom diagnosis, treatment, or therapy is
desired, particularly humans. "Mammal" for purposes of treatment
refers to any animal classified as a mammal, including humans,
domestic and farm animals, and zoo, sports, or pet animals, such as
dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some
embodiments, the mammal is human.
[0137] A therapeutic treatment is one in which the subject is
inflicted prior to administration and a prophylactic treatment is
one in which the subject is not inflicted prior to administration.
In some embodiments, the subject has an increased likelihood of
becoming inflicted or is suspected of being inflicted prior to
treatment. In some embodiments, the subject is suspected of having
an increased likelihood of becoming inflicted.
[0138] In some embodiments, the individual to be treated is an
individual with reduced liver function (e.g., an individual with
liver damage). The term "liver damage" as used herein refers to any
damage (e.g., caused by disease, trauma, unexplained, etc.)
resulting in reduced liver function. An individual with reduced
liver function (e.g., reduced relative to the individual's basal
level of function; reduced relative to a healthy individual of
comparable age, weight, sex, etc.; and the like) is referred to
herein as an individual with a damaged liver (i.e., an individual
with liver damage). Thus, the terms "liver damage" and "reduced
liver function" are used synonymously herein. In some cases, an
individual with "liver damage" is an individual with liver
disease.
[0139] The cause for reduced liver function may be known or
unknown. How to determine whether a given individual has reduced
liver function (and therefore also how to determine whether an
individual receiving treatment is recovering or has recovered liver
function) will be known to one of ordinary skill in the art. Liver
function tests can include liver enzyme tests, also known as liver
function tests (LFTs), a group of blood tests that detect
inflammation and damage to the liver. Blood protein markers of
liver damage may include, for example: increased aspartate
aminotransferase (AST), also known as SGOT; increased alanine
aminotransferase (ALT), also known as SGPT; increased alkaline
phosphatase; increased 5' nucleotidase; decreased albumin;
decreased globulin; and increased gamma-glutamyl transpeptidase
(GGT). If, for example, elevated amounts (and/or activity) of the
above protein markers (or a decreased level for albumin and/or
globulin) are detected in the blood, liver damage may be present.
Additional liver function tests include: (i) prothrombin time (PT),
a test of the time it takes for a blood sample to clot (e.g., if
low levels of clotting factors are present, the prothrombin time is
longer), which can be reported as international normalized ratio
(INR) (a standardized way for laboratories to report PT so that
results can be compared accurately across laboratories); and (ii) a
bilirubin test: bilirubin blood levels may be elevated in people
with impaired bile flow, which can occur in severe liver disease,
gallbladder disease, or other bile system conditions.
[0140] Examples of symptoms, illnesses, and/or diseases that can be
considered to indicate "liver damage" (and that can be treated by
transplanting subject induced hepatocyte-like cells into an
individual having the symptoms, illness, and/or disease), include,
but are not limited to: acute liver failure, alcohol-related liver
disease, Alagille syndrome, alpha 1-antitrypsin deficiency,
alveolar hydatid disease, autoimmune hepatitis, bacillary peliosis,
biliary atresia, Budd-Chiari syndrome, chronic liver disease,
cirrhosis, congenital hepatic fibrosis, congestive hepatopathy,
fatty liver, galactosemia, gastric antral vascular ectasia, Gilbert
syndrome, hemochromatosis, hepatitis (A, B, and/or C), hepatic
encephalopathy, hepato-biliary diseases, hepatolithiasis,
hepatopulmonary syndrome, hepatorenal syndrome, hepatosplenomegaly,
hepatotoxicity, hepatocellular carcinoma, hepatic encephalopathy,
jaundice, Laennec's cirrhosis, liver abscess, liver cysts, liver
cancer, liver failure, Lyngstadaas syndrome, non-alcoholic fatty
liver disease, pediatric end-stage liver disease, peliosis hepatis,
polycystic liver disease, primary biliary cirrhosis, primary
sclerosing cholangitis (PSC), progressive familial intrahepatic
cholestasis, Reye syndrome, type I glycogen storage disease, viral
hepatitis, Wilson disease, Zahn infarct, and Zieve's syndrome.
[0141] In some cases, iHeps are cultured for a period of time prior
to transplantation (e.g., in HCM.TM. for 2 days). Cells (e.g.,
iHeps) can be provided to the individual (i.e., administered into
the individual) alone or with a suitable substrate or matrix, e.g.
to support their growth and/or organization in the tissue to which
they are being transplanted (e.g., liver). In some embodiments, the
matrix is a scaffold (e.g., an organ scaffold). In some
embodiments, 1.times.10.sup.3 or more cells will be administered
(e.g., transplanted), for example 5.times.10.sup.3 or more cells,
1.times.10.sup.4 or more cells, 5.times.10.sup.4 or more cells,
1.times.10.sup.1 or more cells, 5.times.10.sup.1 or more cells,
1.times.10.sup.6 or more cells, 5.times.10.sup.6 or more cells,
1.times.10.sup.7 or more cells, 5.times.10.sup.7 or more cells,
1.times.10.sup.8 or more cells, 5.times.10.sup.8 or more cells,
1.times.10.sup.1 or more cells, 5.times.10.sup.1 or more cells, or
1.times.10.sup.10 or more cells. In some embodiments, subject cells
are administered into the individual on microcarriers (e.g., cells
grown on biodegradable microcarriers).
[0142] The cells induced by the subject methods (iHeps) may be
administered in any physiologically acceptable excipient (e.g.,
William's E medium), where the cells may find an appropriate site
for survival and function (e.g., organ reconstitution). The cells
may be introduced by any convenient method (e.g., injection,
catheter, or the like).
[0143] The cells may be introduced to the subject (i.e.,
administered into the individual) via any of the following routes:
parenteral, subcutaneous, intravenous, intracranial, intraspinal,
intraocular, or into spinal fluid. The cells may be introduced by
injection (e.g., direct local injection), catheter, or the like.
Examples of methods for local delivery (e.g., delivery to the
liver) include, e.g., by bolus injection, e.g. by a syringe, e.g.
into a joint or organ; e.g., by continuous infusion, e.g. by
cannulation, e.g. with convection (see e.g. US Application No.
20070254842, incorporated here by reference); or by implanting a
device upon which the cells have been reversably affixed (see e.g.
US Application Nos. 20080081064 and 20090196903, incorporated
herein by reference).
[0144] In some cases, iHeps are administered into an individual by
ultrasound-guided liver injection. In this way, cells can be placed
directly into a liver lobe (e.g., in humans, or even in mice using
a small animal ultrasound system). Brightness mode (B-mode) can be
used to acquire two-dimensional images for an area of interest with
a transducer and cells can be injected in solution (e.g., 100 .mu.l
to 300 .mu.l, e.g., 200 .mu.l of, for example, William's E medium)
into one site or many sites (e.g., 1-30 sites) in the liver using,
for example, a 30 G needle.
[0145] The number of administrations of treatment to a subject may
vary. Introducing cells into an individual may be a one-time event;
but in certain situations, such treatment may elicit improvement
for a limited period of time and require an on-going series of
repeated treatments. In other situations, multiple administrations
of iHeps may be required before an effect is observed. As will be
readily understood by one of ordinary skill in the art, the exact
protocols depend upon the disease or condition, the stage of the
disease and parameters of the individual being treated.
[0146] A "therapeutically effective dose" or "therapeutic dose" is
an amount sufficient to effect desired clinical results (i.e.,
achieve therapeutic efficacy). A therapeutically effective dose can
be administered in one or more administrations. For purposes of
this disclosure, a therapeutically effective dose of iHeps is an
amount that is sufficient, when administered to (e.g., transplanted
into) the individual, to palliate, ameliorate, stabilize, reverse,
prevent, slow or delay the progression of the disease state (e.g.,
liver damage) by, for example, providing functions normally
provided by a healthy liver. In some cases, transplanted iHeps
integrate into the individual's liver and become part of the liver.
In some cases, transplanted iHeps do not integrate into the
individual's liver, but can still provide functions normally
provided by a healthy liver.
[0147] In some embodiments, a therapeutically effective dose of
iHeps is about 1.times.10.sup.3 or more cells (e.g.,
5.times.10.sup.3 or more, 1.times.10.sup.4 cells, 5.times.10.sup.4
or more, 1.times.10.sup.5 or more, 5.times.10.sup.5 or more,
1.times.10.sup.6 or more, 5.times.10.sup.6 or more,
1.times.10.sup.7 cells, 5.times.10.sup.7 or more, 1.times.10.sup.8
or more, 5.times.10.sup.8 or more, 1.times.10.sup.9 or more,
5.times.10.sup.9 or more, or 1.times.10.sup.10 or more). In some
embodiments, a therapeutically effective dose of iHeps is in a
range of from about 1.times.10.sup.3 cells to about
1.times.10.sup.10 cells (e.g, from about 5.times.10.sup.3 cells to
about 1.times.10.sup.10 cells, from about 1.times.10.sup.4 cells to
about 1.times.10.sup.10 cells, from about 5.times.10.sup.4 cells to
about 1.times.10.sup.10 cells, from about 1.times.10.sup.5 cells to
about 1.times.10.sup.10 cells, from about 5.times.10.sup.5 cells to
about 1.times.10.sup.10 cells, from about 1.times.10.sup.6 cells to
about 1.times.10.sup.10 cells, from about 5.times.10.sup.6 cells to
about 1.times.10.sup.10 cells, from about 1.times.10.sup.6 cells to
about 1.times.10.sup.10 cells, from about 5.times.10.sup.7 cells to
about 1.times.10.sup.10 cells, from about 1.times.10.sup.8 cells to
about 1.times.10.sup.10 cells, from about 5.times.10.sup.8 cells to
about 1.times.10.sup.10, from about 5.times.10.sup.3 cells to about
5.times.10.sup.9 cells, from about 1.times.10.sup.4 cells to about
5.times.10.sup.9 cells, from about 5.times.10.sup.4 cells to about
5.times.10.sup.9 cells, from about 1.times.10.sup.5 cells to about
5.times.10.sup.9 cells, from about 5.times.10.sup.5 cells to about
5.times.10.sup.9 cells, from about 1.times.10.sup.6 cells to about
5.times.10.sup.9 cells, from about 5.times.10.sup.6 cells to about
5.times.10.sup.9 cells, from about 1.times.10.sup.7 cells to about
5.times.10.sup.9 cells, from about 5.times.10.sup.7 cells to about
5.times.10.sup.9 cells, from about 1.times.10.sup.8 cells to about
5.times.10.sup.9 cells, from about 5.times.10.sup.8 cells to about
5.times.10.sup.9, from about 5.times.10.sup.3 cells to about
1.times.10.sup.9 cells, from about 1.times.10.sup.4 cells to about
1.times.10.sup.9 cells, from about 5.times.10.sup.4 cells to about
1.times.10.sup.9 cells, from about 1.times.10.sup.5 cells to about
1.times.10.sup.9 cells, from about 5.times.10.sup.5 cells to about
1.times.10.sup.9 cells, from about 1.times.10.sup.6 cells to about
1.times.10.sup.9 cells, from about 5.times.10.sup.6 cells to about
1.times.10.sup.9 cells, from about 1.times.10.sup.7 cells to about
1.times.10.sup.9 cells, from about 5.times.10.sup.7 cells to about
1.times.10.sup.9 cells, from about 1.times.10.sup.8 cells to about
1.times.10.sup.9 cells, from about 5.times.10.sup.8 cells to about
1.times.10.sup.9, from about 5.times.10.sup.1 cells to about
5.times.10.sup.8 cells, from about 1.times.10.sup.4 cells to about
5.times.10.sup.8 cells, from about 5.times.10.sup.4 cells to about
5.times.10.sup.8 cells, from about 1.times.10.sup.5 cells to about
5.times.10.sup.8 cells, from about 5.times.10.sup.5 cells to about
5.times.10.sup.8 cells, from about 1.times.10.sup.6 cells to about
5.times.10.sup.8 cells, from about 5.times.10.sup.6 cells to about
5.times.10.sup.8 cells, from about 1.times.10.sup.7 cells to about
5.times.10.sup.8 cells, from about 5.times.10.sup.7 cells to about
5.times.10.sup.8 cells, or from about 1.times.10.sup.8 cells to
about 5.times.10.sup.8 cells).
[0148] The cells of this disclosure can be supplied in the form of
a pharmaceutical composition, comprising an isotonic excipient
prepared under sufficiently sterile conditions for human
administration. For general principles in medicinal formulation,
the reader is referred to Cell Therapy: Stem Cell Transplantation,
Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W.
Sheridan eds, Cambridge University Press, 1996; and Hematopoietic
Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill
Livingstone, 2000. Choice of the cellular excipient and any
accompanying elements of the composition will be adapted in
accordance with the route and device used for administration. The
composition may also comprise or be accompanied with one or more
other ingredients that facilitate the engraftment or functional
mobilization of the cells. Suitable ingredients include matrix
proteins that support or promote adhesion of the cells, or
complementary cell types.
[0149] Cells of the subject methods may be genetically altered in
order to introduce genes useful in the differentiated hepatocytes,
e.g. repair of a genetic defect in an individual, selectable
marker, etc. Cells may also be genetically modified to enhance
survival, control proliferation, and the like. Cells may be
genetically altered by transfection or transduction with a suitable
vector, homologous recombination, or other appropriate technique,
so that they express a gene of interest. In some embodiments, a
selectable marker is introduced, to provide for greater purity of
the desired differentiating cell.
[0150] The cells of this disclosure can also be genetically altered
in order to enhance their ability to be involved in tissue
regeneration, or to deliver a therapeutic gene to a site of
administration. A vector is designed using the known encoding
sequence for the desired gene, operatively linked to a promoter
that is either pan-specific or specifically active in
hepatocytes.
[0151] Many vectors useful for transferring exogenous genes into
target mammalian cells are available. The vectors may be episomal,
e.g. plasmids, virus derived vectors such cytomegalovirus,
adenovirus, etc., or may be integrated into the target cell genome,
through homologous recombination or random integration, e.g.
retrovirus derived vectors such MMLV, HIV-1, ALV, etc. For
modification of stem cells, lentiviral vectors are preferred.
Lentiviral vectors such as those based on HIV or FIV gag sequences
can be used to transfect non-dividing cells, such as the resting
phase of human stem cells (see Uchida et al. (1998) P.N.A.S.
95(20):11939-44).
[0152] Combinations of retroviruses and an appropriate packaging
line may also find use, where the capsid proteins will be
functional for infecting the target cells. Usually, the cells and
virus will be incubated for at least about 24 hours in the culture
medium. The cells are then allowed to grow in the culture medium
for short intervals in some applications, e.g. 24-73 hours, or for
at least two weeks, and may be allowed to grow for five weeks or
more, before analysis. Commonly used retroviral vectors are
"defective", i.e. unable to produce viral proteins required for
productive infection. Replication of the vector requires growth in
the packaging cell line.
[0153] The host cell specificity of the retrovirus is determined by
the envelope protein, env (p120). The envelope protein is provided
by the packaging cell line. Envelope proteins are of at least three
types, ecotropic, amphotropic and xenotropic. Retroviruses packaged
with ecotropic envelope protein, e.g. MMLV, are capable of
infecting most murine and rat cell types. Ecotropic packaging cell
lines include BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396).
Retroviruses bearing amphotropic envelope protein, e.g. 4070A
(Danos et al, supra.), are capable of infecting most mammalian cell
types, including human, dog and mouse. Amphotropic packaging cell
lines include PA12 (Miller et al. (1985) Mol. Cell. Biol.
5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol.
6:2895-2902) GRIP (Danos et al. (1988) PNAS 85:6460-6464).
Retroviruses packaged with xenotropic envelope protein, e.g. AKR
env, are capable of infecting most mammalian cell types, except
murine cells.
[0154] The vectors may include genes that must later be removed,
e.g. using a recombinase system such as Cre/Lox, or the cells that
express them destroyed, e.g. by including genes that allow
selective toxicity such as herpesvirus TK, bcl-xs, etc. Suitable
inducible promoters are activated in a desired target cell type,
either the transfected cell, or progeny thereof. By transcriptional
activation, it is intended that transcription will be increased
above basal levels in the target cell by at least about 100 fold,
more usually by at least about 1000 fold.
UTILITY
[0155] The results described in the Examples below demonstrate that
functioning human liver tissue can be generated in vivo after
direct implantation of hepatocyte-like cells, derived from ASCs
using the subject methods, into the liver. The SCi-Heps (iHeps
induced using the subject methods) were produced from ASCs by in
vitro differentiation for a short period in chemically defined
media. SCi-Heps exhibited many of the in vitro functional
properties of mature hepatocytes, and they were able to stably
reconstitute functioning human liver in vivo in a murine model
system, and implantation studies demonstrated that SCi-Heps have a
very low malignant potential. Thus, readily-accessible stem cells
were used to rapidly build functioning human liver tissue in vivo.
The time to generate iHeps from ASCs is greatly reduced by the
subject methods to under 13 days (e.g., 9 days). In addition, the
efficiency of iHep production is greatly increased by the subject
methods to greater that 10% (e.g., 37%). Both of these features
facilitate treatment methods because liver damage (e.g., liver
failure, e.g., due to acetaminophen toxicity) can rapidly cause
death (e.g., in 2 weeks or less).
[0156] These procedures can be scaled to enable autologous liver
regeneration in humans. Since there are .about.140.times.10.sup.6
hepatocytes per gram of human or mouse liver tissue; a 1500 gram
adult human liver has .about.2.times.10.sup.11 hepatocytes, while a
1.8 gram mouse liver has 2.5.times.10.sup.8 cells. After 1 million
human hepatocytes are transplanted into a TK-NOG mouse, .about.50%
replacement of the mouse hepatocytes is routinely obtained, which
amounts to .about.1.2.times.10.sup.8 functioning human hepatocytes
in the mouse liver. This represents a 120-fold increase (or 7
population doublings) in the number of human hepatocytes generated
in vivo relative to the number of transplanted cells. If a similar
regeneration efficiency is achieved in humans, 800 million SCi-Heps
(10.sup.11/120) would be transplanted to replace 50% of the cells
in a human liver. Of relevance to this question, 1 liter (1000 g)
of lipo-aspirate can easily be obtained from a single liposuction
procedure. This volume is estimated to contain 2-10.times.10.sup.9
cells, of which 1-10% are estimated to be ASCs. Thus, one liter of
lipo-aspirate contains between 2.times.10.sup.7 and 10.sup.9 ASCs,
which can provide a sufficient number of cells to enable autologous
human liver replacement. Human liver regenerative procedures,
including ultrasound-guided direct liver Implantation (used in the
examples below), are scalable and appropriate for human clinical
use.
[0157] The examples below demonstrate that spherical culture
coupled with a chemical differentiation process can increase the
yield of SCi-Heps by 7-fold compared to other known methods. Thus,
the spherical culture method helps to ensure that a sufficient
number of SCi-Heps can be obtained to enable liver regeneration in
a human subject. The spherical culture method also reduces the
culture volume by 12-fold and decreases the time in culture by 40%;
these factors dramatically reduce the cost of differentiating ASC
into iHeps. The estimated cost of the medium and growth factors
required for producing Chi-Heps is .about.$49,000 per liter of
lipo-aspirate processed, but the subject method reduces these costs
by over 20-fold (to .about.$2400 per liter of lipo-aspirate
processed).
[0158] The fact that implanted SCi-Heps did not form tumors (see
the Examples below) indicates that these cells have a markedly
reduced risk of causing tumors. In contrast to the results obtained
with SCi-Heps, readily palpable tumors were noted within 3 weeks
after implantation of iPS-Heps. The rapid tumor onset should not be
surprising, since this iPS-derived cell population has a large
percentage of un-differentiated cells. However, .about.10.sup.9
cells must be transplanted to regenerate a human liver, but even as
few as 100-1000 cells with malignant potential in this population
could cause tumor formation. These numbers create a very
significant hurdle for any selective method used to prepare
iPS-derived cells for liver regeneration. The fact that implanted
SCi-Heps did not form tumors indicates that these cells have a
markedly reduced risk of causing tumors to form. Since
gancyclovir-induced liver damage in genetically engineered TK-NOG
mice resembles the acute liver failure in humans that is caused by
exogenous toxicants or drug overdose, methods disclosed here can
provide cells that can be used to treat a damaged liver (e.g., a
patient with acute liver failure).
[0159] Hepatocyte-like cells produced by the subject methods
provide a source of donor cells for cell replacement in damaged
livers. As such, the hepatocyte-like cells may be used for tissue
reconstitution or regeneration in a human patient, an individual in
need of such treatment, and/or for the reconstitution of human
liver tissue in a non-human animal (e.g., a mouse, which can then
be used, for example, for research purposes including drug
screening, drug testing, etc.). The cells are administered in a
manner that permits them to graft or migrate to the intended tissue
site and reconstitute or regenerate the functionally deficient
area.
[0160] The differentiated cells of this disclosure can also be used
to prepare antibodies that are specific for markers of hepatocytes.
Polyclonal antibodies can be prepared by injecting a vertebrate
animal with cells of this disclosure in an immunogenic form.
Production of monoclonal antibodies is described in such standard
references as U.S. Pat. Nos. 4,491,632, 4,472,500 and 4,444,887,
and Methods in Enzymology 73B:3 (1981). Specific antibody molecules
can also be produced by contacting a library of immunocompetent
cells or viral particles with the target antigen, and growing out
positively selected clones. See Marks et al., New Eng. J. Med.
335:730, 1996, and McGuiness et al., Nature Biotechnol. 14:1449,
1996. A further alternative is reassembly of random DNA fragments
into antibody encoding regions, as described in EP patent
application 1,094,108 A.
[0161] Gene expression may be examined before, during, and/or after
the production of hepatocyte-like cells by the subject methods. The
expressed set of genes may be compared against other subsets of
cells, against progentior cells, against terminally differentiated
hepatocytes, against ASCs, and the like, as known in the art. Any
suitable qualitative or quantitative methods known in the art for
detecting specific mRNAs can be used. mRNA can be detected by, for
example, hybridization to a microarray, next-generation sequencing,
in situ hybridization, by reverse transcriptase-polymerase chain
reaction (rtPCR), or in Northern blots containing poly A mRNA. One
of skill in the art can readily use these methods to determine
differences in the size or amount of mRNA transcripts between two
samples.
[0162] Any suitable method for detecting and comparing mRNA
expression levels in a sample can be used in connection with the
methods of the disclosure. For example, the mRNA from a sample can
be sequenced via next-generation sequencing methods known in the
art such as nanopore sequencing (e.g. as described in Soni et al
Clin Chem 53: 1996-2001 2007, or as described by Oxford Nanopore
Technologies), Illumina's reversible terminator method, Roche's
pyrosequencing method (454), Life Technologies' sequencing by
ligation (the SOLiD platform) or Life Technologies' Ion Torrent
platform. Examples of such methods are described in the following
references: Margulies et al (Nature 2005 437: 376-80); Ronaghi et
al (Analytical Biochemistry 1996 242: 84-9); Shendure (Science 2005
309: 1728); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox
et al (Methods Mol Biol. 2009; 553:79-108); Appleby et al (Methods
Mol Biol. 2009; 513:19-39) and Morozova (Genomics. 2008 92:255-64),
which are incorporated by reference for the general descriptions of
the methods and the particular steps of the methods, including all
starting products, reagents, and final products for each of the
steps.
[0163] Alternatively, gene expression in a sample can be detected
using hybridization analysis, which is based on the specificity of
nucleotide interactions. Oligonucleotides or cDNA can be used to
selectively identify or capture DNA or RNA of specific sequence
composition, and the amount of RNA or cDNA hybridized to a known
capture sequence determined qualitatively or quantitatively, to
provide information about the relative representation of a
particular message within the pool of cellular messages in a
sample. Hybridization analysis can be designed to allow for
concurrent screening of the relative expression of hundreds to
thousands of genes by using, for example, array-based technologies
having high density formats, including filters, microscope slides,
or microchips, or solution-based technologies that use
spectroscopic analysis (e.g., mass spectrometry).
[0164] Hybridization to arrays may be performed, where the arrays
can be produced according to any suitable methods known in the art.
For example, methods of producing large arrays of oligonucleotides
are described in U.S. Pat. Nos. 5,134,854, and 5,445,934 using
light-directed synthesis techniques. Using a computer controlled
system, a heterogeneous array of monomers is converted, through
simultaneous coupling at a number of reaction sites, into a
heterogeneous array of polymers. Alternatively, microarrays are
generated by deposition of pre-synthesized oligonucleotides onto a
solid substrate, for example as described in PCT published
application no. WO 95/35505.
[0165] Methods for collection of data from hybridization of samples
with an array are also well known in the art. For example, the
polynucleotides of the cell samples can be generated using a
detectable fluorescent label, and hybridization of the
polynucleotides in the samples detected by scanning the microarrays
for the presence of the detectable label. Methods and devices for
detecting fluorescently marked targets on devices are known in the
art. Generally, such detection devices include a microscope and
light source for directing light at a substrate. A photon counter
detects fluorescence from the substrate, while an x-y translation
stage varies the location of the substrate. A confocal detection
device that can be used in the subject methods is described in U.S.
Pat. No. 5,631,734. A scanning laser microscope is described in
Shalon et al., Genome Res. (1996) 6:639. A scan, using the
appropriate excitation line, is performed for each fluorophore
used. The digital images generated from the scan are then combined
for subsequent analysis. For any particular array element, the
ratio of the fluorescent signal from one sample is compared to the
fluorescent signal from another sample, and the relative signal
intensity determined.
[0166] Methods for analyzing the data collected from hybridization
to arrays are well known in the art. For example, where detection
of hybridization involves a fluorescent label, data analysis can
include the steps of determining fluorescent intensity as a
function of substrate position from the data collected, removing
outliers, i.e. data deviating from a predetermined statistical
distribution, and calculating the relative binding affinity of the
targets from the remaining data. The resulting data can be
displayed as an image with the intensity in each region varying
according to the binding affinity between targets and probes.
[0167] Pattern matching can be performed manually, or can be
performed using a computer program. Methods for preparation of
substrate matrices (e.g., arrays), design of oligonucleotides for
use with such matrices, labeling of probes, hybridization
conditions, scanning of hybridized matrices, and analysis of
patterns generated, including comparison analysis, are described
in, for example, U.S. Pat. No. 5,800,992.
[0168] In vitro induced hepatocyte-like cells (iHeps) produced by
the subject methods provide also provide a source of cells for
novel hepatic drug discovery, development, and safety testing. The
use of in vitro iHeps produced by the subject methods offers the
pharmaceutical industry an invaluable tool for preclinical
screening of candidate drugs to treat cardiomyopathy, arrhythmia,
and heart failure, as well as therapeutics to combat secondary
cardiac toxicities. The development of new screens using In vitro
iHeps produced by the subject methods should reduce the time and
cost of bringing new drugs to market.
[0169] In screening assays for biologically active agents (e.g.,
small molecule compounds, peptides, viruses, etc.) of the subject
hepatocyte-like cells, usually a culture comprising the subject
hepatocyte-like cells, is contacted with the agent of interest, and
the effect of the agent assessed by monitoring output parameters,
such as expression of markers, cell viability, drug metabolism, and
the like.
[0170] Agents of interest for screening include known and unknown
compounds that encompass numerous chemical classes, primarily
organic molecules, which may include organometallic molecules,
inorganic molecules, genetic sequences, etc. An important aspect of
the disclosure is to evaluate candidate drugs, including toxicity
testing; and the like.
[0171] In addition to complex biological agents, such as viruses,
candidate agents include organic molecules comprising functional
groups necessary for structural interactions, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, frequently at least two of the
functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules, including peptides, polynucleotides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof.
[0172] Included are pharmacologically active drugs, genetically
active molecules, etc. Compounds of interest include
chemotherapeutic agents, hormones or hormone antagonists, etc.
Exemplary of pharmaceutical agents suitable for this disclosure are
those described in, "The Pharmacological Basis of Therapeutics,"
Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth
edition, under the sections: Water, Salts and Ions; Drugs Affecting
Renal Function and Electrolyte Metabolism; Drugs Affecting
Gastrointestinal Function; Chemotherapy of Microbial Diseases;
Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Forming
organs; Hormones and Hormone Antagonists; Vitamins, Dermatology;
and Toxicology, all incorporated herein by reference.
[0173] Test compounds include all of the classes of molecules
described above, and may further comprise samples of unknown
content. Of interest are complex mixtures of naturally occurring
compounds derived from natural sources such as plants. While many
samples will comprise compounds in solution, solid samples that can
be dissolved in a suitable solvent may also be assayed. Samples of
interest include manufacturing samples, pharmaceuticals, libraries
of compounds prepared for analysis, and the like. Samples of
interest include compounds being assessed for potential therapeutic
value, i.e. drug candidates.
[0174] Compounds, including candidate agents, are obtained from a
wide variety of sources including libraries of synthetic or natural
compounds. For example, numerous means are available for random and
directed synthesis of a wide variety of organic compounds,
including biomolecules, including expression of randomized
oligonucleotides and oligopeptides. Alternatively, libraries of
natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means, and may be used to produce combinatorial
libraries. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification, etc. to produce
structural analogs.
[0175] Agents are screened for biological activity by adding the
agent to at least one and usually a plurality of cell samples,
usually in conjunction with cells lacking the agent. The change in
parameters in response to the agent is measured, and the result
evaluated by comparison to reference cultures, e.g. in the presence
and absence of the agent, obtained with other agents, etc.
[0176] The agents are conveniently added in solution, or readily
soluble form, to the medium of cells in culture. The agents may be
added in a flow-through system, as a stream, intermittent or
continuous, or alternatively, adding a bolus of the compound,
singly or incrementally, to an otherwise static solution. In a
flow-through system, two fluids are used, where one is a
physiologically neutral solution, and the other is the same
solution with the test compound added. The first fluid is passed
over the cells, followed by the second. In a single solution
method, a bolus of the test compound is added to the volume of
medium surrounding the cells. The overall concentrations of the
components of the culture medium should not change significantly
with the addition of the bolus, or between the two solutions in a
flow through method.
[0177] Preferred agent formulations do not include additional
components, such as preservatives, that may have a significant
effect on the overall formulation. Thus preferred formulations
consist essentially of a biologically active compound and a
physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc.
However, if a compound is liquid without a solvent, the formulation
may consist essentially of the compound itself.
[0178] A plurality of assays may be run in parallel with different
agent concentrations to obtain a differential response to the
various concentrations. As known in the art, determining the
effective concentration of an agent typically uses a range of
concentrations resulting from 1:10, or other log scale, dilutions.
The concentrations may be further refined with a second series of
dilutions, if necessary. Typically, one of these concentrations
serves as a negative control, i.e. at zero concentration or below
the level of detection of the agent or at or below the
concentration of agent that does not give a detectable change in
the phenotype.
[0179] The cells may be freshly isolated, cultured, genetically
altered as described above, or the like. The cells may be
environmentally induced variants of clonal cultures: e.g. split
into independent cultures and grown under distinct conditions, for
example with or without virus; in the presence or absence of other
biological agents. The manner in which cells respond to an agent,
particularly a pharmacologic agent, including the timing of
responses, is an important reflection of the physiologic state of
the cell.
[0180] Parameters are quantifiable components of cells,
particularly components that can be accurately measured, desirably
in a high throughput system. A parameter can be any cell component
or cell product including cell surface determinant, receptor,
protein or conformational or posttranslational modification
thereof, lipid, carbohydrate, organic or inorganic molecule,
nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a
cell component or combinations thereof. While most parameters will
provide a quantitative readout, in some instances a
semi-quantitative or qualitative result will be acceptable.
Readouts may include a single determined value, or may include
mean, median value or the variance, etc. Characteristically a range
of parameter readout values will be obtained for each parameter
from a multiplicity of the same assays. Variability is expected and
a range of values for each of the set of test parameters will be
obtained using standard statistical methods with a common
statistical method used to provide single values.
[0181] For further elaboration of general techniques useful in the
practice of this disclosure, the practitioner can refer to standard
textbooks and reviews in cell biology, tissue culture, and
embryology. With respect to tissue culture and stem cells, the
reader may wish to refer to Teratocarcinomas and embryonic stem
cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd.
1987); Guide to Techniques in Mouse Development (P. M. Wasserman et
al. eds., Academic Press 1993); Embryonic Stem Cell Differentiation
in Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties
and uses of Embryonic Stem Cells: Prospects for Application to
Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod.
Fertil. Dev. 10:31, 1998).
Kits
[0182] Also provided are kits for use in the subject methods. The
subject kits include any combination of components for used in
stage 1 and/or stage 2 media. For example, in some cases, a kit
includes a Wnt signaling agonist (e.g., Wnt3a), activin A, an FGF
(e.g., FGF4, FGF5, FGF6, etc.), and HGF. In some embodiments, a kit
can further include oncostatinM, dexamethasone, and/or dimethyl
sulfoxide. In some embodiments, a subject kit includes a basal
media (e.g., RPMI 1640). In some embodiments, a subject kit
includes an antibody specific for a hepatocyte marker protein for
use in a verifying step (or an enrichment step). In some such
embodiments, the hepatocyte marker protein is selected from the
group consisting of: glucose-6-phosphatase, albumin (ALB),
alpha-1-antitrypsin (AAT, also known as SERPINA1), cytokeratin 8
(CK8), cytokeratin 18 (CK18), cytokeratin 8/18 (CK8/18),
asialoglycoprotein receptor 1 (ASGR1), alcohol dehydrogenase 1,
arginase Type I, cytochrome p450 3A4 (CYP3A4), liver-specific
organic anion transporter (LST-1), forkhead box protein A2 (FoxA2),
alpha-fetoprotein (AFP), tryptophan 2,3-dioxygenase (TDO2), and a
combination thereof. In some embodiments, a subject kit includes
assay reagents (e.g, for measuring bile, albumin, and/or urea; for
performing hepatocyte assays such as LDL endocytosis assays,
glycogen synthesis assays, cytochrome P450 1A2 detoxification
activity assays; and the like) for use in a verifying step.
[0183] In addition to the above components, the subject kits may
further include (in certain embodiments) instructions for
practicing the subject methods. These instructions may be present
in the subject kits in a variety of forms, one or more of which may
be present in the kit. One form in which these instructions may be
present is as printed information on a suitable medium or
substrate, e.g., a piece or pieces of paper on which the
information is printed, in the packaging of the kit, in a package
insert, and the like. Yet another form of these instructions is a
computer readable medium, e.g., diskette, compact disk (CD), flash
drive, and the like, on which the information has been recorded.
Yet another form of these instructions that may be present is a
website address which may be used via the internet to access the
information at a removed site.
EXAMPLES
[0184] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., room temperature (RT); base pairs (bp); kilobases (kb);
picoliters (pl); seconds (s or sec); minutes (m or min); hours (h
or hr); days (d); weeks (wk or wks); nanoliters (nl); microliters
(ul); milliliters (ml); liters (L); nanograms (ng); micrograms
(ug); milligrams (mg); grams ((g), in the context of mass);
kilograms (kg); equivalents of the force of gravity ((g), in the
context of centrifugation); nanomolar (nM); micromolar (uM),
millimolar (mM); molar (M); amino acids (aa); kilobases (kb); base
pairs (bp); nucleotides (nt); intramuscular (i.m.); intraperitoneal
(i.p.); subcutaneous (s.c.); and the like.
[0185] Disclosed here is an efficient, high-yield, cost-effective
method for producing hepatocyte-like cells from adipose-derived
stem cells. The hepatocyte-like cells produced by this method have
many of the functional properties of mature hepatocytes in vitro,
and can stably reconstitute a functioning human liver in vivo in a
murine model system. The following materials and methods were used
for Examples 1 and 2 below.
Materials and Methods
[0186] ASC preparation and differentiation. Lipo-aspirates were
obtained as de-identified samples from human donors undergoing
liposuction at Stanford University Medical Center according to
protocol that was approved by the Stanford University Medical
Center IRB. ASC were prepared from these samples as described
(Banas et al, J Gastroenterol Hepatol. 2009 January; 24(1):70-7,
which is hereby incorporated by reference in its entirety). The ASC
were cultured in MESENPRO RS.TM. Medium (Gibco, 12746-012),
passaged at 90% confluence at a 1:4 ratio, and the medium was
changed every other day. For preparation of Chi-Heps, after 2-5
passages, the ASC cells were plated on Martrigel (BD Biosciences,
Cat: 354277) coated dishes. After the cells reached 50% confluence,
endodermal trans-differentiation was induced over 3 days of culture
in the stage 1 medium: RPMI1640 (Gibco, Cat: 12633-012)
supplemented with 100 ng/mL ActivinA (R&D, 338-AC-010), 50
ng/mL Wnt3a (StemRD, Cat: W3A-H-100), 20 ng/mL FGF4 (PeproTech,
Cat: 100-31) and 20% B27 (Gibco Cat: 17504-044). Hepatic
differentiation was then induced over a 13-15 day period by culture
in the stage 2 medium: hepatocyte culture medium (HCM) (Lonza, Cat:
cc-3198) supplemented with 150 ng/mL hepatocyte growth factor (HGF)
(PeproTech, Cat: 100-39), 25 ng/mL FGF4, 30 ng/mL oncostatinM (OSM,
PeproTech, Cat: 300-10), 2.times.10.sup.-5 M dexamethasone (Dex,
Sigma, Cat: D4902), and 0.1% DMSO (Sigma, Cat: C6164). The cells
were then cultured in HCM alone for 2 days prior to transplantation
into TK-NOG mice.
[0187] Preparation of SCi-Hep. Primary ASC were isolated from
lipo-aspirates and suspended in MesenPRO RS.TM. Medium (Gibco, Cat:
12746-012) at 5.times.10.sup.4 cells/ml. Then, the cells were
cultured by the hanging drop method, which resulted in the
formation of spherical cellular aggregates (`spheres`) that
resembled embryoid bodies. Briefly, ASC were plated as individual
drops (each .about.40 ul) in rows on 150 mm petri dishes (BD
Biosciences, Cat 354551) using an 8-channel pipette. After two
days, the dishes were rinsed with PBS, and the spheres were
collected by centrifugation at 150.times.g at 37.degree. C.,
re-suspended at 30 spheres/ml in the stage 1 medium, and seeded on
matrigel-coated dishes (BD Biosciences, Cat: 354262). The sphere
derived cells were then induced to differentiate into hepatocytes
by a 2-stage process. Endodermal transdifferentiation was induced
over a 2-day period by culturing the spheres in the stage 1 medium.
Hepatic differentiation was induced over the subsequent 2-9 day
period by culture in the stage 2 medium. The cells were then
cultured in HCM alone for 2 days prior to transplantation into
TK-NOG mice.
[0188] Preparation of iPS-Heps. ASCs were transfected with
Lentivirus encoding Oct4, Sox2, Klf4 and c-MYC to generate iPS
cells. Four days after infection, the cells were re-plated at
5.times.10.sup.4 cells per 100-mm matrigel (BD Biosciences, Cat:
354277) coated dish in mTeSR-1 medium (StemCell Technologies Inc,
Cat: 05850), and the media was changed every other day. Fifteen
days after infection, individual colonies were manually picked, and
passaged onto new matrigelcoated dishes. After the cells reached
50% confluence, a three-stage hepatic differentiation protocol was
then used. (i) iPS cells were cultured for 4 days in the stage 1
medium. (ii) Over the next 7 days, the cells were induced in
hepatocyte progenitor (HP) medium containing RPM11640 (Gibco, Cat:
12633-012) with 20% B27, 20 ng/ml BMP4 (Pepro Tech, Cat: 120-05ET)
and 20 ng/ml FGF4 (PeproTech, Cat: 100-31). (iii) The cells were
then placed for 15 days in a hepatocyte maturation (HM) medium,
which had hepatocyte growth medium (Lonza, Cat: cc-3198) with 10
ng/ml HGF (PeproTech, Cat: 100-39), 10 ng/ml Oncostatin M (OSM,
PeproTech, Cat: 300-10) and 0.1 uM dexamethasone (Dex, Sigma, Cat:
D4902). The iPS cells used for differentiation were between
passages 10 and 20.
[0189] Immunofluorescence staining. Cells were fixed in 4%
paraformaldehyde for 10 min, followed by blocking with 10% chicken
serum for 30 min. After washing 3 times in PBS with 0.05% Triton
X-100) (TPBS), the cells were incubated with anti-human albumin
(Bethyl Laboratories, Cat: A80-129A, 1:100) or anti-human CK8/18
(abcam, Cat: ab17139, 1:100) primary antibodies overnight at
4.degree. C., and then washed three times with TPBS. Alexa Fluor
488 (Invitrogen, Cat: A21200, 1:1000) or Alexa Fluor 594
(Invitrogen, Cat: A21201, 1:1000)-conjugated secondary antibodies
were then applied for one hour in the dark. Nuclear staining was
assessed using 4,6-diamidino-2-phenylindole (DAPI, Sigma, Cat:
D9542). The images were acquired using a Nikon Eclipse Ni-E imaging
system.
[0190] For assessment of ASGR1 expression in vitro, the cells were
permeabalized with 0.2% Triton X-100 for 10 min followed by washing
three times in PBS with 0.05% Triton X-100 (TPBS). The cells were
pre-incubated with 10% chicken serum for 30 min, followed by
incubation with a 1:50 dilution of Anti-ASGR1 antibody
(Sigma-Aldrich cat #: HPA012852) primary antibody overnight at
4.degree. C., and then washed three times with TPBS. Then, a 1:1000
dilution of Alexa Fluor 488 (Invitrogen, Cat #: A21200,)-conjugated
secondary antibody was applied for one hr in the dark. Nuclear
staining was assessed using 4,6-diamidino-2-phenylindole (DAPI,
Sigma-Aldrich, Cat #: D9542). The images were acquired using a
Nikon Eclipse Ni-E imaging system.
[0191] Periodic Acid-Schiff (PAS) Staining. PAS staining was
performed according to the manufacturer's (Sigma, 395B)
instructions. Briefly, the cells were fixed with 4%
paraformaldehyde for 1 min. After rinsing with distilled water, the
cells were stained with PAS solution for 5 min, then washed with
distilled water, and stained using Schiff's reagent for 15 min. The
cells were then washed once with water and counter-stained in
hematoxylin solution for 90 sec; followed by rinsing 3 times with
distilled water prior to analysis.
[0192] LDL endocytosis. LDL uptake was assessed using the Dil-LDL
assay (Biomedical Technologies Inc., BT-904) according to the
manufacturer's instruction. The cells were pre-incubated in
serum-free medium with 1% BSA for 24 hr. Then 10 ug/mL Dil-LDL was
added for at least five hours at 37.degree. C. After washing 3
times with DPBS, the cells were imaged using were imaged using
Nikon Eclipse Ni-E imaging system.
[0193] Flow cytometry. CK8/18 expression was analyzed by flow
cytometry (FACS, Aida, Software Flowjo). The cells were first
blocked with an Fc receptor-blocking reagent (Miltenyi Biotech,
Germany) according to the manufacturer's instructions, and then
stained with primary antibody anti-human CK8/18 (abcam, Cat:
ab17139, 1:200) for 30 min at 4.degree. C., which was followed by
incubation with a secondary antibody Alexa Fluor 488 (Invitrogen,
Cat: A21200, 1:1000). Appropriately diluted isotype-matched
antibodies (Ebioscience) were used as controls. The data from
10,000 analyzed events were stored and analyzed.
[0194] Human albumin production. Human albumin production was
evaluated using enzyme linked immunosorbent assay (ELISA, E80-129;
Bethyl Laboratories, Montgomery, Tex., USA), which uses a
human-specific antibody that does not cross-react with mouse
albumin. Briefly, cell culture supernatants were collected on day 0
and every three days afterwards. The supernatants were concentrated
using Amicon.RTM. Ultra-4 centrifugal filter units (EMD Millipore,
UFC810024). Blood samples were first centrifuged at 2000 g for 5
min to isolate sera, and then diluted at 1:10000 for assay. Each
data point is the mean.+-.SE of 4 biologically independent samples
analyzed.
[0195] Urea production and CYP450 activity. Cellular urea
production was detected in un-diluted cell culture supernatants
using a urea assay kit (abcam, ab83362) according to the
manufacturer's instructions. Cellular CYP450 activity was measured
using the P450-Glo.TM. CYP3A4 assay (Promega, Cat: V9002) according
to the manufacturer's instructions. In brief, the cells were
cultured with medium containing a luminogenic CYP substrate at
37.degree. C. for 60 min. Then, 25 .mu.l of supernatant was
transferred to 96-well opaque white luminometer plate at room
temperature, and 25 .mu.l of luciferin detection reagent was added
to initiate the luminescent reaction. After a 20 min incubation,
luminescence was measured using an IVIS 100 Imaging System.
[0196] RT-PCR analysis. Total RNA was extracted using RNeasy Plus
kit (Qiagen, Cat: 74134) and (0.5 .mu.g) was reverse-transcribed
using the SuperScript III Reverse Transcriptase (Invitrogen, Cat
#11754-050) according to the manufacturer's guidelines. PCR
analyses were performed using the following TaqMan gene expression
assays: FOXA2 (Hs00232764_m1), AFP (Hs00173490_m1), ALB
(Hs99999922_s1), AAT (Hs01097800_m1), A1AT (Hs00165475_m1), TDO2
(Hs00194611_m1), CD37 (Hs01099648_m1), and CD29 (Hs00559595_m1),
and CD105 (Hs00923996_m1). For statistical analysis of the RT-PCR
data, the measured expression levels for each of the analyzed genes
on the indicated day were normalized relative to its level of
expression in ASC. Then, a one-sample t-test was applied using the
log-transformed expression level to assess the statistical
significance of the expression changes.
[0197] Microarray analysis of gene expression. Total RNA was
harvested and separately processed from 3 independently prepared
cultures of each cell type according to the manufacturer's
instructions. In brief, cells were detached from the dish using the
StemPro Accutase.RTM. Cell Dissociation Reagent (Invitrogen, Cat:
A11105-01). RNA was extracted using the RNeasy Mini Kit (QIAGEN,
Cat: 74104), and 10 ug RNA was labeled, and hybridized to
Affymetrix Human Genome U219 Arrays, and processed according to the
manufacturer's instructions. The resulting image files were
analyzed using Affymetrix Microarray Analysis Suite version 5.0
software. Three biological replicates were analyzed for ASC,
Chi-Hep, SCi-Hep, iPS-Hep and hepatocyte cells. The probe intensity
data generated from all arrays were read into the R software
environment ("""www." followed by "R-project.org", version 2.15.1)
directly from the CEL files using the R/affy package (Gautier et
al, Bioinformatics. 2004 Feb. 12; 20(3):307-15), which was also
used to extract and manipulate probe level data to assess data
quality and to create expression summary measures. Normalization
was carried out using the robust multiarray average (RMA) method
(Irizarry et al, Biostatistics. 2003 April; 4(2):249-64) to
generate one expression measure for each probe set on each array.
Student's t-test was applied to identify the expression changes in
iHep, iPS-Hep and hepatocyte cells as compared to the ASC cells.
The empirical Bayes method (Smyth et al, Stat Appl Genet Mol Biol.
2004; 3:Article3) was applied to adjust the standard deviation
estimation for each probe, and a multipletesting adjustment was
applied to generate the final the p-values using the R/limma
package. Gene expression changes meeting pre-determined criteria
(fold-change >2 and adjusted pvalue <0.01) were selected for
further analysis.
[0198] For illustration of the spatial relationship of the gene
expression profiles of these cells, we defined the distance between
any pair of array data as the squared root of the sum of the
squares of the difference in the level of expression for each gene
on the microarray:
Dist .function. ( Array .times. .times. 1 , Array .times. .times. 2
) = i = 1 N .times. ( E 1 .times. .times. i - E 2 .times. .times. i
) 2 ##EQU00001##
where N represents the total number of probes on the array and
E.sub.1i and E.sub.2i present the log 2 of the normalized
expression level for probe i on array 1 and 2, respectively. In
other words, the distance for a pair of profiles is the Euclidean
distance between the two profiles treated as a vector of size N.
Since there are 3 replicated arrays for each cell type, we defined
the distance between any two cell types as the average of the
distances measured when each of the 3 arrays for one cell type was
compared with each of the 3 arrays for the other cell type (which
produces a total of 9 array pairs) The 2-dimensional illustration
of the spatial relationship among the 4 cell types was created by
putting two triangles, which share the common ASC-hepatocyte axis
next to each other. The length of each side of a triangle is
proportional to the distance between the cell types at the
indicated vertices.
[0199] Principle component analysis (PCA) was also used to
visualize the relatedness of the overall patterns of the gene
expression in the different types of cells analyzed. In brief, the
dimension that explained the largest amount of the variation in the
profiles was identified, and this became the 1st principle
component (PC) shown on the x-axis. Then the dimension that was
orthogonal to PC 1 and explained the largest amount of the
remaining variation was identified, which became the 2nd PC shown
on the y-axis. As a result, the first two components captured the
majority of the variation in the data. Therefore, visualization
could be focused on these components to reduce dimension without
losing a significant amount of information present in the original
data.
[0200] Preparation of TK-NOG mice. All animal experiments were
performed according to protocols approved by the Stanford
Institutional Animal Care and Use Committee. To prepare TK-NOG mice
for transplantation, 8-10 week old TK-NOG mice were treated (i.p.)
with 25 mg/kg ganciclovir (GCV) on day -7 and -5 prior to
transplantation. Then, a 20 .mu.l blood sample was collected 6 days
after the 1st GCV treatment, diluted 1:3 into water, and 10 .mu.l
of the diluted sample was used to measure the serum ALT level using
a Fuji dry-chem 7000 instrument as described (Hasegawa et al,
Biochem Biophys Res Commun. 2011 Feb. 18; 405(3):405-10, which is
hereby incorporated by reference in its entirety). Only mice with
an ALT level >200 U/L were used for cellular transplantation on
day 7. Mice with ALT levels <200 U/L were treated with an
additional dose of 25 mg/kg GCV on day 7, the ALT level was
re-examined on day 13, only mice whose repeat ALT level was >200
U/L were used for cell transplantation on day 14.
[0201] Ultrasound-guided liver injection. Transplanted cells were
directly placed into a liver lobe under ultrasound-guided injection
using a small animal ultrasound system Vevo 2100 (Visualsonics
Canada). Brightness mode (B-mode) was used to acquire
two-dimensional images for an area of interest with MS550s
transducer. The mice were placed under 1.5% isofluorane anesthesia
during this procedure using a single animal vaporizer unit
(EZ-Systems Corp, EZ-108SA). Then, 5.times.10.sup.1 cells were
suspended in 200 .mu.l of William's E medium (Invitrogen,
A12176-01), which was injected into 10 distinct sites in the liver
using a 30 G needle.
[0202] Analysis of tumor formation. To assess tumor formation,
5.times.10.sup.4 Chi-Hep, SCi-Hep or iPS-Hep were harvested and
mixed with 50 .mu.l of matrigel (BD Biosciences, Cat: 354277). NOG
mice were anesthetized using 1.5% isoflurane using individual
vaporizer unit (EZ-Systems Corp, EZ-108SA). An incision was made,
and a slight pressure to both sides of the incision was applied to
expose the kidney. The cells were then injected under the kidney
capsule using a syringe with a 27-gauge needle. After slowly
delivering the cells, a dry swab was placed over the injection site
to prevent leakage. During the procedure, the kidney was kept moist
by application of saline with a cotton-tipped swab. After three to
8 weeks, the mice were sacrificed and the injected kidney was
harvested for histological analysis.
Results
Example 1--Production of Hepatocyte-Like Cells from
Adipocyte-Derived Stem Cells
[0203] Provided is a method for differentiating adipocyte-derived
stem cells (ASCs) into hepatocyte-like cells (iHeps) (FIG. 1A).
ASCs are first cultured using the `hanging drop` method to produce
spherical cellular aggregates (`spheres`) (FIG. 1B). As shown by
analysis of SRY-related HMGbox transcription factor 17 (Sox17)
expression, spherical culture doubles the number of ASCs in a
lipo-aspirate that differentiate into endodermal precursor cells
(FIG. 5). Since the ability of mesenchymal-derived ASCs to
differentiate into endoderm may be rate limiting for hepatocyte
generation, wnt3a was added to the (stage 1) differentiation
medium. Relative to a previous described method of generating iHeps
(Banas et al., J Gastroenterol Hepatol. 2009 January; 24(1):70-7),
the subject method produces a 2.3-fold increase in the number of
ASCs obtained from a lipo-aspirate and a 3-fold increase in the
efficiency (% of hepatocyte-like cells obtained, as assayed using
the CK8/18 marker) after the 2-stage differentiation process is
completed (FIG. 1C and Table 1). This method increases the number
of iHeps obtained from a liter of lipo-aspirate by 7-fold and
reduces the period of in vitro culture required to obtain
biochemically defined hepatocytes at >37% purity to 9 days or
less. Like Chi-Heps, the cells produced by spheroid culture, which
we refer to as SCi-Heps, developed a hepatocyte-like morphology
(FIG. 1B), and exhibited many properties of mature hepatocytes.
They expressed proteins (CK8/18) that are found on hepatocytes
(Figure iC), and had multiple metabolic properties of hepatocytes,
including: LDL endocytosis, glycogen synthesis (FIG. 1D), albumin
secretion, and urea production (FIG. 1E). Moreover, SCi-Heps
expressed multiple hepatocyte specific mRNAs, and had markedly
reduced levels of expression of multiple adipocyte-specific mRNAs
(FIG. 2A), along with reduced expression of an ASC-specific cell
surface protein (CD105) (FIG. 6; FIG. 12).
TABLE-US-00006 TABLE 1 ASCs prepared from 3 different donors were
induced to differentiate into Chi-Heps using a previous method
(Banas et al., J Gastroenterol Hepatol. Jan. 2009; 24 (1): 70-7) or
into SCi-Heps (Spherical Culture iHeps) using the subject method.
The number of ASCs obtained after 3 days (.+-.SEM), the percentage
of cells (.+-.SEM) expressing a hepatocyte marker (CK8/18+) after
differentiation for 12 (Chi-Hep) or 9 (SCi-Hep) days, the number of
days required to complete the hepatocyte differentiation process
and the estimated number of iHeps obtained per liter of
lipo-aspirate are shown. Days of Estimated Differen- Days Cells/
Method Day 3 Cell # % CK8/18.sup.+ tiation Total Liter Chi-Hep (2.7
.+-. 0.3) .times. 10.sup.4 12.3 .+-. 2.8 12 18-30 3.3 .times.
10.sup.8 SCi-Hep (6.1 .+-. 0.2) .times. 10.sup.4 37.7 .+-. 7.7 9 12
2.3 .times. 10.sup.9
[0204] We also compared the properties of Chi- and SCi-Heps with
iPS-Heps, which are ASCs (obtained from the same donor) that were
re-programed into iPS cells after transfection of four genes (Oct4,
Sox2, KIf4 and c-Myc), and then induced to differentiate into
hepatocytes using the Ochiya protocol (Banas et al., J
Gastroenterol Hepatol. 2009 January; 24(1):70-7). The iPS-Heps
expressed a protein (CK8/18) found on hepatocytes (FIG. 1C); could
endocytose LDL, synthesize glycogen (FIG. 7) secrete albumin,
produce urea, and had CYP450 activity (FIG. 8). Of importance,
SCi-Heps produced albumin and urea after only 3 days of in vitro
differentiation, which was 3-6 days before Chi-Heps (FIG. 1E) and
12 days before iPS-Heps (FIG. 8) produced these analytes. ASCs must
first be reprogramed into and then exit from the pluripotent state
before they can differentiate into iPSHeps. In contrast, SCi-Heps
are produced by direct differentiation of ASCs into endoderm, which
explains why they can more quickly produce these analytes.
Consistent with the more rapid differentiation process, SCi-Heps
expressed mRNAs for endodermal (epithelial cell adhesion molecule,
Epcam) and hepatocyte-specific (Albumin, Foxa2) genes within 3 days
after initiation of hepatic differentiation (FIG. 2B). Also,
SCi-Heps had the highest level of albumin production (on a per cell
basis) among the 3 types of ASC-derived cells tested. Their
increased level of albumin production is consistent with the FACS
results indicating that 37% of SCi-Heps expressed a mature
hepatocyte marker, while only .about.12% and 20% of Chi-Heps and
iPS-Heps, respectively, expressed this marker (Figure iC). Thus,
the SCi-Heps method produces an increased number of hepatocyte like
cells from a lipoaspirate, and the cells are prepared within a
timeframe that makes it possible that they could be used in an
acute clinical situation, such as would occur after an overdose of
acetaminophen.
[0205] To further characterize these cells, gene expression
profiling was performed in ASCs, Chi-Heps, SCi-Heps, iPS-Heps and
hepatocytes using microarrays, and the data analysis is described
in the supplemental information. In brief, multiple comparisons
indicated that in vitro differentiation significantly altered the
gene expression pattern in ASCs, and that Chi- and SCi-Heps
expressed a very large number of hepatocyte-specific genes (e.g.,
as published in Table S2 in Xu et al., Cell Transplantation, 2013
Oct. 21; DOI: 10.3727/096368913X432; "Enabling Autologous Human
Liver Regeneration With Differentiated Adipocyte Stem Cells"; the
entirety of which, including figures, tables, supplementary
figures, supplementary tables, etc., is hereby incorporated by
reference). Moreover, two analyses (a space diagram of the gene
expression differences (FIG. 2C) and principal component analysis
(FIG. 9)) indicate that the Chi- and SCi-Heps had a gene expression
profile that was closer to that of hepatocytes than the iPS-Hep
profile. iPS-Heps expressed a larger number of genes that were not
expressed in adipocytes or hepatocytes (FIG. 11). In summary,
SCi-Heps have a gene expression pattern that resembles, but it does
not fully mirror, that of hepatocytes. Since only 37% of the
Sci-Hep cells expressed a mature hepatocyte marker (FIG. 1C), it is
possible that the gene expression pattern in fully differentiated
SCi-Heps could more closely resemble that of hepatocytes than is
suggested by this analysis, since some of the gene expression
changes could be masked (diluted) by the preponderance of less
differentiated cells in the population. Although SCi-Heps were
produced by a different method and were cultured for a much shorter
time period than were Chi-Heps, their gene expression profiles were
extremely similar: 48165 of 49395 probes did not show a significant
expression difference (adjusted p-value >0.01 or fold-change
<2) between these two types of iHeps. The level of expression of
306 genes (0.6%) had a >3-fold (adjusted p<0.01) and only 44
genes (0.08%) had a >10-fold (adjusted p<0.01) difference in
expression in these two cell types (e.g., as published in Table S4
in Xu et al., Cell Transplantation, 2013 Oct. 21; DOI:
10.3727/096368913.times.673432; "Enabling Autologous Human Liver
Regeneration With Differentiated Adipocyte Stem Cells"; the
entirety of which, including figures, tables, supplementary
figures, supplementary tables, etc., is hereby incorporated by
reference). Although a few liver-specific genes (e.g. CYP3A4) were
differentially expressed, the vast majority of genes had a similar
level of expression in Chi-Heps and SCi-Heps, which indicates that
both types of cells have a similar liver-specific gene expression
profile.
[0206] Analysis of gene expression data. A micro-array-based
analysis of global gene expression was performed on ASCs, Chi-Heps,
SCi-Heps, iPS-Heps and hepatocytes. To eliminate the effect that
inter-individual differences could have on the gene expression
profile, ASCs, Chi-Heps, SCi-Heps and iPS-Heps were prepared from
the same individuals. For our initial analysis, the hepatocyte and
ASC gene expression profiles were compared to enable the selection
of a set of genes that met pre-determined criteria (fold-change
>5 and adjusted p value <0.01) for differential expression.
To ensure that the subsequent comparisons were evaluating robust
expression differences between ASCs and hepatocytes, we selected
genes whose expression differences were greater than 5-fold. Based
upon these criteria, this analysis identified 1,129 and 1,437 genes
whose expression was increased or decreased, respectively, in
hepatocytes relative to ASCs (e.g., as published in Table S2 in Xu
et al., Cell Transplantation, 2013 Oct. 21; DOI:
10.3727/096368913.times.673432; "Enabling Autologous Human Liver
Regeneration With Differentiated Adipocyte Stem Cells"; the
entirety of which, including figures, tables, supplementary
figures, supplementary tables, etc., is hereby incorporated by
reference). To quantitatively assess the similarity between the 3
different types of iHeps and hepatocytes, we investigated how many
of these selected genes also had an altered expression pattern
after ASCs underwent the 3 different differentiation processes. For
example, we found that the expression of 494 (or 44%) and 341 (or
24%) of the selected up- or down-regulated genes, respectively,
were similarly altered in Chi-Heps (fold change >2 and adjusted
p value <0.01) (FIG. 11). However, this result indicates that
the level of expression of 1731 (or 67%) of these selected 2566
genes was either not significantly changed in Chi-Heps (relative to
ASCs) or changed in a different direction (compared to
hepatocytes). Similarly, we found using the same criteria that the
expression of 565 (or 50%) and 449 (or 31%) of the selected up- or
down-regulated genes, respectively, were similarly altered in
SCi-Heps (FIG. 11); which indicates that the level of expression of
1552 (or 60%) of the selected 2566 genes was either not
significantly changed in SCi-Heps or changed in a different
direction. These results indicate that Chi- and SCi-Heps have a
similar gene expression profile, which resembles, but certainly
does not fully mirror, that of hepatocytes. Similar results emerged
when this type of comparison was made using the iPS-Hep gene
expression profile (FIG. 11). We also used a second approach to
compare the global gene expression profiles measured in the 3
different types of iHeps. This time, we directly compared the gene
expression profiles in Chi-Heps, SCi-Heps and iPS-Heps with that in
ASCs, and examined the number of gene expression changes that were
also consistently present in hepatocytes (relative to ASCs). This
approach differs from that used above, since this comparison
examines all genes that are differentially expressed in iHeps
relative to ASCs. Out of 1487 genes that were differentially
expressed (fold-change >2 and adjusted p value <0.01) in
Chi-Heps relative to ASCs, 835 (56%) genes also showed consistent
(and significant) expression changes in hepatocyte cells relative
to ASCs (fold change >5 and p value <0.01). Similarly, out of
2372 genes that were differentially expressed in SCi-Heps, 1014
(43%) of these genes also showed consistent (and significant)
expression changes in hepatocyte cells relative to ASCs. However,
the same comparison performed using the iPS-Hep expression data
revealed that only 31% (1372 out of 4456) of the differentially
expressed genes in iPS-Heps showed consistent changes in
hepatocytes. The differences in the iPS-Hep gene expression pattern
are also exemplified by analysis of the genes whose expression
pattern was not changed in hepatocytes relative to ASCs. Of the
16446 genes whose expression pattern was unchanged in hepatocytes
relative to ASCs, the expression of 96% and 92% of these genes were
also unchanged in Chi- and SCi-Heps, respectively (FIG. 11).
However, the level of expression of 18% (or 2969) of these 16446
genes was altered in iPS-Heps. In summary, these analyses indicate
that although their expression pattern does not fully reflect that
of hepatocytes, Chi- and SCi-Heps have a gene expression pattern
that better resembles that of hepatocytes than iPS-Heps. Moreover,
there are a significant number of gene expression changes in
iPS-Heps that are not reflective of that associated with hepatocyte
differentiation.
Example 2--Human Liver Regeneration Using SCi-Heps
[0207] To determine whether SCi-Heps could reconstitute human liver
in vivo, 5.times.10.sup.6 SCi-Heps were transplanted by
ultrasound-guided injection directly into the liver of four
gancyclovir-conditioned TK-NOG mice (FIG. 3A). We previously
demonstrated that the amount of human albumin in the sera of
chimeric mice is an indicator of the extent of liver humanization
(Hasegawa et al, Biochem Biophys Res Commun. 2011 Feb. 18;
405(3):405-10), so the serum human albumin level was serially
assessed over an 8-week period after SCi-Heps transplantation. All
four of these mice produced substantial and increasing amounts of
human serum albumin when monitored over an 8-week period after
transplantation. They had an average human albumin concentration of
0.29 (.+-.0.09) mg/ml in their sera at 4 weeks, which increased to
0.82 (.+-.0.41) at 8 weeks after SCi-Heps transplantation (FIG.
3B). In contrast, none of the 3 mice that were transplanted with
the same number of undifferentiated ASCs produced detectable human
serum albumin.
[0208] Liver histology revealed that the transplanted SCi-Heps
integrated into the liver, produced human albumin, and expressed
markers found on mature human hepatocytes (ASGR1, CK8/18) (FIG.
3C). Since SCi-Heps did not express ASGR1 in vitro just prior to
transplantation (FIG. 10), ASGR1 expression indicated that the
SCi-Heps continued to differentiate after liver engraftment. Since
it was important to determine if the engrafted SCi-Heps would
proliferate in the liver, we investigated whether the SCi-Heps
expressed the Ki67 nuclear protein, which is a specific marker for
cellular proliferation. A significant percentage of the engrafted
SCi-Heps were Ki67 positive (FIG. 3D), which indicates that they
proliferate in situ in the liver. In addition, a significant
percentage of the engrafted SCi-Heps cells also expressed tight
junction protein ZO-1 (FIG. 3D). This indicates that they have
established tight junction interactions with one another, which is
required for human bile duct formation. In contrast, livers
obtained from mice that were transplanted with undifferentiated
ASCs did not have any Ki67 or ZO-1 positive cells (FIG. 3D).
[0209] SCi-Heps do not form tumors. Because malignant potential is
a critical determinant of whether SCi-Heps could be used for liver
regeneration in human subjects, we examined whether SCi-Heps would
form tumors after implantation under the kidney capsule of
immunocompromised NOG mice. No tumors were formed over a 2-month
observation period after 5.times.104 Chi- or SCi-Heps were
implanted into each of 5 NOG mice. Moreover, analysis of tissue
sections indicated that only normal tissue was present in the area
of implantation. In contrast, implantation of the same number of
iPS-Heps into each of 4 NOG mice resulted in the formation of
multiple tumors within 3 weeks, which could be palpated through the
body wall (FIG. 4A). Analysis of the tumor tissue obtained two
months after iPS-Heps transplantation revealed that the tumors
contained tissue derived from all 3 germ cell layers from all 4
mice (FIG. 4B).
Example 3--ASC Culture and iHep Formation Using Stirred Suspension
Culture
[0210] ASCs were placed in spinner flask culture (in the absence of
microcarriers) using numerous different rates of rotation, ranging
from 0 to 150 rotations per minute (rpm). Culture conditions were
identified that allowed ASCs to form aggregates and to proliferate.
After 24 hours in the spinner flask system, the number of ASCs
increased from 5.times.10.sup.5 (starting cell number) to
7.7.+-.0.3.times.10.sup.6 (n=4 independent replicates) which is
approximately a 14-fold increase in the number of cells. In
contrast, a 6-fold increase in the number of ASCs (from
1.times.10.sup.4 to 6.1+0.2.times.10.sup.4) was obtained when ASCs
were cultured for a 48-hour period using the hanging drop method
(i.e., hanging drop suspension culture). Moreover, the morphology
of the ASC cellular aggregates in the spinner flasks resembled the
`spheres` formed using the hanging drop method (FIG. 13).
[0211] Furthermore, the cellular aggregates were able to be
differentiated to endoderm at similar efficiency as using the
hanging drop method. These results indicate that high density
culture (e.g., stirred suspension culture, e.g., spinner flask
culture, bioreactor culture, etc.) will support the growth and
proliferation of ASCs, that ASCs form cellular aggregates (e.g.,
spheres) even in the absence of microcarriers, and that ASCs can
proliferate at a substantially greater rate in stirred suspension
culture (e.g., spinner flasks). Thus, stirred suspension culture
can provide a greater starting number of ASCs (e.g., prior to
contact with a differentiation media), and a similar (or greater)
differentiation efficiency as seen with hanging drop suspension
culture (e.g., spinner flask culture, bioreactor culture, etc.),
thereby producing a much larger total number of iHeps.
[0212] ASCs cultured by attachment, and cellular aggregates
(spheres) produced using (i) spinner flask culture (one day
culture, in the absence of microcarriers), or (ii) hanging drop
suspension culture (two days culture), were either: (1) stained
with the ASC surface marker CD34, then analyzed for CD34 positive
cells using fluorescent activated cell sorting (FACS) (FIG. 14A,
Table 4); or (2) cultured in stage 1 medium for two days, them
stained with endoderm marker Sox17 and analyzed for positive cells
using FACS (FIG. 14B, Table 4). FIGS. 14 A-B demonstrate that ASCs
cultured by stirred suspension culture (in this case, spinner flask
culture in the absence of microcarriers) form a greater percentage
(A) of CD34+ cells (adipose stem cells) and a roughly equal
percentage (B) of SOX17+ cells (endodermal precursor cells)
compared to ASCs cultured by the hanging-drop method.
TABLE-US-00007 TABLE 4 This table demonstrates that ASCs cultured
by stirred suspension culture (spinner flask culture in this case)
form a greater percentage of CD34+ cells (adipose stem cells) and a
roughly equal percentage of SOX17+ cells (endodermal precursor
cells) compared to ASCs cultured by the hanging-drop method.
Percentage is the percent of cells positive for expression of the
marker. [Table 4] Attached ASC Hanging Drop Spinner flask CD34+
cells 52.4% 62.8% 87.5% SOX17+ cells 2.4% 63.3% 61.2%
[0213] FIGS. 15 A-B demonstrate that iHeps produced using spinner
flask culture (SS-Hep) had the specific cytochrome CYP enzymes
CYP3A4 and CYP1A1, and the activity of CYP3A4 was strongly induced
by dexamethasone (Dex) treatment. YJM is a human hepatocyte cell
line. CYP activity was normalized to cell viability. Results are
presented with (+) and without (-) Dex induction.
[0214] FIG. 16 and Table 5 demonstrate that iHeps produced using
spinner flask culture (SS-Hep) secreted an increased level of human
albumin (hAlb) compared to Chi-Heps and SCi-Heps (approximately
16-fold relative to SCi-Heps and approximately 96-fold relative to
Chi-Heps).
TABLE-US-00008 TABLE 5 This table demonstrates that iHeps produced
using spinner flask culture (SS-Hep) secreted an increased level of
human albumin (hAlb) compared to Chi-Heps and SCi-Heps (hAlb is in
units of ng/ml/10.sup.4 cells). [Table 5] ASCs Chi-Heps SCi-Heps
SS-Heps hAlb secretion 0 22 124 2019
[0215] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of the present invention is embodied by the
appended claims.
Sequence CWU 1
1
191426PRTHomo sapiens 1Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu
Ala Ser Cys Trp Ile1 5 10 15Ile Val Arg Ser Ser Pro Thr Pro Gly Ser
Glu Gly His Ser Ala Ala 20 25 30Pro Asp Cys Pro Ser Cys Ala Leu Ala
Ala Leu Pro Lys Asp Val Pro 35 40 45Asn Ser Gln Pro Glu Met Val Glu
Ala Val Lys Lys His Ile Leu Asn 50 55 60Met Leu His Leu Lys Lys Arg
Pro Asp Val Thr Gln Pro Val Pro Lys65 70 75 80Ala Ala Leu Leu Asn
Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95Glu Asn Gly Tyr
Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110Met Asn
Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120
125Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly
130 135 140Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe
Leu Lys145 150 155 160Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val
Thr Ile Arg Leu Phe 165 170 175Gln Gln Gln Lys His Pro Gln Gly Ser
Leu Asp Thr Gly Glu Glu Ala 180 185 190Glu Glu Val Gly Leu Lys Gly
Glu Arg Ser Glu Leu Leu Leu Ser Glu 195 200 205Lys Val Val Asp Ala
Arg Lys Ser Thr Trp His Val Phe Pro Val Ser 210 215 220Ser Ser Ile
Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val225 230 235
240Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu
245 250 255Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys
Lys Lys 260 265 270Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu
Lys Glu Gln Ser 275 280 285His Arg Pro Phe Leu Met Leu Gln Ala Arg
Gln Ser Glu Asp His Pro 290 295 300His Arg Arg Arg Arg Arg Gly Leu
Glu Cys Asp Gly Lys Val Asn Ile305 310 315 320Cys Cys Lys Lys Gln
Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn 325 330 335Asp Trp Ile
Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly 340 345 350Glu
Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe 355 360
365His Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe
370 375 380Ala Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro
Met Ser385 390 395 400Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile
Lys Lys Asp Ile Gln 405 410 415Asn Met Ile Val Glu Glu Cys Gly Cys
Ser 420 4252155PRTHomo sapiens 2Met Ala Glu Gly Glu Ile Thr Thr Phe
Thr Ala Leu Thr Glu Lys Phe1 5 10 15Asn Leu Pro Pro Gly Asn Tyr Lys
Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30Asn Gly Gly His Phe Leu Arg
Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40 45Thr Arg Asp Arg Ser Asp
Gln His Ile Gln Leu Gln Leu Ser Ala Glu 50 55 60Ser Val Gly Glu Val
Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu65 70 75 80Ala Met Asp
Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95Glu Cys
Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105
110Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys
115 120 125Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln
Lys Ala 130 135 140Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp145
150 1553288PRTHomo sapiens 3Met Val Gly Val Gly Gly Gly Asp Val Glu
Asp Val Thr Pro Arg Pro1 5 10 15Gly Gly Cys Gln Ile Ser Gly Arg Gly
Ala Arg Gly Cys Asn Gly Ile 20 25 30Pro Gly Ala Ala Ala Trp Glu Ala
Ala Leu Pro Arg Arg Arg Pro Arg 35 40 45Arg His Pro Ser Val Asn Pro
Arg Ser Arg Ala Ala Gly Ser Pro Arg 50 55 60Thr Arg Gly Arg Arg Thr
Glu Glu Arg Pro Ser Gly Ser Arg Leu Gly65 70 75 80Asp Arg Gly Arg
Gly Arg Ala Leu Pro Gly Gly Arg Leu Gly Gly Arg 85 90 95Gly Arg Gly
Arg Ala Pro Glu Arg Val Gly Gly Arg Gly Arg Gly Arg 100 105 110Gly
Thr Ala Ala Pro Arg Ala Ala Pro Ala Ala Arg Gly Ser Arg Pro 115 120
125Gly Pro Ala Gly Thr Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala
130 135 140Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His
Phe Lys145 150 155 160Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly
Phe Phe Leu Arg Ile 165 170 175His Pro Asp Gly Arg Val Asp Gly Val
Arg Glu Lys Ser Asp Pro His 180 185 190Ile Lys Leu Gln Leu Gln Ala
Glu Glu Arg Gly Val Val Ser Ile Lys 195 200 205Gly Val Cys Ala Asn
Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu 210 215 220Leu Ala Ser
Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu225 230 235
240Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp
245 250 255Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser
Lys Thr 260 265 270Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met
Ser Ala Lys Ser 275 280 2854239PRTHomo sapiens 4Met Gly Leu Ile Trp
Leu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp1 5 10 15Pro Ala Ala Gly
Pro Gly Ala Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 25 30Gly Gly Val
Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45Tyr Cys
Ala Thr Lys Tyr His Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60Asn
Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala65 70 75
80Val Glu Val Gly Ile Val Ala Ile Arg Gly Leu Phe Ser Gly Arg Tyr
85 90 95Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr
Ser 100 105 110Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly
Tyr Asn Thr 115 120 125Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser
Thr Pro Gly Ala Arg 130 135 140Arg Gln Pro Ser Ala Glu Arg Leu Trp
Tyr Val Ser Val Asn Gly Lys145 150 155 160Gly Arg Pro Arg Arg Gly
Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser 165 170 175Leu Phe Leu Pro
Arg Val Leu Asp His Arg Asp His Glu Met Val Arg 180 185 190Gln Leu
Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gln Pro 195 200
205Arg Arg Arg Arg Gln Lys Gln Ser Pro Asp Asn Leu Glu Pro Ser His
210 215 220Val Gln Ala Ser Arg Leu Gly Ser Gln Leu Glu Ala Ser Ala
His225 230 2355206PRTHomo sapiens 5Met Ser Gly Pro Gly Thr Ala Ala
Val Ala Leu Leu Pro Ala Val Leu1 5 10 15Leu Ala Leu Leu Ala Pro Trp
Ala Gly Arg Gly Gly Ala Ala Ala Pro 20 25 30Thr Ala Pro Asn Gly Thr
Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 35 40 45Ser Leu Val Ala Leu
Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro 50 55 60Lys Glu Ala Ala
Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile65 70 75 80Lys Arg
Leu Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu 85 90 95Gln
Ala Leu Pro Asp Gly Arg Ile Gly Gly Ala His Ala Asp Thr Arg 100 105
110Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser Ile
115 120 125Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys
Gly Lys 130 135 140Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr
Phe Lys Glu Ile145 150 155 160Leu Leu Pro Asn Asn Tyr Asn Ala Tyr
Glu Ser Tyr Lys Tyr Pro Gly 165 170 175Met Phe Ile Ala Leu Ser Lys
Asn Gly Lys Thr Lys Lys Gly Asn Arg 180 185 190Val Ser Pro Thr Met
Lys Val Thr His Phe Leu Pro Arg Leu 195 200 2056268PRTHomo sapiens
6Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu Ile Leu1 5
10 15Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gln
Pro 20 25 30Gly Pro Ala Ala Thr Asp Arg Asn Pro Arg Gly Ser Ser Ser
Arg Gln 35 40 45Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser
Ser Pro Ala 50 55 60Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln
Ser Ser Phe Gln65 70 75 80Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser
Leu Tyr Cys Arg Val Gly 85 90 95Ile Gly Phe His Leu Gln Ile Tyr Pro
Asp Gly Lys Val Asn Gly Ser 100 105 110His Glu Ala Asn Met Leu Ser
Val Leu Glu Ile Phe Ala Val Ser Gln 115 120 125Gly Ile Val Gly Ile
Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met 130 135 140Ser Lys Lys
Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys145 150 155
160Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser
165 170 175Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val
Ala Leu 180 185 190Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro
Arg Val Lys Pro 195 200 205Gln His Ile Ser Thr His Phe Leu Pro Arg
Phe Lys Gln Ser Glu Gln 210 215 220Pro Glu Leu Ser Phe Thr Val Thr
Val Pro Glu Lys Lys Lys Pro Pro225 230 235 240Ser Pro Ile Lys Pro
Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250 255Asn Ser Val
Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 2657208PRTHomo sapiens 7Met
Ala Leu Gly Gln Lys Leu Phe Ile Thr Met Ser Arg Gly Ala Gly1 5 10
15Arg Leu Gln Gly Thr Leu Trp Ala Leu Val Phe Leu Gly Ile Leu Val
20 25 30Gly Met Val Val Pro Ser Pro Ala Gly Thr Arg Ala Asn Asn Thr
Leu 35 40 45Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu Ser Arg Ser Arg
Ala Gly 50 55 60Leu Ala Gly Glu Ile Ala Gly Val Asn Trp Glu Ser Gly
Tyr Leu Val65 70 75 80Gly Ile Lys Arg Gln Arg Arg Leu Tyr Cys Asn
Val Gly Ile Gly Phe 85 90 95His Leu Gln Val Leu Pro Asp Gly Arg Ile
Ser Gly Thr His Glu Glu 100 105 110Asn Pro Tyr Ser Leu Leu Glu Ile
Ser Thr Val Glu Arg Gly Val Val 115 120 125Ser Leu Phe Gly Val Arg
Ser Ala Leu Phe Val Ala Met Asn Ser Lys 130 135 140Gly Arg Leu Tyr
Ala Thr Pro Ser Phe Gln Glu Glu Cys Lys Phe Arg145 150 155 160Glu
Thr Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Asp Leu Tyr 165 170
175Gln Gly Thr Tyr Ile Ala Leu Ser Lys Tyr Gly Arg Val Lys Arg Gly
180 185 190Ser Lys Val Ser Pro Ile Met Thr Val Thr His Phe Leu Pro
Arg Ile 195 200 2058194PRTHomo sapiens 8Met His Lys Trp Ile Leu Thr
Trp Ile Leu Pro Thr Leu Leu Tyr Arg1 5 10 15Ser Cys Phe His Ile Ile
Cys Leu Val Gly Thr Ile Ser Leu Ala Cys 20 25 30Asn Asp Met Thr Pro
Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45Pro Glu Arg His
Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 55 60Arg Val Arg
Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp65 70 75 80Lys
Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90
95Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly
100 105 110Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys
Leu Tyr 115 120 125Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys
Glu Leu Ile Leu 130 135 140Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala
Lys Trp Thr His Asn Gly145 150 155 160Gly Glu Met Phe Val Ala Leu
Asn Gln Lys Gly Ile Pro Val Arg Gly 165 170 175Lys Lys Thr Lys Lys
Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190Ile
Thr9233PRTHomo sapiens 9Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu
Leu Leu His Leu Leu1 5 10 15Val Leu Cys Leu Gln Ala Gln Glu Gly Pro
Gly Arg Gly Pro Ala Leu 20 25 30Gly Arg Glu Leu Ala Ser Leu Phe Arg
Ala Gly Arg Glu Pro Gln Gly 35 40 45Val Ser Gln Gln His Val Arg Glu
Gln Ser Leu Val Thr Asp Gln Leu 50 55 60Ser Arg Arg Leu Ile Arg Thr
Tyr Gln Leu Tyr Ser Arg Thr Ser Gly65 70 75 80Lys His Val Gln Val
Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu 85 90 95Asp Gly Asp Pro
Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly 100 105 110Ser Arg
Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met 115 120
125Asn Lys Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp
130 135 140Cys Val Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala
Leu Gln145 150 155 160Asn Ala Lys Tyr Glu Gly Trp Tyr Met Ala Phe
Thr Arg Lys Gly Arg 165 170 175Pro Arg Lys Gly Ser Lys Thr Arg Gln
His Gln Arg Glu Val His Phe 180 185 190Met Lys Arg Leu Pro Arg Gly
His His Thr Thr Glu Gln Ser Leu Arg 195 200 205Phe Glu Phe Leu Asn
Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser 210 215 220Gln Arg Thr
Trp Ala Pro Glu Pro Arg225 23010208PRTHomo sapiens 10Met Ala Pro
Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala1 5 10 15Val Pro
Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 20 25 30Leu
Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40
45Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg
50 55 60Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn
Gly65 70 75 80Thr Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly
Ile Leu Glu 85 90 95Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg
Gly Val Asp Ser 100 105 110Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly
Glu Leu Tyr Gly Ser Glu 115 120 125Lys Leu Thr Gln Glu Cys Val Phe
Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140Tyr Asn Thr Tyr Ser Ser
Asn Leu Tyr Lys His Val Asp Thr Gly Arg145 150 155 160Arg Tyr Tyr
Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175Arg
Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185
190Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser
195 200 20511208PRTHomo sapiens 11Met Trp Lys Trp Ile Leu Thr His
Cys Ala Ser Ala Phe Pro His Leu1 5 10 15Pro Gly Cys Cys Cys Cys Cys
Phe Leu Leu Leu Phe Leu Val Ser Ser 20
25 30Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro
Glu 35 40 45Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser
Ala Gly 50 55 60Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val
Arg Trp Arg65 70 75 80Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys
Ile Glu Lys Asn Gly 85 90 95Lys Val Ser Gly Thr Lys Lys Glu Asn Cys
Pro Tyr Ser Ile Leu Glu 100 105 110Ile Thr Ser Val Glu Ile Gly Val
Val Ala Val Lys Ala Ile Asn Ser 115 120 125Asn Tyr Tyr Leu Ala Met
Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140Glu Phe Asn Asn
Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly145 150 155 160Tyr
Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170
175Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr
180 185 190Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val
His Ser 195 200 20512207PRTHomo sapiens 12Met Ala Glu Val Gly Gly
Val Phe Ala Ser Leu Asp Trp Asp Leu His1 5 10 15Gly Phe Ser Ser Ser
Leu Gly Asn Val Pro Leu Ala Asp Ser Pro Gly 20 25 30Phe Leu Asn Glu
Arg Leu Gly Gln Ile Glu Gly Lys Leu Gln Arg Gly 35 40 45Ser Pro Thr
Asp Phe Ala His Leu Lys Gly Ile Leu Arg Arg Arg Gln 50 55 60Leu Tyr
Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly Thr65 70 75
80Val His Gly Thr Arg His Asp His Ser Arg Phe Gly Ile Leu Glu Phe
85 90 95Ile Ser Leu Ala Val Gly Leu Ile Ser Ile Arg Gly Val Asp Ser
Gly 100 105 110Leu Tyr Leu Gly Met Asn Glu Arg Gly Glu Leu Tyr Gly
Ser Lys Lys 115 120 125Leu Thr Arg Glu Cys Val Phe Arg Glu Gln Phe
Glu Glu Asn Trp Tyr 130 135 140Asn Thr Tyr Ala Ser Thr Leu Tyr Lys
His Ser Asp Ser Glu Arg Gln145 150 155 160Tyr Tyr Val Ala Leu Asn
Lys Asp Gly Ser Pro Arg Glu Gly Tyr Arg 165 170 175Thr Lys Arg His
Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp 180 185 190Pro Ser
Lys Leu Pro Ser Met Ser Arg Asp Leu Phe His Tyr Arg 195 200
20513216PRTHomo sapiens 13Met Gly Ala Ala Arg Leu Leu Pro Asn Leu
Thr Leu Cys Leu Gln Leu1 5 10 15Leu Ile Leu Cys Cys Gln Thr Gln Gly
Glu Asn His Pro Ser Pro Asn 20 25 30Phe Asn Gln Tyr Val Arg Asp Gln
Gly Ala Met Thr Asp Gln Leu Ser 35 40 45Arg Arg Gln Ile Arg Glu Tyr
Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60His Val Gln Val Thr Gly
Arg Arg Ile Ser Ala Thr Ala Glu Asp Gly65 70 75 80Asn Lys Phe Ala
Lys Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg 85 90 95Val Arg Ile
Lys Gly Ala Glu Ser Glu Lys Tyr Ile Cys Met Asn Lys 100 105 110Arg
Gly Lys Leu Ile Gly Lys Pro Ser Gly Lys Ser Lys Asp Cys Val 115 120
125Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala Phe Gln Asn Ala
130 135 140Arg His Glu Gly Trp Phe Met Ala Phe Thr Arg Gln Gly Arg
Pro Arg145 150 155 160Gln Ala Ser Arg Ser Arg Gln Asn Gln Arg Glu
Ala His Phe Ile Lys 165 170 175Arg Leu Tyr Gln Gly Gln Leu Pro Phe
Pro Asn His Ala Glu Lys Gln 180 185 190Lys Gln Phe Glu Phe Val Gly
Ser Ala Pro Thr Arg Arg Thr Lys Arg 195 200 205Thr Arg Arg Pro Gln
Pro Leu Thr 210 21514207PRTHomo sapiens 14Met Tyr Ser Ala Pro Ser
Ala Cys Thr Cys Leu Cys Leu His Phe Leu1 5 10 15Leu Leu Cys Phe Gln
Val Gln Val Leu Val Ala Glu Glu Asn Val Asp 20 25 30Phe Arg Ile His
Val Glu Asn Gln Thr Arg Ala Arg Asp Asp Val Ser 35 40 45Arg Lys Gln
Leu Arg Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 50 55 60His Ile
Gln Val Leu Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly65 70 75
80Asp Lys Tyr Ala Gln Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln
85 90 95Val Arg Ile Lys Gly Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn
Arg 100 105 110Lys Gly Lys Leu Val Gly Lys Pro Asp Gly Thr Ser Lys
Glu Cys Val 115 120 125Phe Ile Glu Lys Val Leu Glu Asn Asn Tyr Thr
Ala Leu Met Ser Ala 130 135 140Lys Tyr Ser Gly Trp Tyr Val Gly Phe
Thr Lys Lys Gly Arg Pro Arg145 150 155 160Lys Gly Pro Lys Thr Arg
Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175Arg Tyr Pro Lys
Gly Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190Thr Val
Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Ala 195 200
20515211PRTHomo sapiens 15Met Ala Pro Leu Ala Glu Val Gly Gly Phe
Leu Gly Gly Leu Glu Gly1 5 10 15Leu Gly Gln Gln Val Gly Ser His Phe
Leu Leu Pro Pro Ala Gly Glu 20 25 30Arg Pro Pro Leu Leu Gly Glu Arg
Arg Ser Ala Ala Glu Arg Ser Ala 35 40 45Arg Gly Gly Pro Gly Ala Ala
Gln Leu Ala His Leu His Gly Ile Leu 50 55 60Arg Arg Arg Gln Leu Tyr
Cys Arg Thr Gly Phe His Leu Gln Ile Leu65 70 75 80Pro Asp Gly Ser
Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly 85 90 95Ile Leu Glu
Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly 100 105 110Val
Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu Leu Tyr 115 120
125Gly Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu Gln Phe Glu
130 135 140Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His
Gly Asp145 150 155 160Thr Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys
Asp Gly Thr Pro Arg 165 170 175Asp Gly Ala Arg Ser Lys Arg His Gln
Lys Phe Thr His Phe Leu Pro 180 185 190Arg Pro Val Asp Pro Glu Arg
Val Pro Glu Leu Tyr Lys Asp Leu Leu 195 200 205Met Tyr Thr
21016170PRTHomo sapiens 16Met Arg Arg Arg Leu Trp Leu Gly Leu Ala
Trp Leu Leu Leu Ala Arg1 5 10 15Ala Pro Asp Ala Ala Gly Thr Pro Ser
Ala Ser Arg Gly Pro Arg Ser 20 25 30Tyr Pro His Leu Glu Gly Asp Val
Arg Trp Arg Arg Leu Phe Ser Ser 35 40 45Thr His Phe Phe Leu Arg Val
Asp Pro Gly Gly Arg Val Gln Gly Thr 50 55 60Arg Trp Arg His Gly Gln
Asp Ser Ile Leu Glu Ile Arg Ser Val His65 70 75 80Val Gly Val Val
Val Ile Lys Ala Val Ser Ser Gly Phe Tyr Val Ala 85 90 95Met Asn Arg
Arg Gly Arg Leu Tyr Gly Ser Arg Leu Tyr Thr Val Asp 100 105 110Cys
Arg Phe Arg Glu Arg Ile Glu Glu Asn Gly His Asn Thr Tyr Ala 115 120
125Ser Gln Arg Trp Arg Arg Arg Gly Gln Pro Met Phe Leu Ala Leu Asp
130 135 140Arg Arg Gly Gly Pro Arg Pro Gly Gly Arg Thr Arg Arg Tyr
His Leu145 150 155 160Ser Ala His Phe Leu Pro Val Leu Val Ser 165
17017352PRTHomo sapiens 17Met Ala Pro Leu Gly Tyr Phe Leu Leu Leu
Cys Ser Leu Lys Gln Ala1 5 10 15Leu Gly Ser Tyr Pro Ile Trp Trp Ser
Leu Ala Val Gly Pro Gln Tyr 20 25 30Ser Ser Leu Gly Ser Gln Pro Ile
Leu Cys Ala Ser Ile Pro Gly Leu 35 40 45Val Pro Lys Gln Leu Arg Phe
Cys Arg Asn Tyr Val Glu Ile Met Pro 50 55 60Ser Val Ala Glu Gly Ile
Lys Ile Gly Ile Gln Glu Cys Gln His Gln65 70 75 80Phe Arg Gly Arg
Arg Trp Asn Cys Thr Thr Val His Asp Ser Leu Ala 85 90 95Ile Phe Gly
Pro Val Leu Asp Lys Ala Thr Arg Glu Ser Ala Phe Val 100 105 110His
Ala Ile Ala Ser Ala Gly Val Ala Phe Ala Val Thr Arg Ser Cys 115 120
125Ala Glu Gly Thr Ala Ala Ile Cys Gly Cys Ser Ser Arg His Gln Gly
130 135 140Ser Pro Gly Lys Gly Trp Lys Trp Gly Gly Cys Ser Glu Asp
Ile Glu145 150 155 160Phe Gly Gly Met Val Ser Arg Glu Phe Ala Asp
Ala Arg Glu Asn Arg 165 170 175Pro Asp Ala Arg Ser Ala Met Asn Arg
His Asn Asn Glu Ala Gly Arg 180 185 190Gln Ala Ile Ala Ser His Met
His Leu Lys Cys Lys Cys His Gly Leu 195 200 205Ser Gly Ser Cys Glu
Val Lys Thr Cys Trp Trp Ser Gln Pro Asp Phe 210 215 220Arg Ala Ile
Gly Asp Phe Leu Lys Asp Lys Tyr Asp Ser Ala Ser Glu225 230 235
240Met Val Val Glu Lys His Arg Glu Ser Arg Gly Trp Val Glu Thr Leu
245 250 255Arg Pro Arg Tyr Thr Tyr Phe Lys Val Pro Thr Glu Arg Asp
Leu Val 260 265 270Tyr Tyr Glu Ala Ser Pro Asn Phe Cys Glu Pro Asn
Pro Glu Thr Gly 275 280 285Ser Phe Gly Thr Arg Asp Arg Thr Cys Asn
Val Ser Ser His Gly Ile 290 295 300Asp Gly Cys Asp Leu Leu Cys Cys
Gly Arg Gly His Asn Ala Arg Ala305 310 315 320Glu Arg Arg Arg Glu
Lys Cys Arg Cys Val Phe His Trp Cys Cys Tyr 325 330 335Val Ser Cys
Gln Glu Cys Thr Arg Val Tyr Asp Val His Thr Cys Lys 340 345
35018728PRTHomo sapiens 18Met Trp Val Thr Lys Leu Leu Pro Ala Leu
Leu Leu Gln His Val Leu1 5 10 15Leu His Leu Leu Leu Leu Pro Ile Ala
Ile Pro Tyr Ala Glu Gly Gln 20 25 30Arg Lys Arg Arg Asn Thr Ile His
Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45Thr Leu Ile Lys Ile Asp Pro
Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60Asn Thr Ala Asp Gln Cys
Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu65 70 75 80Pro Phe Thr Cys
Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95Leu Trp Phe
Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110Gly
His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120
125Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His
Glu His145 150 155 160Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp
Leu Gln Glu Asn Tyr 165 170 175Cys Arg Asn Pro Arg Gly Glu Glu Gly
Gly Pro Trp Cys Phe Thr Ser 180 185 190Asn Pro Glu Val Arg Tyr Glu
Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205Val Glu Cys Met Thr
Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220His Thr Glu
Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro225 230 235
240His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp
245 250 255Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp
Cys Tyr 260 265 270Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala
Ile Lys Thr Cys 275 280 285Ala Asp Asn Thr Met Asn Asp Thr Asp Val
Pro Leu Glu Thr Thr Glu 290 295 300Cys Ile Gln Gly Gln Gly Glu Gly
Tyr Arg Gly Thr Val Asn Thr Ile305 310 315 320Trp Asn Gly Ile Pro
Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335His Asp Met
Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350Tyr
Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360
365Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp
370 375 380Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn
Tyr Met385 390 395 400Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr
Cys Ser Met Trp Asp 405 410 415Lys Asn Met Glu Asp Leu His Arg His
Ile Phe Trp Glu Pro Asp Ala 420 425 430Ser Lys Leu Asn Glu Asn Tyr
Cys Arg Asn Pro Asp Asp Asp Ala His 435 440 445Gly Pro Trp Cys Tyr
Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460Pro Ile Ser
Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu465 470 475
480Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val
485 490 495Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser
Leu Arg 500 505 510Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile
Lys Glu Ser Trp 515 520 525Val Leu Thr Ala Arg Gln Cys Phe Pro Ser
Arg Asp Leu Lys Asp Tyr 530 535 540Glu Ala Trp Leu Gly Ile His Asp
Val His Gly Arg Gly Asp Glu Lys545 550 555 560Cys Lys Gln Val Leu
Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575Ser Asp Leu
Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590Phe
Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600
605Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn
610 615 620Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly
Asn Glu625 630 635 640Lys Cys Ser Gln His His Arg Gly Lys Val Thr
Leu Asn Glu Ser Glu 645 650 655Ile Cys Ala Gly Ala Glu Lys Ile Gly
Ser Gly Pro Cys Glu Gly Asp 660 665 670Tyr Gly Gly Pro Leu Val Cys
Glu Gln His Lys Met Arg Met Val Leu 675 680 685Gly Val Ile Val Pro
Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 700Ile Phe Val
Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile705 710 715
720Leu Thr Tyr Lys Val Pro Gln Ser 72519252PRTHomo sapiens 19Met
Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala1 5 10
15Leu Leu Phe Pro Ser Met Ala Ser Met Ala Ala Ile Gly Ser Cys Ser
20 25 30Lys Glu Tyr Arg Val Leu Leu Gly Gln Leu Gln Lys Gln Thr Asp
Leu 35 40 45Met Gln Asp Thr Ser Arg Leu Leu Asp Pro Tyr Ile Arg Ile
Gln Gly 50 55 60Leu Asp Val Pro Lys Leu Arg Glu His Cys Arg Glu Arg
Pro Gly Ala65 70 75 80Phe Pro Ser Glu Glu Thr Leu Arg Gly Leu Gly
Arg Arg Gly Phe Leu 85 90 95Gln Thr Leu Asn Ala Thr Leu Gly Cys Val
Leu His Arg Leu Ala Asp 100 105 110Leu Glu Gln Arg Leu Pro Lys Ala
Gln Asp Leu Glu Arg Ser Gly Leu 115 120 125Asn Ile Glu Asp Leu Glu
Lys Leu Gln Met Ala Arg Pro Asn Ile Leu 130 135 140Gly Leu Arg Asn
Asn Ile Tyr Cys Met Ala Gln Leu Leu Asp Asn Ser145 150 155 160Asp
Thr Ala Glu Pro Thr Lys Ala Gly Arg Gly Ala Ser Gln Pro Pro 165 170
175Thr Pro Thr Pro Ala Ser Asp Ala Phe Gln Arg Lys
Leu Glu Gly Cys 180 185 190Arg Phe Leu His Gly Tyr His Arg Phe Met
His Ser Val Gly Arg Val 195 200 205Phe Ser Lys Trp Gly Glu Ser Pro
Asn Arg Ser Arg Arg His Ser Pro 210 215 220His Gln Ala Leu Arg Lys
Gly Val Arg Arg Thr Arg Pro Ser Arg Lys225 230 235 240Gly Lys Arg
Leu Met Thr Arg Gly Gln Leu Pro Arg 245 250
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