U.S. patent application number 13/460315 was filed with the patent office on 2012-09-06 for compositions, methods, and devices for treating liver disease.
This patent application is currently assigned to Massachusetts Institute of Technology. Invention is credited to Biju Parekkadan, Martin L. Yarmush.
Application Number | 20120225130 13/460315 |
Document ID | / |
Family ID | 39402354 |
Filed Date | 2012-09-06 |
United States Patent
Application |
20120225130 |
Kind Code |
A1 |
Yarmush; Martin L. ; et
al. |
September 6, 2012 |
COMPOSITIONS, METHODS, AND DEVICES FOR TREATING LIVER DISEASE
Abstract
Described are compositions and methods for treating liver
disease, e.g., acute liver disease, using bone marrow-derived stem
cells and bone marrow-derived stem cell conditioned media.
Inventors: |
Yarmush; Martin L.; (Newton,
MA) ; Parekkadan; Biju; (Cambridge, MA) |
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
The General Hospital Corporation d/b/a massachusetts General
Hospital
Boston
MA
|
Family ID: |
39402354 |
Appl. No.: |
13/460315 |
Filed: |
April 30, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11871071 |
Oct 11, 2007 |
8172784 |
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13460315 |
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60850778 |
Oct 11, 2006 |
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60851234 |
Oct 12, 2006 |
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60923138 |
Apr 12, 2007 |
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60995316 |
Sep 26, 2007 |
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Current U.S.
Class: |
424/572 |
Current CPC
Class: |
A61K 35/28 20130101;
A61P 37/00 20180101; G01N 33/5073 20130101; A61M 1/3472 20130101;
G01N 33/5067 20130101; C12N 5/0663 20130101; C12N 2502/1358
20130101; A61P 1/16 20180101; A61M 1/3489 20140204; C12M 29/16
20130101; C12N 5/067 20130101; G01N 2500/00 20130101; A61M 1/3689
20140204; G01N 2800/085 20130101; A61M 1/3687 20130101 |
Class at
Publication: |
424/572 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 1/16 20060101 A61P001/16; A61P 37/00 20060101
A61P037/00 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Grant
Nos. R01 DK43371, K18 DK076819, and K08 DK66040 awarded by the
National Institutes of Health. The Government has certain rights in
the invention.
Claims
1.-31. (canceled)
32. A method comprising preparing a multipotent stromal cell (MSC)
lysate, and formulating the MSC lysate with a pharmaceutically
acceptable carrier.
33. The method of claim 32, wherein the MSC lysate is prepared by
sonicating MSC.
34. The method of claim 32, wherein the MSC lysate is formulated
for parenteral administration.
35. A pharmaceutical composition comprising a multipotent stromal
cell (MSC) lysate, and a pharmaceutically acceptable carrier.
36. A method comprising administering an effective amount of a
multipotent stromal cell (MSC) lysate to a subject in need
thereof.
37. The method of claim 36, wherein the subject is a human
subject.
38. The method of claim 36, wherein the subject has an autoimmune
disease.
39. The method of claim 36, wherein the subject has liver
disease.
40. The method of claim 36, wherein the subject has acute liver
failure.
41. The method of claim 36, wherein the MSC lysate is xenogeneic to
the subject.
42. The method of claim 36, wherein the subject is a farm animal, a
sport animal, a primate, a horse, a dog, a cat, a rat, or a mouse.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit under 35 USC
.sctn.119(e) of U.S. Provisional Patent Application Ser. No.
60/850,778, filed on Oct. 11, 2006, U.S. Provisional Patent
Application Ser. No. 60/851,234, filed on Oct. 12, 2006, U.S.
Provisional Patent Application Ser. No. 60/923,138, filed on Apr.
12, 2007, and U.S. Provisional Patent Application entitled
"Compositions and Methods for Treating Liver Disease," filed on
Sep. 26, 2007, under Attorney Docket No. 00786-667P04.sup.-MGH
3241/3436, Ser. No. ______. The entire contents of all of the
foregoing applications are hereby incorporated by reference.
TECHNICAL FIELD
[0003] This invention relates to compositions and methods for
treating diseases and disorders related to degeneration of the
liver.
BACKGROUND
[0004] Liver failure is the inability of the liver to perform its
normal synthetic and metabolic function as part of normal
physiology. Acute liver failure can occur in as little as 48 hours,
and typically coincides with the loss or dysfunction of 80-90% of
liver cells. Liver failure is a life-threatening condition that
demands urgent medical care.
[0005] Acute liver failure has an estimated prevalence of 2000
cases per year and a mortality rate of approximately 80
percent.
[0006] In many cases, orthotopic liver transplantation is the only
effective treatment for acute liver failure. The use of such
transplants, however, is limited due to donor shortages, high cost,
and the requirement for life-long immunosuppression. Thus, there is
a clear need for alternative treatments for the treatment of liver
failure.
SUMMARY
[0007] The present invention is based, at least in part, on the
surprising discovery that multipotent stromal cell (MSC)-based
therapy provides trophic support to the injured liver. Thus,
administration of MSC conditioned media (MSC-CM), or the use of a
bioartificial liver device (BAL) with MSCs, can be used to treat
subjects with liver disease.
[0008] In one aspect, the present invention features methods of
preparing a pharmaceutical composition for the treatment of liver
disease. In some embodiments, the methods include providing a
population of undifferentiated multipotent stromal cells (MSCs),
culturing the undifferentiated MSCs in a medium, e.g., at about 80%
confluency, e.g., using about 1.times.10.sup.2 cells/cm.sup.2 to
1.times.10.sup.4 cells/cm.sup.2, e.g., for a time sufficient for a
desired amount of active factors to be produced, e.g., 6, 12, 18,
24, 36, or 48 hours, e.g., 12-48 hours, 12-36 hours, 24-36 hours,
e.g., 24 hours, obtaining the medium (MSC-conditioned medium
(MSC-CM)), fractionating the medium using known fractionation
methods, e.g., by one or more of (1) charge, (2) size, and/or (3)
heparin sulfate binding, selecting a fraction of the medium that is
capable of one or both of promoting hepatocyte proliferation and/or
inhibiting hepatocyte death, and optionally, formulating the
selected fraction for administration to a mammal, e.g., for
systemic administration, e.g., by intravenous administration.
Preferably, the MSCs are maintained in culture in an
undifferentiated state. In general, the MSC-CM and active fractions
thereof will be cell-free. In some embodiments, the composition is
concentrated 25-fold. In some embodiments, the composition includes
a serum free tissue culture medium or PBS. In some aspects, the
methods include lyophilizing the composition.
[0009] In another aspect, the present invention features
pharmaceutical compositions including MSC-CM or an active fraction
thereof, e.g., produced by a method described herein.
[0010] In a further aspect, the present invention features
extracorporeal liver support devices. In some embodiments, the
devices include a purified population of undifferentiated MSCs. In
some embodiments, the devices also include a population of primary
hepatocytes. Thus, the invention provides systems for treating
blood or plasma from a mammal. The systems include an
extracorporeal bioreactor (EB) including a fluid treatment
compartment and a cell compartment, and a selectively permeable
barrier separating the fluid treatment compartment and the cell
compartment, wherein the cell compartment comprises a population of
undifferentiated multipotent stromal cells (MSCs). In addition to
the MSCs, the cell compartment can also include a population of
primary hepatocytes. The selectively permeable barrier can be, for
example, a bundle of hollow fibers, or a flat membrane; suitable
barriers are known in the art.
[0011] In general, the EB also includes a biological fluid inlet
and a biological fluid outlet, wherein the biological fluid inlet
and outlet permit fluid communication between the fluid treatment
compartment and a bloodstream of the mammal.
[0012] The systems will preferably also include one or a plurality
of pumps for circulating the blood or plasma, e.g., from the
subject through the fluid treatment compartment of the EB and back
to the subject.
[0013] The systems can also include an ultrafiltration cartridge in
fluid communication with the subject and/or fluid treatment
compartment, such that blood from the subject is separated into
ultrafiltrate/plasma and cellular components, and the UF/plasma is
circulated through the fluid treatment compartment of the EB while
the cellular components of the blood are returned to the
subject.
[0014] The systems described herein can be used in methods of
treating liver disease in a subject. The methods include
identifying a subject having a liver disease; providing a system
for treating blood or plasma from a mammal that includes an
extracorporeal bioreactor (EB) including a fluid treatment
compartment and a cell compartment, and a selectively permeable
barrier separating the fluid treatment compartment and the cell
compartment, wherein the cell compartment includes a population of
undifferentiated multipotent stromal cells (MSCs) (and optionally a
population of primary hepatocytes); and exposing the subject's
plasma or blood to the MSCs in the EB.
[0015] The systems can also be used in methods for treating blood
or plasma from a subject having a liver disease. The methods
include identifying a subject having a liver disease; providing a
system as described herein; removing blood or plasma from the
subject; introducing the blood or plasma into the fluid treatment
compartment of the EB; and allowing the blood or plasma to flow
through and exit the fluid treatment compartment, thereby treating
the blood or plasma.
[0016] In an additional aspect, the present invention provides
methods of treating liver disease in a subject. In some
embodiments, these methods include identifying a subject in need of
treatment, and administering to the subject an effective amount of
a pharmaceutical composition including MSC-CM or an active fraction
thereof, e.g., produced by a method described herein. The subject
can be identified, for example, by evaluating the level of a serum
marker of liver function. The methods can include selecting a
subject on the basis that they have a liver disease, e.g., as
determined by evaluating the level of a serum marker of liver
function.
[0017] A number of serum markers of liver function are known in the
art, including, but not limited to, lactate dehydrogenase (LDH),
alkaline phosphatase (ALP), aspartate aminotransferase (AST),
alanine aminotransferase (ALT), serum bilirubin, albumin and/or
globulins.
[0018] In certain embodiments, treatment continues until the
subject's level of a serum marker of liver function is within the
normal range as determined by the subject's clinician, e.g., for a
time sufficient to ameliorate a symptom or improve a clinical
parameter of the liver disease, e.g., a time selected by a
clinician or health care provider. In some embodiments, the liver
disease to be treated is acute liver failure. In some embodiments,
the acute liver failure is fulminant hepatic failure. In some
embodiments, the liver disease to be treated is liver fibrosis. In
some aspects, the composition is administered using an intravenous
bolus technique.
[0019] In yet another aspect, the present invention provides
additional methods for treating liver disease in a subject. These
methods include identifying a subject in need of the treatment,
providing a system including an extracorporeal bioreactor (EB)
including a purified population of undifferentiated multipotent
stromal cells (MSCs), and exposing the subject's plasma or blood to
the MSCs in the EB. In some embodiments, treatment can continue for
a time sufficient to ameliorate a symptom or improve a clinical
parameter of the liver disease, e.g., a time selected by a
clinician or health care provider. In some embodiments, the subject
is identified by evaluating the level of a serum marker of liver
function, e.g., lactate dehydrogenase (LDH), alkaline phosphatase
(ALP), aspartate aminotransferase (AST), alanine aminotransferase
(ALT), serum bilirubin, albumin, and/or globulins. In some
embodiments, the liver support device also includes a purified
population of primary hepatocytes.
[0020] In another aspect, the present invention provides methods
for identifying a biologically active component for the treatment
of a liver disease, by (i) obtaining a sample of a medium
containing factors secreted from a purified undifferentiated
multipotent stromal cell population (MSC), (ii) obtaining a
fraction of the medium using fractionation methods known in the
art, (iii) assaying the ability of the fraction to promote
hepatocyte proliferation or inhibit hepatocyte death, e.g., in
vitro or in vivo, (iv) selecting a fraction of the medium that is
capable of promoting hepatocyte proliferation or inhibiting
hepatocyte death, (v) optionally repeating the steps of (i) to
(iv), and (vi) identifying one or more molecules present in the
selected fraction. In some embodiments, distinct fractions are
obtained according to one or both of size or charge. In some
embodiments, the sample of the medium is a heparin sulfate binding
fraction of the medium.
[0021] The term "acute liver failure" includes, but is not limited
to, the conditions referred to by the terms hyperacute liver
failure, acute liver failure, subacute liver failure, and fulminant
hepatic failure (FHF).
[0022] To "treat" means to reduce one or more symptoms of liver
disease, e.g., to promote an improvement in liver function as
evaluated using one or more serum markers of liver function (e.g.,
lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate
aminotransferase (AST), alanine aminotransferase (ALT), serum
bilirubin, albumin and globulins), and, thus, to ameliorate at
least one symptom, e.g., a symptom associated with parenchymal cell
loss in an organ, for example, the liver.
[0023] An "effective amount" is an amount sufficient to produce a
beneficial or desired result. For example, a therapeutically
effective amount is one that achieves a desired therapeutic effect,
e.g., to ameliorate a symptom or improve a clinical parameter of a
disease, e.g., a liver disease. This amount can be the same or
different from a prophylactically effective amount, which is an
amount necessary to prevent onset of disease or disease
symptoms.
[0024] An "active fraction" is a fraction that can promote
hepatocyte proliferation or inhibit hepatocyte death, e.g., in a
culture of primary hepatocytes.
[0025] As used herein, the terms "patient," "subject," and
"individual" are used interchangeably to refer to a mammal,
including, without limitation, humans, farm animals, sport animals,
pets, primates, horses, dogs, cats, rats, and mice. In some
embodiments, the subject is a non-human mammal, e.g., an
experimental animal or veterinary subject.
[0026] Unless otherwise defined, 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. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0027] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0028] FIG. 1 is a bar graph showing Bromodeoxyuridine
(5-bromo-2-deoxyuridine, BrdU) incorporation in activated SCs as a
function of MSC:SC ratio. Proliferation of activated SCs is
inhibited by indirect co-culture with MSCs. Data represent the mean
of two experiments performed in triplicate. Error bars are standard
deviation. *p<0.05 compared to SCs alone (MSC:SC=0).
[0029] FIG. 2 is an image of a representative RT-PCR agarose gel
showing IL-10 mRNA expression levels. GAPDH served as an internal
control for RT-PCR analysis.
[0030] FIG. 3 is a bar graph showing mean ELISA results from two
independent experiments, each of which was performed in triplicate.
Error bars are standard deviation. **p<0.01 compared to
unstimulated MSCs.
[0031] FIG. 4 is a bar graph showing cytokine secretion in
monoculture and direct co-culture of SCs and MSCs (1:1 ratio). IL-6
and TNF-.alpha. concentrations were measured by ELISA with
species-specific antibodies after 2 days of culture. Results are
the mean of two experiments performed in triplicate. Error bars are
standard deviation.
[0032] FIG. 5 is a bar graph showing ELISA measurement of IL-10
secretion after 2 days of monoculture or 1:1 direct co-culture of
MSCs and SCs.
[0033] FIG. 6 is a bar graph showing IL-10 secretion as a function
of MSC number after 4 days of 1:1 indirect co-culture with
activated SCs, determined by RT-PCR. Results are the mean of two
independent experiments performed in triplicate. Error bars are
standard deviation. **p<0.01 compared to SCs alone
(MSC:SC=0).
[0034] FIG. 7 is a bar graphs showing the inhibition of IL-10
secretion by MSCs in direct (1:1) co-culture with activated SCs
after treatment with anti-IL-6 neutralizing antibody. Results are
the mean of two experiments performed in triplicate. Error bars are
standard deviation. **p<0.01 compared to no antibody.
[0035] FIG. 8 is a bar graph showing Hepatocyte Growth Factor (HGF)
secretion after 2 days of monoculture or 1:1 direct co-culture of
SCs and MSCs. Results are the mean of two experiments performed in
triplicate.
[0036] FIG. 9 is a schematic model of the paracrine effects of
MSC-derived factors on activated SCs. Autocrine factors synthesized
by SCs are not represented in this model. Release of IL-6 by
activated SCs leads to the secretion of IL-10 by MSCs. Induced
IL-10, along with constitutively secreted TNF-.alpha., inhibit of
SC proliferation and collagen synthesis. The marginal effect of
IL-10 on SC proliferation is denoted by the smaller font size. SCs
undergo apoptosis after co-culture with MSCs due to increased
levels of HGF.
[0037] FIG. 10 is a Kaplan-Meier survival analysis of Gal-N rats
after MSC-CM treatment. *p=0.017 (Log Rank). MSC-CM, mesenchymal
stem cell-conditioned medium; FHF, fulminant hepatic failure;
Gal-N, D-galactosamine.
[0038] FIGS. 11A-11F are bar graphs showing systemic levels of
(11A) IL-1.beta.; (11B) TNF-.alpha.; (11C) IL-6; (11D) IL-2; (11E)
IL-1ra; and (11F) IL-10 following exposure to Gal-N. Data shown are
mean.+-.standard deviation of experiments performed in triplicate.
*p<0.05, **p<0.001.
[0039] FIG. 12A is a bar graph showing scores determined by
semi-quantitative histological exam as described in Example 6. Data
shown are mean.+-.standard error of the mean of 10 random high
power fields per animal. Solid Bar=100 .mu.m. *p=0.024,
**p=0.004.
[0040] FIG. 12B is a bar graph showing quantification of
infiltrating immune cells by digital image analysis. Data shown are
mean.+-.standard error of the mean of 10 random high power fields
per animal. Solid Bar=100 .mu.m. *p=0.024, **p=0.004.
[0041] FIG. 13 is a bar graph showing the results of quantification
of TUNEL-positive nuclei using digital image analysis. Data are
reported as mean.+-.standard error of the mean for 10 random fields
per animal. *p=0.009.
[0042] FIG. 14 is a bar graph showing isolated primary rat
hepatocyte apoptosis using in vitro culture. Data are shown as
mean.+-.standard deviation. *p=0.005.
[0043] FIG. 15 is a bar graph showing PCNA-reactive nuclei were
quantified by digital image analysis. Data are reported as
mean.+-.standard error of the mean for 10 random fields per animal.
*p=0.04.
[0044] FIG. 16 is a bar graph showing the results of quantification
of changes in gene expression by real time PCR(RT-PCR) after MSC-CM
treatment. Abbreviations are: oncostatin M (OSM); .alpha.-1.beta.
adrenergic receptor (AR); transforming growth factor-.alpha.
(TGF-.alpha.); hepatocyte growth factor (HGF); tumor necrosis
factor-.alpha. (TNF-.alpha.); epidermal growth factor (EGF);
interleukin 6 (IL-6); stem cell factor (SCF); heparin-binding
epidermal growth factor-like growth factor (HB-EGF); and tissue
metalloproteinase 3 (TIMP3).
[0045] FIG. 17A is a bar graph showing quantification of
BrdU-positive hepatocytes by image analysis. Data are shown as
mean.+-.standard deviation of two separate experiments in
duplicate. *p<0.05, **p<0.01; ns=not significant.
[0046] FIG. 17B is a bar graph showing albumin secretion. Data are
shown as mean.+-.standard deviation of two separate experiments in
duplicate. *p<0.05, **p<0.01; ns=not significant.
[0047] FIG. 17C is a bar graph showing urea synthesis. Data are
shown as mean.+-.standard deviation of two separate experiments in
duplicate. *p<0.05, **p<0.01; ns=not significant.
[0048] FIG. 18 is a line graph showing the results of Kaplan-Meier
survival analysis of Gal-N administered rats treated with cell
transplants or lysates. Time points of interventions are stated
above survival plots. Results are cumulative data of two
independent experiments (N=8 per each group) using different
batches of MSCs. P-value determined by Log Rank Test.
[0049] FIG. 19A is a line graph showing Kaplan-Meier survival
analysis of Gal-N administered rats treated with concentrated
MSC-CM.
[0050] FIG. 19B is a bar graph showing dose response of animal
survival 72 hours after liver failure induction as a function of
MSC mass from which MSC-CM was derived. Controls received vehicle
or fibroblast conditioned medium (fibroblast-CM). Time points of
interventions are stated above survival plots. Results for both
panels are cumulative data of two independent experiments using
different batches of MSC-CM (N=8 per each group). P-value
determined by Log Rank Test.
[0051] FIG. 20 is a schematic representation of an exemplary
extracorporeal circuit. Briefly, circuits were primed with sterile,
heparinized Sprague-Dawley rat plasma, which was filtered
immediately before use. The rat was administered 100 U heparin (0.1
ml) systemically through the venous line 5 minutes before the start
of perfusion. The arterial and venous lines were subsequently
connected to the extracorporeal circuit. Arterial blood was pumped
at 0.55-0.85 ml/minute through #13 MASTERFLEX silicone tubing
(Cole-Palmer Instrument Co.) by a digital variable-speed
peristaltic pump (Cole-Palmer Instrument Co.) drive. A plasma
separator (MICROKROS, membrane material: mixed esters of cellulose,
membrane pore size: 0.2 um, membrane surface area: 16 cm.sup.2;
Spectrum Laboratories Inc., Laguna Hills, Calif.) was placed after
the pump. Separated plasma was pumped though the BAL by means of
second and third peristaltic pump at a flow rate of 0.1 ml/minute.
The separated plasma and remaining blood components were reunited
before entering a bubble trap. Reconstituted blood returned to the
animal through the venous cannula. During perfusion, the heparin
(41.5 U/mL) with 5% dextrose solution was administered continuously
through the venous line at a rate of 0.2 ml/hour via a syringe
infusion pump. In this configuration, the dead volume of the entire
perfusion system was 12 ml, of which, 6 ml were accounted for by
the BAL. Oxygenating gas (21% O.sub.2, 5% CO.sub.2, 74% N.sub.2)
flow was established through the chamber above the internal
membrane of the BAL.
[0052] FIGS. 21A-21B are bar graphs showing data collected from
extracorporeal bioreactor (EB) studies. Animals were treated with
an MSC-EB, using a 3T3 fibroblast-based bioreactor (fibroblast-EB)
and an acellular bioreactor (acellular-EB) as controls. Serum
biomarkers of liver injury, aspartate aminotransferase (AST, 21A)
and alanine aminotransferase (ALT, 21B) preceding and 24 hours
after treatment with a MSC-EB (n=5) or an acellular-EB (n=3). Due
to mortality, n=1 in the acellular group after treatment. P-value
determined by student's t-test analysis.
[0053] FIG. 21C is a line graph showing the results of Kaplan-Meier
survival analysis of Gal-N administered rats treated with EBs. Time
points of interventions are stated above survival plots. Each
result shown was from an independent experiment using different
batches of MSCs. P-value determined by Log Rank Test.
[0054] FIG. 22A is a schematic illustration of the experimental
design of an adoptive transfer study. Gal-N injured rats were
treated with vehicle or MSC-CM followed by infusion of
In.sup.111-labeled leukocytes.
[0055] FIG. 22B is a bar graph illustrating leukocyte count and
differentials. Whole blood was harvested after cannulation and
analyzed for peripheral blood cells using a commercially available
flow cytometer (white blood cells (WBC); neutrophils (NE);
lymphocytes (LY); monocytes (MO); basophils (BA); eosinophils
(EO).
[0056] FIG. 22C is a bar graph showing the in vivo distribution of
radiolabeled leukocytes following sub-lethal Gal-N treatment.
[0057] FIG. 23A-C shows that MSC-CM is composed of high levels of
chemokines that correlate with survival benefit seen in FHF.
Serum-free MSC-CM was analyzed using an antibody array for 174
specified proteins. FIG. 23A is a bar graphs showing the results o
densitometry analysis of spotted antibody array results. Data are
presented as spot intensity relative to the negative control and
normalized to positive control. FIG. 23B is a pie chart showing
cluster analysis of MSC secreted proteins based on reported
function. MSC-CM was fractionated over a heparin-agarose column
into heparin bound and unbound fractions. FIG. 23C is a line graph
showing the results of Kaplan-Meier survival analysis of Gal-N
administered rats treated with the (+) heparin MSC-CM and (-)
heparin MSC-CM. Time points of interventions are stated above
survival plots. Results for (C) are cumulative data of two
independent experiments using different batches of MSC-CM (N=8 per
each group). P-value determined by Log Rank Test.
[0058] FIG. 24 is a bar graph of Urea synthesis in hepatocytes
cultured on growth-arrested NIH-3T3-J2 fibroblasts for 14 days
supplemented with 5 ng/ml SDF-1a or AMD3100, a CXCR4 antagonist.
Data represent mean.+-.standard error of the mean (s.e.m.) of two
independent experiments performed in triplicate.
[0059] FIGS. 25 and 26 are schematic illustrations of examples of
bioartificial liver systems.
[0060] FIG. 27 is a schematic illustration of a hollow fiber
bioreactor.
[0061] FIG. 28 is a schematic illustration of a cross section at
line A-A through the hollow fiber bioreactor shown in FIG. 27.
[0062] FIG. 29 is a schematic illustration of a flat-plate or
two-compartment bioreactor
DETAILED DESCRIPTION
[0063] The present invention is based, at least in part, on the
surprising discovery that multipotent stromal cells (MSCs), and
agents secreted therefrom, provide trophic support to the injured
liver. Without wishing to be bound by theory, it is believed that
this therapeutic effect is a result of inhibiting hepatocellular
death and stimulating hepatocellular regeneration. Thus, methods of
treatment that include parenteral administration of a cell-free
MSC-conditioned media composition, or the use of artificial liver
devices including MSCs, can be used to treat subjects with liver
disease, as described herein.
[0064] At least in part, the data presented herein demonstrate that
MSCs, and agents secreted therefrom, are capable of modulating the
function of activated stellate cells via paracrine mechanisms,
treating an inflammatory condition, and providing trophic support
to the injured liver by inhibiting hepatocellular death and
stimulating hepatocyte regeneration. Accordingly, MSC-based
therapy, as described herein, is useful in the treatment of any
acute and/or chronic condition, including autoimmune and
inflammatory conditions, in which the inhibition of cell death and
stimulation of tissue repair would be beneficial. Such conditions
include, but are not limited to, for example, liver disease (e.g.,
fibrosis, cirrhosis, acute liver failure, fulminant hepatic failure
(FHF), and any other degenerative liver disease), ischemic injury
(e.g., myocardial infarction and stroke), kidney failure, acute
pancreatitis, and autoimmune disease. MSC based therapy may also be
beneficial in any condition or disorder in which the action of
trophic secreted molecules are beneficial.
MSC Therapy
[0065] As described herein, MSCs and agents secreted therefrom,
e.g., in an MSC conditioned media composition, can be used to treat
liver diseases or disorders involving the loss or damage of
parenchymal liver cells in a subject. In general, the etiology of
these diseases or disorders can be a local or systemic inflammatory
response. However, other liver diseases or disorders associated
with the loss or damage of parenchymal liver cells are also
included.
[0066] As described herein, MSCs and agents secreted therefrom,
e.g., in an MSC conditioned media composition, are particularly
useful in the treatment of acute liver failure in a subject, for
example, FHF. In some embodiments, a subject will be selected for
treatment using the compositions and methods described herein based
on a positive diagnosis of liver disease. Liver disease can be
diagnosed using methods known in the art, for example, using one or
more of the liver function assays described herein, or based on the
judgment of a clinician.
[0067] In some embodiments, treatment is performed using
administration of a MSC conditioned medium (MSC-CM) composition, or
an active fraction thereof. In some embodiments, treatment is
performed using a MSC-based extracorporeal bioreactor (MSC-EB).
MSC Isolation
[0068] Multipotent stromal cells (MSCs) are also referred to in the
art as bone marrow-, adipose-, umbilical cord-, and
placental-derived mesenchymal stem cells, and bone marrow-,
adipose-, umbilical cord-, and placental-derived stromal cells.
MSCs can be isolated using methods known in the art, e.g., from
bone marrow mononuclear cells, umbilical cord blood, adipose
tissue, placental tissue, based on their adherence to tissue
culture plastic. For example, MSCs can be isolated from
commercially available bone marrow aspirates (see Example 1).
Purification of MSCs can be achieved using methods known in the
art, e.g., the methods described herein, including but not limited
to FACS.
[0069] In some embodiments, an MSC preparation will include a
solution. This solution can contain those components required to
support MSC survival and growth. Such components are routinely
present in commercially available tissue culture media, for
example, Dulbecco's modified Eagle's medium (DMEM). Such components
include, for example, amino acids, vitamins, a carbon source
(natural and non-natural), salts, sugars, plant derived
hydrolysates, sodium pyruvate, surfactants, ammonia, lipids,
hormones or growth factors, buffers, non-natural amino acids, sugar
precursors, indicators, nucleosides and/or nucleotides, butyrate or
organics, DMSO, animal derived products, gene inducers, non-natural
sugars, regulators of intracellular pH, betaine or osmoprotectant,
trace elements, minerals, non-natural vitamins. Additional
components that can be used to supplement a commercially available
tissue culture medium include, for example, animal serum (e.g.,
fetal bovine serum (FBS), fetal calf serum (FCS), horse serum
(HS)), antibiotics (e.g., including but not limited to, penicillin,
streptomycin, neomycin sulfate, amphotericin B, blasticidin,
chloramphenicol, amoxicillin, bacitracin, bleomycin, cephalosporin,
chlortetracycline, zeocin, and puromycin), and glutamine (e.g.,
L-glutamine). MSC survival and growth also depends on the
maintenance of an appropriate aerobic environment, pH, and
temperature. In some embodiments, the solution will not include
animal serum, e.g., when the MSCs are placed into a bioartificial
liver device.
[0070] In some embodiments, an MSC preparation will be essentially
free of non-MSC cells or cellular material. In some embodiments, an
MSC preparation will contain at least 80% MSCs identified by the
criteria described herein, e.g., at least 85%, 90%, 95%, 98%, 99%
and above MSCs. In some embodiments, the cells in an MSC
preparation will be 100% MSCs. A population of cells that is at
least 80% MSCs can be termed a "purified" population of MSCs. In
general, the methods, compositions, and devices described herein
will use purified populations of MSCs.
[0071] As one example, MSCs can be cultured using Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 100
U/ml penicillin, 100 .mu.g/ml streptomycin, 0.1 mM non-essential
amino acids and 1 ng/ml of basic fibroblast growth factor (Life
Technologies, Rockville, Md.). After 4 days of culture,
non-adherent hematopoietic cells can be removed by washing with
PBS. Monolayers of adherent MSCs are then cultured with medium
changes 2-3 times per week. MSCs can be passaged using 0.25%
trypsin/0.1% EDTA. MSCs can be routinely subcultured at a density
of 5.times.10.sup.3 cells/cm.sup.2. MSCs can be maintained using
methods known in the art (see, e.g., Pittenger et al., Science,
284:143-147, 1999).
[0072] In some embodiments, MSCs can be used according to the
methods described herein during passages 1-7, e.g., during passages
4-7, where passage one is the first passage following isolation. In
general, MSCs in culture should not be allowed to exceed a cell
density great than 80% confluency. Confluency can be estimated
visually, for example, using a standard light microscope.
Alternatively, cell confluency can be determined by performing cell
counts using, for example, a standard haemocytometer. In some
embodiments, 80% confluence corresponds to about 1.times.10.sup.6
MSCs per 175 cm.sup.2 tissue culture grade flask. In some
embodiments, 80% confluence corresponds to about 5.times.10.sup.3
cells/cm.sup.2.
[0073] In general, it will be desirable to maintain the MSCs in an
undifferentiated state. MSC differentiation status can be evaluated
using methods known in the art, e.g., the MSC characterization
methods described herein.
MSC Characterization
[0074] An MSC preparation (i.e., an undifferentiated MSC
preparation) can be characterized prior to therapeutic use to
confirm the identity, purity, and differentiation status of the
cells.
[0075] MSCs display a unique and easily identifiable fibroblastoid
morphology and express a unique array of antigens that react with
SH2 (CD105) and SH3 (CD73) monoclonal antibodies. MSCs also have a
unique ability to reproducibly give rise to adipocytes,
osteoblasts, and chondrocytes in vitro (Pittenger et al., Science,
284:143-147, 1999).
[0076] As described herein, an MSC preparation (e.g., an
undifferentiated MSC preparation) can be characterized by
performing one or more of the following tests, which can be
performed in vivo or in vitro (see Example 2):
[0077] (1) evaluating the ability of an MSC or an MSC preparation
to differentiate into adipocytes, osteoblasts, and chondrocytes.
Undifferentiated MSCs can give rise to all of these cell types.
[0078] (2) detecting the presence or absence of one or more of the
following cell surface markers: CD29, CD44, CD73, CD90, CD105,
CD106, CD11b, CD14, CD18, CD34, CD36, and CD45 on an isolated MSC
or in an MSC preparation, e.g., using an array. Undifferentiated
MSCs express CD105, CD106, and CD44 and do not express CD14, CD34,
and CD45.
[0079] In general, positive identification of an MSC preparation
(e.g., an undifferentiated MSC preparation) requires that at least
80%, e.g., at least 85%, 90%, 95%, 98%, 99% and above, or 100% of
the cells in the preparation test positive for cell surface
expression of CD105, CD106, and CD44 and test negative for cell
surface expression of CD14, CD34, and CD45 (i.e., CD105+; CD106+;
CD44+; CD14-; CD34-; CD44-).
[0080] In some embodiments, positive identification of an
undifferentiated MSC preparation requires only one of the two above
described tests to be performed. However, both tests can be
performed.
[0081] In some embodiments, additional tests can be performed,
e.g., prior to therapeutic use of an MSC preparation to confirm the
absence of contaminants including, for example, bacteria, viruses,
fungus, infectious proteins, and unwanted immunological molecules.
Such tests are known in the art.
MSC-Conditioned Media (MSC-CM)
[0082] An MSC-CM composition can be prepared using a population of
undifferentiated MSCs between passages 4-7, where passage one is
the first passage following isolation. As described herein, an
MSC-CM composition can be prepared using about 1.times.10.sup.5 to
1.times.10.sup.7 cells, e.g., about 1.times.10.sup.5 to
1.times.10.sup.6 cells, 1.times.10.sup.6 to 1.times.10.sup.7 cells,
1.times.10.sup.6 to 9.times.10.sup.6 cells, 1.times.10.sup.6 to
8.times.10.sup.6 cells, 1.times.10.sup.6 to 7.times.10.sup.6 cells,
1.times.10.sup.6 to 6.times.10.sup.6 cells, 1.times.10.sup.6 to
5.times.10.sup.6 cells, 1.times.10.sup.6 to 4.times.10.sup.6 cells,
1.times.10.sup.6 to 3.times.10.sup.6 cells, and 1.times.10.sup.6 to
2.times.10.sup.6 cells. In some embodiments, an MSC-CM composition
can be prepared using 2.times.10.sup.6 cells.
[0083] In some embodiments, an MSC-CM composition is prepared using
about 1.times.10.sup.2 cells/cm.sup.2 to 1.times.10.sup.4
cells/cm.sup.2, e.g., about 1.times.10.sup.2 cells/cm.sup.2 to
1.times.10.sup.3 cells/cm.sup.2, 1.times.10.sup.3 cells/cm.sup.2 to
1.times.10.sup.4 cells/cm.sup.2, 1.times.10.sup.3 cells/cm.sup.2 to
9.times.10.sup.3 cells/cm.sup.2, 1.times.10.sup.3 cells/cm.sup.2 to
8.times.10.sup.3 cells/cm.sup.2, 1.times.10.sup.3 cells/cm.sup.2 to
7.times.10.sup.3 cells/cm.sup.2, 1.times.10.sup.3 cells/cm.sup.2 to
6.times.10.sup.3 cells/cm.sup.2, 1.times.10.sup.3 cells/cm.sup.2 to
5.times.10.sup.3 cells/cm.sup.2, 1.times.10.sup.3 cells/cm.sup.2 to
4.times.10.sup.3 cells/cm.sup.2, 1.times.10.sup.3 cells/cm.sup.2 to
3.times.10.sup.3 cells/cm.sup.2, and 1.times.10.sup.3
cells/cm.sup.2 to 2.times.10.sup.3 cells/cm.sup.2. In some
embodiments, an MSC-CM composition can be prepared using about
5.times.10.sup.3 cells/cm.sup.2. In some embodiments, an MSC-CM
composition can be prepared using two populations of
1.times.10.sup.6 MSCs per 175 cm.sup.2, i.e., an MSC-CM composition
can be prepared using 2.times.10.sup.6 cells.
[0084] In some embodiments, an MSC-CM composition is prepared as
follows (see also Example 4):
[0085] (1) Wash 70-80% confluent MSCs thoroughly with phosphate
buffered saline (PBS);
[0086] (2) Culture MSCs from (1) for about 12, 24, 36, or 48 hours,
e.g., 24 hours in an appropriate volume of serum free culture
medium containing DMEM, or an equivalent thereof, supplemented with
0.05% bovine serum albumin (BSA) (note; media volume will vary
depending on the size of the cell culture vessel) in a suitable
vessel, e.g., a T175 cm.sup.2 flask, with each vessel/flask at 80%
confluency, equivalent to about 5.times.10.sup.3 cells/cm.sup.2;
and
[0087] (3) Collect MSC culture media from (2).
[0088] The collected MSC-CM composition can be concentrated, e.g.,
using methods known in the art, for example, ultrafiltration units
with a 3 kD cutoff (AMICON Ultra-PL 3, Millipore, Bedford, Mass.,
USA). For example, the MSC-CM composition can be concentrated at
least 2-fold to 10-fold, 10-fold to 20-fold, 20-fold to 30-fold,
30-fold to 49-fold, and above. As one example, an MSC-CM
composition is concentrated 25-fold.
[0089] In some embodiments, the MSC-CM composition comprises
culture medium containing DMEM supplemented with 0.05% bovine serum
albumin (BSA). In some embodiments, the MSC-CM composition does not
contain any animal serum. In some embodiments, the MSC-CM
composition comprises PBS. Alternatively, the MSC-CM composition is
provided in lyphophilized form.
[0090] In some embodiments, an MSC-CM composition can be
fractionated by size or by charge. In some embodiments, for
example, an MSC-CM composition can be fractionated into heparin
sulfate binding and non heparin binding fractions. For example, in
heparin sulfate fractionation experiments, a concentrated MSC-CM
composition can be passed over a heparin column, or other columns
e.g., an ion-exchange, size, reverse-phase or other chromatographic
separation methods per vendor's instructions. Flow-through and
eluted fractions can then be collected separately. The eluted
fractions (i.e., the heparin-binding fraction) can then be
collected and optionally concentrated, as described above.
[0091] In some embodiments, an MSC-CM composition is at least 50%,
60%, 70%, 80%, 90%, and 100% free of non-heparin binding
material.
Methods of Treatment
[0092] The methods described herein can be used for treating liver
disease in a subject. In some embodiments, the etiology of the
liver disease to be treated may be a local or systemic inflammatory
response. The methods described herein are of particular use for
the treatment of FHF in a subject. The methods include treating a
subject diagnosed as having FHF, or suspected as having FHF, for
example, a subject that presents to a clinician with symptoms that
are typical of FHF. In some embodiments, the methods and
compositions described herein are of use in the treatment of liver
fibrosis.
[0093] Generally, the methods described herein include (1)
administering a therapeutically effective amount of an MSC-CM
composition (or active fraction thereof) to a subject who is need
of, or who has been determined to be in need of such treatment;
and/or (2) treating a subject identified as in need of, or who has
been determined to be in need of such treatment, with a
extracorporeal liver support device or a liver assist device
containing MSCs (e.g., an extracorporeal bioreactor (EB))
containing MSCs (MSC-EB).
[0094] MSC-CM Therapy
[0095] The methods described herein can include parenteral
administration of a composition including one or more of (A) a
purified fraction of an MSC-CM composition; (B) one or more
purified fractions of an MSC-CM composition; (C) MSC-CM composition
fractions of particular molecular weights, for example, 1-3 kDa,
3-6 kDa, 6-50 kDa, and 50 kDa and above, including combinations of
these fractions; (D) an unfractionated MSC-CM composition; and (E)
any combination of A-D. Each of these are referred to individually
and collectively as the "composition" or the "MSC-CM composition."
Biologically active fractions can be identified in vitro by
exposing hepatocytes to apoptotic stimuli, such as actinomyosin D
and TNF-.alpha.. The level of apoptosis can then be determined
using, for example, TUNEL assay according to the manufacturer's
instructions. Biologically active fractions will inhibit apoptosis
by a statistically significant amount in this system, e.g., by 25%,
30%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, or more. These in vitro
observations may be complemented using in vivo assays in an acute
liver failure animal model.
[0096] In some embodiments, a therapeutically effective dose of an
MSC-CM composition is administered to a subject using systemic
intravenous bolus injection. Bolus administration (also referred to
as bolus infusion) includes administration of a dose of drug over a
short period of time, e.g., by injection into a blood vessel.
[0097] In some embodiments, a therapeutically effective dose of an
MSC-CM composition is administered to a subject using an
intravenous drip. Other modes of administration can include any
number of different routes including, but not limited to,
intravenous, intradermal, subcutaneous, and percutaneous
injection.
[0098] A therapeutically effective amount of an MSC-CM composition
can be given to the subject in one or more administrations,
applications, or dosages. The compositions can be administered from
one or more times per day to one or more times per week; including
once every other day. The skilled artisan will appreciate that
certain factors can influence the dosage and timing required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of the compositions described herein can include a
single treatment or a series of treatments.
[0099] In some embodiments, an MSC-CM composition is administered
to a subject as a single daily dose or as multiple daily doses, for
example, 1, 2, or 3 daily doses. Doses can be administered with or
without regard to food intake.
[0100] In some embodiments, an MSC-CM composition is administered
to a subject for a period of 1 to 7 days. In some embodiments, an
MSC-CM composition is administered to a subject for a period of at
least 1 week to 1 month, for example, at least 1, 2, 3, or 4 weeks.
In a further alternative embodiment, an MSC-CM composition is
administered to a subject for a period of at least 1 month to 1
year or longer. In some embodiments, an MSC-CM composition is
administered until the desired therapeutic effect (e.g., functional
recovery of the liver, e.g., to normal or near-normal levels, or to
levels manageable by other therapeutic means) has been achieved, as
decided by the subject or the subject's healthcare provider. In
some embodiments, liver function can be assessed using known tests
of liver function, for example, serum lactate dehydrogenase (LDH),
alkaline phosphatase (ALP), aspartate aminotransferase (AST),
alanine aminotransferase (ALT), bilirubin, protein levels,
prothrombin time, activated clotting time (ACT), partial
thromboplastin time (PTT), and prothrombin consumption time (PCT)
(discussed further below).
[0101] Dosage, toxicity, and therapeutic efficacy of the
composition can be determined, e.g., by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and
the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Compounds that exhibit high therapeutic indices are
generally preferred. While compounds that exhibit toxic side
effects can be used, care should be taken to design a delivery
system that targets such compounds to the site of affected tissue
to minimize potential damage to uninfected cells and, thereby,
reduce side effects.
[0102] In some embodiments, an MSC-CM composition can be validated
and/or the therapeutic efficacy of an MSC-CM composition, e.g., of
a given batch of an MSC-CM composition, can be determined using an
experimental model (e.g., an animal model or an in vitro model),
e.g., as described herein.
[0103] MSC-EB Therapy and Devices
[0104] The methods described herein can include the use of
extracorporeal liver support devices with MSCs to treat subjects
with liver disease. Such devices are also within the scope of the
present disclosure.
[0105] Extracorporeal liver support devices are analogous to the
devices used to perform kidney dialysis. The devices described
herein are bioartificial liver (BAL) devices that include
extracorporeal bioreactors (EBs), which are cartridges or vessels
having at least a perfusion inlet and a perfusion outlet, and a
cell compartment, e.g., a matrix, within the vessel that provides a
suitable environment for living cells while allowing perfusion of
the cell compartment with suitable media for maintaining the cells.
Such cell compartments can be, e.g., hollow fibers, with
circulation of blood or plasma outside the fibers, or flat plates;
see, e.g., U.S. Pat. No. 6,759,245.
[0106] Generally, systems comprising EBs also continuously separate
plasma from cellular components of blood using an ultrafiltrate
generator. The ultrafiltrate (e.g., plasma) is circulated through
the cartridge containing cultured cells, i.e., an EB.
Alternatively, whole blood can be treated in the EB.
[0107] As noted above, EB cartridges generally contain a
semi-permeable barrier made of a material that allows the passage
of macromolecules and other cell derived products to and from the
subject's plasma. However, the cells themselves do not leave the
EB. After circulation and one or multiple passes through the
bioreactor, the treated ultrafiltrate (e.g., plasma) is recombined
with the cellular components of the subject's blood and returned to
the subject via venous access. Generally, the subject's blood or
plasma is supplemented with heparin to prevent clotting. This
circulation is maintained continuously for, e.g., a 10 hour support
period of therapy. In current BAL systems, blood or plasma carries
toxins from the patient to a bioreactor containing hepatocytes.
See, e.g., Yarmush et al., Cell Transplant, 1:323 (1992), and
Arkadopoulos et al., Int'l J. Artif. Organs, 21:781 (1998).
[0108] The present devices include a) a bioreactor comprising a
fluid treatment compartment and a cell compartment, and optionally
a selectively permeable barrier separating the fluid treatment
compartment and the cell compartment; and b) a cell reservoir in
fluid communication with the cell compartment of the bioreactor,
wherein the cell reservoir comprises a population of
undifferentiated undifferentiated multipotent stromal cells (MSCs).
Blood or ultrafiltrate from a subject is passed into the fluid
treatment compartment, where agents secreted by the MSCs pass into
the blood or ultrafiltrate, either by direct contact between the
MSCs and the blood or ultrafiltrate, or by passage of the agents
across the optional selectively permeable barrier, when it is
present. In the present devices and methods, MSCs in an EB are used
in an undifferentiated state, as assessed using techniques
described herein.
[0109] Extracorporeal liver support devices including bioreactors
are also commonly referred to as bioartificial liver devices (BALs)
or bioartificial liver assist devices (BLADs). A number of such
devices are known in the art and can be adapted for use with MSCs.
Exemplary commercially available extracorporeal liver support
device that can be used as described herein include, but are not
limited to, the ELAD.RTM. system currently marketed by Vital
Therapies, Incorporated (shown in FIG. 1 of U.S. Pat. App. Pub. No.
2005/0182349), Circe's HEPATASSIST.RTM., Gerlach's BELS, and Excorp
Medical's BLSS. Additional suitable exemplary devices are described
in U.S. Pat. Nos. 6,472,200, 5,605,835; 7,160,719; 7,273,465;
6,858,146; 6,582,955; 5,270,192; 6,759,245; and U.S. Pat. App. Pub.
Nos. 2003-0017142.
[0110] FIG. 25 shows a schematic diagram of an exemplary
extracorporeal liver support system in which the EBs containing
MSCs as described herein can be used. The system includes an
exemplary bioreactor 1 with multiple cartridges 2. The bioreactor 1
includes an oxygenated fluid inlet 3 for introducing an oxygenated
fluid from an oxygenated fluid supply 4, an oxygenated fluid outlet
3', a liquid inlet 5 for introducing a biological liquid, supplied
by pump 6 from immunoisolation unit 7, into the bioreactor, and a
liquid outlet 5' for removing the biological liquid from the
bioreactor for return to the immunoisolation unit 7. Blood from a
patient 9 flows via pump 6' into a plasmapheresis unit 8, from
which a portion of the plasma then flows into the immunoisolation
unit 7, via pump 6''. Treated plasma flows from the immunoisolation
unit 7 and is mixed with blood from the plasmapheresis unit 8 prior
to flowing back into the patient 9. For further details, see U.S.
Pat. No. 6,759,245.
[0111] Referring to FIG. 26, another example of a bioartificial
liver system is shown in schematic form (see U.S. Pat. No.
7,160,719). System 10 includes an EB 42 that includes MSCs, and
optionally hepatocytes. A biological fluid to be treated (e.g.,
blood or plasma) can be introduced into EB 42 via biological fluid
inlet pathway 32 and can exit via biological fluid outlet pathway
33. For example, a venovenous catheter can be used to place a
mammal's bloodstream in fluid communication with EB 42 via
biological fluid inlet path 32. In an alternative embodiment, a
biological fluid can be treated in vitro using a biological fluid
reservoir (not shown) in place of the mammal 51. In some
embodiments, the device includes an ultrafiltrate (UF) generator
41. In those cases, blood from the mammal 51 flows along blood path
31 into UF generator 41, where the cellular components are
separated from plasma. The ultrafiltered plasma then flows along
path 32 into EB 42, while the cellular components rejoin the
treated UF via path 34. The biological fluid then returns to mammal
51 or to the biological fluid reservoir via path 35.
[0112] Although FIGS. 25 and 26 depict the various components in a
specific orientation and having similar dimensions, the components
can be in any orientation, size, or shape.
[0113] In some situations, to minimize MSC differentiation,
individual MSC-containing EB devices can be operated for a maximum
of 24 hours. In some embodiments, MSC cells can be combined with
primary hepatocytes in a conventional EB.
[0114] Thus, according to the present methods, a subject in need of
therapy can be connected to a BAL device having an EB containing
MSCs (MSC-EB), or containing a mixture of hepatocytes and MSCs.
These methods can be used for treating blood or plasma from the
subject. The method includes providing a system, as described
herein, that contains an EB that includes a cell compartment
containing a population of MSCs for treating the blood or plasma;
removing the blood or plasma from the subject; introducing the
blood or plasma into the fluid treatment compartment of the EB; and
allowing the blood or plasma to flow through and exit the fluid
treatment compartment, thereby treating the blood or plasma.
[0115] The flow rate of a subject's plasma through an EB can be
adjusted as needed, e.g., to a rate of about 50-500 ml/minute,
e.g., 50, 100, 200, 300, 400, and 500 ml/minute. In some
embodiments, the flow rate will be adjusted to optimize passage of
secreted agents from the undifferentiated MSCs to the
ultrafiltrate. In some embodiments, the target flow rate will be
175 ml/minute. Treatment of the subject (e.g., circulation of the
subject's plasma through a device) can continue for a
therapeutically effective time, e.g., between 1 hour and 24 hours,
e.g., about 2, 3, 4, 5, 10, 12, 15, 18, 20, or 23 hours. Subjects
can undergo multiple rounds of MSC-EB therapy with each round
lasting for, e.g., between 1 hour and 24 hours. MSC-EB therapy can
be continued, e.g., until a desired therapeutic effect (e.g.,
recovery of sufficient liver function to acceptable or near-normal
levels) has been achieved, as decided by the subject or the
subject's healthcare provider, or until a donor liver is available
for transplantation. In some embodiments, liver function can be
assessed using standard tests of liver function, for example, serum
LDH, ALP, AST, ALT, bilirubin, protein levels, prothrombin time,
ACT, PTT, and PCT. In some embodiments, tests of liver function are
performed prior to and/or following MSC-EB therapy, to assess the
effectiveness of the MSC-EB therapy. Such assessments can also be
made before and/or after each round of MSC-EB therapy, as well as
before therapy begins and after therapy has been completed due to
the desired therapeutic effect being achieved or the procurement of
a donor organ.
[0116] Extracorporeal liver support devices including bioreactors
are also commonly referred to as bioartificial liver devices (BALs)
or bioartificial liver assist devices (BLADs). A number of such
devices are known in the art and can be adapted for use with MSCs.
Exemplary commercially available extracorporeal liver support
device that can be used as described herein include, but are not
limited to, the ELAD.RTM. system currently marketed by Vital
Therapies, Incorporated (shown in FIG. 1 of U.S. Pat. App. Pub. No.
2005/0182349), Circe's HEPATASSIST.RTM., Gerlach's BELS, and Excorp
Medical's BLSS. Additional suitable exemplary devices are described
in U.S. Pat. Nos. 6,472,200, 5,605,835; 7,160,719; 7,273,465;
6,858,146; 6,582,955; 5,270,192; 6,759,245; and U.S. Pat. App. Pub.
Nos. 2003-0017142.
Subject Selection
[0117] The methods described herein are of particular use for the
treatment of liver disease (e.g., acute liver failure) in a subject
in need thereof. The methods include: identifying a subject with
liver disease (e.g., acute liver failure), and treating the subject
with the compositions described herein using the methods described
herein.
[0118] In some embodiments, the methods of treatment described
herein include a step of selecting a subject on the basis that they
have a liver disease, e.g., FHF or liver fibrosis. In some
embodiments, a test of liver function, e.g., as known in the art or
described herein, is administered, and the subject is selected for
treatment using a method described herein on the basis of the
result of that test, e.g., a test result indicating that the
subject has a liver disease (e.g., a liver disease associated with
the loss of liver function and/or the loss or damage of
hepatocytes, e.g., of parenchymal liver cells).
[0119] Thus, in some embodiments, a subject in need of treatment
with the compositions and methods described herein can be selected
based on, for example, serum markers of liver function. A subject
in need of treatment with the methods described herein can also be
selected based on diagnosis by clinician of liver disease (e.g.,
acute liver disease) in a subject.
Liver Disease
[0120] The term "liver disease" applies to many diseases and
disorders that cause the liver to function improperly or to cease
functioning, and this loss of liver function is indicative of liver
disease. Thus, liver function tests are frequently used to diagnose
liver disease. Examples of such tests include, but are not limited
to, the following;
[0121] (1) Assays to determine the levels of serum enzymes such as
lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate
aminotransferase (AST), and alanine aminotransferase (ALT), where
an increase in enzyme levels indicates liver disease. One of skill
in the art will reasonably understand that these enzyme assays
indicate only that the liver has been damaged. They do not assess
the liver's ability to function. Other tests can be used to assay a
liver's ability to function.
[0122] (2) Assays to determine serum bilirubin levels. Serum
bilirubin levels are reported as total bilirubin and direct
bilirubin. Normal values of total serum bilirubin are 0.1-1.0 mgdl
(e.g., about 2-18 mmol/L). Normal values of direct bilirubin are
0.0-0.2 mg/dl (0-4 mmol/L). Increases in serum bilirubin are
indicative of liver disease.
[0123] (3) Assays to determine serum protein levels, for example,
albumin and the globulins (e.g., alpha, beta, gamma). Normal values
for total serum proteins are 6.0-8.0 g/dl (60-80 g/L). A decrease
in serum albumin is indicative of liver disease. An increase in
globulin is indicative of liver disease.
[0124] Other tests include prothrombin time, international
normalized ratio, activated clotting time (ACT), partial
thromboplastin time (PTT), prothrombin consumption time (PCT),
fibrinogen, coagulation factors; alpha-fetoprotein, and
alpha-fetoprotein-L3 (percent).
[0125] A clinically important type of liver disease is hepatitis.
Hepatitis is an inflammation of the liver that can be caused by
viruses (e.g., hepatitis virus A, B and C (HAV, HBV, and HCV,
respectively), chemicals, drugs, alcohol, inherited diseases, or
the patient's own immune system (autoimmune hepatitis). This
inflammation can be acute and resolve within a few weeks to months,
or chronic, and persist over many years. Chronic hepatitis can
persist for decades before causing significant symptoms, such as
cirrhosis (scarring and loss of function), liver cancer, or
death.
[0126] Other important examples of the different diseases and
disorders encompassed by the term "liver disease" include, but are
not limited to amebic liver abscess, biliary atresia, fibrosis,
cirrhosis, coccidioidomycosis, delta agent, hepatocellular
carcinoma (HCC), alcoholic liver disease, primary biliary
cirrhosis, pyogenic liver abscess, Reye's syndrome, sclerosing
cholangitis, and Wilson's disease. In some embodiments, the
compositions and methods described herein are suitable for the
treatment of liver disease characterized by the loss or damage of
parenchymal liver cells. In some aspects, the etiology of this can
be a local or systemic inflammatory response.
Liver Failure
[0127] Liver failure occurs when large parts of the liver become
damaged and the liver is no longer able to perform its normal
physiological function. In some aspects, liver failure can be
diagnosed using the above described assays of liver function. In
some embodiments, liver failure can be diagnosed (e.g., initially
diagnosed) based on a subject's symptoms. Symptoms that are
associated with liver failure include, for example, one or more of
the following, nausea, loss of appetite, fatigue, diarrhea,
jaundice, abnormal/excessive bleeding (e.g., coagulopathy), swollen
abdomen, mental disorientation or confusion (e.g., hepatic
encephalopathy), sleepiness, and coma.
[0128] Chronic liver failure occurs over months to years and is
most commonly caused by viruses (e.g., HBV and HCV),
long-term/excessive alcohol consumption, cirrhosis,
hemochromatosis, and malnutrition.
[0129] Acute liver failure is the appearance of severe
complications after the first signs of liver disease (e.g.,
jaundice). Acute liver failure includes a number of conditions, all
of which involve severe hepatocyte injury or necrosis. In most
cases of acute liver failure, massive necrosis of hepatocytes
occurs; however, hepatocellular failure without necrosis is
characteristic of fatty liver of pregnancy and Reye's syndrome.
Altered mental status (hepatic encephalopathy) and coagulopathy in
the setting of a hepatic disease generally define acute liver
failure. Consequently, acute liver failure is generally clinically
defined as the development of coagulopathy, usually an
international normalized ratio (a measure of the time it takes
blood to clot compared to an average value--INR) of greater than
1.5, and any degree of mental alteration (encephalopathy) in a
patient without preexisting cirrhosis and with an illness of less
than 26 weeks' duration. Acute liver failure indicates that the
liver has sustained severe damage resulting in the dysfunction of
80-90% of liver cells.
[0130] Acute liver failure occurs when the liver fails rapidly.
Hyperacute liver failure is characterized as failure of the liver
within one week. Acute liver failure is characterized as the
failure of the liver within 8-28 days. Subacute liver failure is
characterized as the failure of the liver within 4-12 weeks.
[0131] In some embodiments, the compositions and methods described
herein are particularly suitable for the treatment of hyperacute,
acute, and subacute liver failure, all of which are referred to
herein as "acute liver failure." Common causes for acute liver
failure include, for example, viral hepatitis, exposure to certain
drugs and toxins (e.g., fluorinated hydrocarbons (e.g.,
trichloroethylene and tetrachloroethane), amanita phalloides (e.g.,
commonly found in the "death-cap mushroom"), acetaminophen
(paracetamol), halothanes, sulfonamides, henytoins),
cardiac-related hepatic ischemia (e.g., myocardial infarction,
cardiac arrest, cardiomyopathy, and pulmonary embolism), renal
failure, occlusion of hepatic venous outflow (e.g., Budd-Chiari
syndrome), Wilson's disease, acute fatty liver of pregnancy, amebic
abscesses, and disseminated tuberculosis.
[0132] Acute liver failure encompasses both fulminant hepatic
failure (FHF) and subfulminant hepatic failure (or late-onset
hepatic failure). FHF is generally used to describe the development
of encephalopathy within 8 weeks of the onset of symptoms in a
patient with a previously healthy liver. Subfulminant hepatic
failure is reserved for patients with liver disease for up to 26
weeks prior to the development of hepatic encephalopathy.
[0133] FHF is usually defined as the severe impairment of hepatic
functions in the absence of pre-existing liver disease. FHF may
result from exposure of a susceptible individual to an agent
capable of producing serious hepatic injury. Examples of such
agents include infectious agents, excessive alcohol, hepatotoxic
metabolites, and hepatotoxic compounds (e.g., drugs). Other causes
include congenital abnormalities, autoimmune disease, and metabolic
disease. In many cases the precise etiology of the condition is
unknown (e.g., idiopathic). FHF may be diagnosed, for example,
using the liver function assays described above.
Liver Fibrosis
[0134] Liver fibrosis is the excessive accumulation of
extracellular matrix proteins including collagen that occurs in
most types of chronic liver diseases. Advanced liver fibrosis
results in cirrhosis, liver failure, and portal hypertension, and
often requires liver transplantation. A key event in the etiology
of liver fibrosis is inappropriate or excessive hepatic stellate
cell activation (Abdel-Aziz et al., Am. J. Pathol., 137:1333-1342,
1990; Iredale et al., J. Clin. Invest., 102:538-549, 1998).
Multiple Organ Failure
[0135] Organ failure is generally defined as parenchymal cell loss
associated with a local and systemic inflammatory response. More
specifically, organ failure is the failure of an essential system
in the body requiring medical intervention. Multiple organ
dysfunction syndrome (MODS) is altered organ function in an acutely
ill patient requiring medical intervention to perform homeostasis.
MODS usually involves two or more organs.
[0136] MODS typically results from infection, injury (accident,
surgery), hypoperfusion and hypermetabolism. Following an
initiating event, an uncontrolled inflammatory response ensues,
which causes tissue injury and triggers local and systemic
responses. Respiratory failure is common in the first 72 hours
after the original insult, hepatic failure is common in the first
5-7 days, gastrointestinal bleeding may occur at 10-15 days, and
renal failure is common at 11-17 days. Mortality rates for MODS
vary from 30% to 100%. There is currently no effective therapeutic
regimen available to reverse established MODS.
[0137] In some embodiment, the compositions and methods described
herein can be used for the treatment of organ failure, e.g.,
multiple organ failure.
Pharmaceutical Formulations
[0138] The compositions described herein, e.g., an MSC-CM
composition and active agents isolated therefrom, can be
incorporated into pharmaceutical compositions suitable for
administration to a subject, e.g., a mammal, e.g., a human. As used
herein the language "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances are known. Except insofar as any
conventional media or agent is incompatible with the active
compound, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions.
[0139] A pharmaceutical composition will generally be formulated to
be compatible with its intended route of administration. Solutions
or suspensions used for parenteral, application can include the
following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0140] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0141] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an agent described herein)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0142] In one embodiment, the MSC-CM compositions are prepared with
carriers that will protect the compositions against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0143] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration. In one aspect, the pharmaceutical compositions can
be included as a part of a kit. Such kits are also within the scope
of the present invention.
[0144] Methods of Screening
[0145] The invention provides methods for identifying the active
compounds contained in an MSC-CM composition, e.g., in the heparin
binding fraction of an MSC-CM composition.
[0146] In some embodiments, the active compounds contained in an
MSC-CM composition, e.g., in the heparin binding fraction of an
MSC-CM composition, can be obtained by systematically analyzing
each of the components contained in an MSC-CM composition.
Alternatively, an MSC-CM composition can be fractionated, e.g., by
size and/or charge, and the active fractions can be identified.
Then, each of the active fractions can be further fractionated, and
the active fractions thereof identified. This can be repeated a
desired number of times, and then the components of the action
fraction can be identified, e.g., using methods known in the art
(e.g., HPLC, mass spectrometry), and some or all of the components
can then be tested for activity in the assay system. If two or more
active compounds are identified, they can be combined to form a
combined composition to achieve some or all of the efficacy of the
complete MSC-CM, e.g., a combined composition with at least 40%,
e.g., 50%, 60%, 70%, 80%, 90%, or more of the efficacy of the
MSC-CM. In some embodiments, the therapeutic activity of individual
components of an MSC-CM composition, or different sized fractions
of an MSC-CM composition are tested using in vitro and/or in vivo
assays, e.g., of hepatocyte growth, proliferation, survival,
morphology, and function; a number of such assays are known in the
art. For example, suitable in vitro assays can include assays to
analyze hepatocyte proliferation, e.g., via BrdU incorporation;
hepatocyte apoptosis, for example, by analyzing morphological
changes associated with apoptosis/necrosis, or by using, e.g.,
TUNEL assay; RT-PCR to detect alterations in the mRNA expression
levels of IL-10, IL-6, HGF, EGF, and TNF-.alpha.; stellate cell
proliferation; stellate cell apoptosis; ELISA to detect altered
IL-10, TNF-.alpha., IL-1.beta., IL-6, IL-2, IL-1ra expression, and
immune cell chemotaxis. Such in vivo assays include, analysis of
liver histologies, e.g., following liver biopsy or following
sacrifice of an animal model and animal model survival studies. The
aim of such screening experimentation is to identify the
biologically active component of an MSC-CM composition. The term
"biologically active component of an MSC-CM composition," as used
herein, refers to a component of an MSC-CM composition that
produces one or more of the beneficial effects described herein,
for example, modulates stellate cell signaling, promotes hepatocyte
proliferation, inhibits hepatocyte cell death, or induces any
change in hepatocyte mRNA or protein levels as described herein. In
vitro and in vivo assays to detect such effects are known in the
art and described herein.
[0147] Kits
[0148] The present invention also includes kits. In some
embodiments, the kits comprise one or more doses of an MSC-CM
composition. The composition, shape, and type of dosage form for
the induction regimen and maintenance regimen can vary depending on
a subjects requirements. For example, dosage form can be a
parenteral dosage form (e.g., intravenous bolus administration), an
oral dosage form, a delayed or controlled release dosage form, a
topical, and a mucosal dosage form, including any combination
thereof.
[0149] In a particular embodiment, a kit can contain one or more of
the following in a package or container: (1) one or more doses of
an MSC-CM composition, e.g., in liquid or frozen solution form or
lyophilized; (2) one or more pharmaceutically acceptable buffers
(3) one or more vehicles for administration of the dose, such as
one or more syringes, a catheter, a pump, a hydrogel, and a depot
formulation form of administration; (4) one or more additional
bioactive agents for concurrent or sequential administration with
an MSC-CM composition, such as supplemental active ingredients
(SAI); and (5) instructions for administration. Kits in which two
or more, including all, of the components (1)-(5), are found in the
same container can also be used.
[0150] When a kit is supplied, the different components of the
compositions included can be packaged in separate containers and
admixed immediately before use. Such packaging of the components
separately can permit long term storage without losing the active
components' functions. When more than one bioactive agent is
included in a particular kit, the bioactive agents can be (1)
packaged separately and admixed separately with appropriate
(similar of different, but compatible) adjuvants or excipients
immediately before use, (2) packaged together and admixed together
immediately before use, or (3) packaged separately and admixed
together immediately before use. If the chosen compounds will
remain stable after admixing, the compounds can be admixed at a
time before use other than immediately before use, including, for
example, minutes, hours, days, months, years, and at the time of
manufacture.
[0151] The compositions included in particular kits of the present
invention can be supplied in containers of any sort such that the
life of the different components are optimally preserved and are
not adsorbed or altered by the materials of the container. Suitable
materials for these containers can include, for example, glass,
organic polymers (e.g., polycarbonate and polystyrene), ceramic,
metal (e.g., aluminum), an alloy, or any other material typically
employed to hold similar reagents. Exemplary containers can
include, without limitation, test tubes, vials, flasks, bottles,
syringes, and the like.
[0152] As stated above, the kits can also be supplied with
instructional materials. These instructions can be printed and/or
can be supplied, without limitation, as an electronic-readable
medium, such as a floppy disc, a CD-ROM, a DVD, a Zip disc, a video
cassette, an audiotape, and a flash memory device. Alternatively,
instructions can be published on a internet web site or can be
distributed to the user as an electronic mail.
[0153] MSC Extracorporeal Bioreactor Cartridges
[0154] Also included within the present invention are EBs, and
cartridges for use therein including MSCs (MSC-EB cartridges). Each
cartridge can be supplied as a single cartridge for use in a
extracorporeal liver support device, described above. MSC-EB
cartridges can be supplied for single use with a patient for a time
frame of up to 24 hours. Multiple cartridges can be supplied for
use in a single subject, as dictated by a subject's treatment
regimen. A number of suitable configurations of cartridges are
known in the art, see, e.g., U.S. Pat. Nos. 5,270,192; 7,160,719;
6,858,146; 6,582,955; 6,759,245; Dixit and Gitnick, Eur. J. Surg.
Suppl. (582):71-6 (1998); and Legallais et al., J. Memb. Sci.
181:81-95 (2001). Such cartridges can include, e.g., hollow fibers
or flat plates. Generally, a semi-permeable membrane will separate
the biological fluid to be treated from the cells, and such a
membrane can form part of the cartridges, e.g., an exterior wall of
the cartridge. The cartridges are configured to be inserted into a
BAL device, e.g., as part of a bioreactor or as an entire
bioreactor.
[0155] FIG. 27 is a schematic illustration of a hollow core
bioreactor cartridge 100, containing a number of hollow fibers 140,
and an inlet 110 and outlet 120. The hollow fibers will be
semi-permeable, allowing passage of the active factors secreted by
the MSCs into the blood or plasma. FIG. 28 is a cross-sectional
view of bioreactor 100 at line A-A, illustrating the hollow fibers
140, which have an interior capillary lumen 130 and are surrounded
by extracapillary space 150. In hollow fiber bioreactors, the MSCs
can be either in the lumen 130, while the blood or plasma flows
through the extracapillary space 150, or vice-versa. In this case,
the compartment including the MSCs is considered the cell
compartment, while the compartment through with the blood or plasma
flows is the fluid treatment compartment.
[0156] FIG. 29 is a schematic illustration of a flat-plate or
two-compartment bioreactor 200, including a fluid treatment
compartment 210 and a cell compartment 220, separated by a
semi-permeable membrane 230. Blood or plasma flows through fluid
treatment compartment 210 along path 250. Cell compartment 220
includes MSCs 240 (and, optionally, hepatocytes). This type of
bioreactor is described in further detail in U.S. Pat. No.
6,759,254.
[0157] Also provided are kits that can contain one or more of the
following in a package or container: (1) an MSC-EB cartridge; (2)
one or more pharmaceutically acceptable buffers (3) instructions
for installing the MSC-EB into a specific BAL. Embodiments in which
two or more, including all, of the components (1)-(3), are found in
the same container can also be used.
EXAMPLES
[0158] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
MSC Isolation, Culture and Ex Vivo Expansion
[0159] Human MSCs were isolated from commercially available bone
marrow aspirates (bone marrow-derived MSCs) obtained from a single,
male donor of 25 years of age (Clonetics-Poietics, Walkersville,
Md.), as previously described (Mauney et al., Biomaterials,
26:6167-6175, 2005). Briefly, whole bone marrow aspirates were
plated at a density of 8-10 .mu.l aspirate/cm.sup.2 on 175 cm.sup.2
tissue culture flasks and grown to confluence in expansion medium
at 37.degree. C. and 5% carbon dioxide. Expansion medium consisted
of Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum, 100 U/ml penicillin, 100 (.1 g/ml streptomycin, 0.1 mM
non-essential amino acids and 1 ng/ml of basic fibroblast growth
factor (Life Technologies, Rockville, Md.). After 4 days of
culture, non-adherent hematopoietic cells were removed by washing
with PBS, and monolayers of adherent cells were cultured with
medium changes 2-3 times per week. Cells were passaged using 0.25%
trypsin/0.1% EDTA, subcultured at a density of 5.times.10.sup.3
cells/cm.sup.2 and used for experiments during passages 4-7.
Isolated MSCs were maintained as previously described (Pittenger et
al., Science, 284:143-147, 1999).
Example 2
Bone Marrow-Derived MSC Characterization
[0160] Mesenchymal stem cells (MSCs) posses several unique and well
established characteristics that allow these cells to be
distinguished from other cells. Such characteristics include the
ability of the cells to reproducibly give rise (i.e.,
differentiate) to adipocytes, osteoblasts, and chondrocytes in
vitro (Pittenger et al., Science, 284:143-147, 1999); and the
ability of the cells to rescue mesenchymal tissue disorders in
humans (Horwitz et al., Proc. Natl. Acad. Sci. USA, 99:8932-8937,
2002). In addition, human MSCs can be further identified by
evaluating the cell surface expression of CD29, CD44, CD73, CD90,
CD105, CD106, CD11b, CD14, CD18, CD34, CD36, and CD45 (Pittenger et
al., Circ. Res., 95:9-20, 2004).
[0161] MSCs isolated using the methods described above were
characterized using two distinct techniques.
[0162] First, the multipotency of bone marrow-derived MSCs was
assessed in vitro by culturing the cells in (1) osteogenesis, (2)
adipogenesis or (3) chondrogenesis induction medium for 2-3 weeks,
with medium changes every 3 days, as follows.
[0163] (1). Osteogenic medium consisted of Iscove's modified
Dulbecco's medium (IMDM) supplemented with 0.1 .mu.M dexamethasone,
10 mM .beta.-glycerol phosphate, 0.2 mM ascorbic acid (AsA), 100
U/ml penicillin and 100 .mu.g/ml streptomycin.
[0164] (2). Adipogenesis medium consisted of IMDM supplemented with
0.5 mM 3-isobutyl-1-methylxanthine, 1 .mu.M hydrocortisone, 0.1 mM
indomethacin, 10% rabbit serum, 100 U/ml penicillin and 100
.mu.g/ml streptomycin.
[0165] (3). For chondrogenesis studies, cells were transferred into
a 15-mL polypropylene tube and centrifuged at 1000 rpm for 5
minutes to form a pelleted micromass that was then treated with
chondrogenic medium. Chondrogenesis medium consisted of
high-glucose DMEM (Chemicon International, Temecula, Calif.)
supplemented with 0.1 .mu.M dexamethasone, 50 .mu.g/mL AsA, 100
.mu.g/mL sodium pyruvate, 40 .mu.g/mL praline, 10 ng/mL
TGF-.beta..sub.1, 50 mg/mL ITS+ premix (Becton Dickinson; 6.25
.mu.g/mL insulin, 6.25 .mu.g/mL transferring, 6.25 ng/mL selenious
acid, 1.25 mg/mL bovine serum albumin, and 5.35 mg/mL linoleic
acid), 100 U/ml penicillin and 100 .mu.g/ml streptomycin.
[0166] The phenotype of differentiated cells was evaluated after
2-3 weeks of induction, as follows.
[0167] (1). Mineral content under osteogenic conditions was
determined using the Von Kossa stain, as follows. Briefly, cells
were fixed with 4% paraformaldehyde for 15 minutes, washed twice
with PBS, stained with 1% silver nitrate under a 100 W light for 60
minutes and washed with deionized (DI) water.
[0168] (2). Lipid accumulation after adipogenic conditions was
determined by oil Red O staining, as follows. Briefly, cells were
fixed with 4% paraformaldehyde, washed twice with PBS, stained with
oil-Red 0 for 15 minutes and washed with DI water.
[0169] (3). Proteoglycan content after chondrogenic conditions was
assessed by safranin-O staining, as follows. Briefly, the
micropellet was fixed in 4% paraformaldehyde, serially diluted in
ethanol and embedded in paraffin blocks. Blocks were sectioned and
stained with safranin-O.
[0170] All images were captured on an Nikon Eclipse E800 Upright
Microscope.
[0171] The results showed that osteogenesis, adipogenesis, and
chondrogenesis were induced in their respective induction mediums.
The staining for osteogenesis, adipogenesis, and chondrogenesis is
visualized by mineral deposition, lipid droplets, and chondroitin
sulfate, respectively.
[0172] Second, MSCs were immunophenotyped by flow cytometry (FACS
Calibur, Becton Dickinson, Franklin Lakes, N.J.). The surface
antigen panel included CD14, CD34, CD44, CD45, and CD106 (BD
Pharmingen, Franklin Lakes, N.J.).
[0173] As expected, the surface antigen profile of the isolated
MSCs was CD 14-, CD105+, CD34-, CD45-, CD106+ and CD44+. These
observations are consistent with previous reports of
undifferentiated MSC surface marker expression (Pittenger et al.,
Circ. Res., 95:9-20, 2004).
[0174] Thus, the methods used were suitable for isolation of
MSCs.
Example 3
Immunomodulation of Stellate Cells by MSCs
[0175] This example demonstrates that MSCs are capable of
modulating activated hepatic stellate cells (SCs), likely via
paracrine mechanisms.
[0176] Liver fibrosis, the precursor to cirrhosis, is generally
thought to be the result of an imbalance in extracellular matrix
(ECM) synthesis and degradation mediated primarily by SCs.
[0177] SCs are pericytes frequently found in the perisinusoidal
space located between the sinusoids and hepatocytes in the liver.
In a normal and healthy liver, SCs are quiescent and primarily
involved vitamin A storage. Following liver injury, SCs undergo a
phenotypic switch into proliferative, .alpha.-smooth muscle actin
positive, myofibroblast-like cells capable of increased collagen
synthesis. SCs can be easily distinguished by the large lipid
droplets observed in the cytoplasm of these cells.
[0178] In vivo activation of SCs is divided into a fibrogenic and
hyperplastic response that is mediated by many autocrine and
paracrine signals. Spontaneous resolution of liver fibrosis has
been reported in different rat models of chronic liver injury
(Abdel-Aziz, et al., Am. J. Pathol., 137:1333-1342, 1990; Iredale
et al., J. Clin. Invest. 102:538-549, 1998). This resolution has
been correlated with decreased synthesis of type I collagen and
tissue inhibitor of matrix metalloproteinases (TIMP) 1 and 2
transcripts, with a concomitant decrease in the number of
.alpha.-SMA positive SCs (Iredale et al., J. Clin. Invest.
102:538-549, 1998). It is unclear whether the decrease in the
number of activated SCs is due to selective apoptosis or reversion
to a quiescent state by microenvironmental cues.
[0179] These data demonstrate that MSC therapy is useful in the
treatment of disorders caused by activated SCs in a subject, for
example, liver fibrosis.
[0180] 3A--SC Isolation
[0181] Primary rat SCs were isolated from 2-3 month-old adult
female Lewis rats (Charles River Laboratories, Wilmington, Mass.)
weighing 180-200 g, as previously reported in detail for hepatocyte
isolation (Dunn et al., FASEB J., 3:174-177, 1989). Briefly, the
supernatant from the hepatocyte purification steps, containing the
non-parenchymal liver cells were obtained and treated with DNAse I
for 15 minutes at 37.degree. C., centrifuged at 300 g for 20
minutes, and then resuspended in PBS. This cell suspension was
subjected to a density gradient separation by gently layering the
suspension on top of a discontinuous 40%-60% isotonic Percoll
gradient. The layers were then centrifuged at 900 g for 25 minutes.
The lowest density layer, enriched in SCs, was then removed,
diluted with PBS and centrifuged at 900 g for 25 minutes. The cells
were resuspended in medium (Dulbecco's modified Eagle's medium
supplemented with 10% FBS, 100 U/ml penicillin and 100 .mu.g/ml
streptomycin) and cultured on 175 cm.sup.2 tissue culture flasks.
SCs were cultured for 10-14 days on tissue culture plastic, which
led to their activation, before use in experiments. The purity and
differentiation of SC cultures were assessed by performing
immunofluorescence for desmin, a myofibroblast marker, and a-SMA, a
marker for myofibroblasts in a more advanced state of
differentiation.
[0182] Immunofluorescent detection was performed after a 15 minute
fixation in 4% paraformaldehyde solution prepared in phosphate
buffered saline (PBS), followed by a single wash with PBS. All
steps were performed at room temperature. Cells were permeabilized
by incubating with blocking buffer (10% normal horse serum, 0.025%
Triton X-100 and 0.5% dimethylsulfoxide in PBS) for 45 minutes.
Subsequently, the cells were incubated with mouse monoclonal
anti-.alpha.-SMA antibody (GeneTex Inc, San Antonio, Tex.; 1:100
dilution) or goat polyclonal anti-desmin antibody (Santa Cruz
Biotechnology-clone C18; 1:100 dilution) in blocking buffer for 60
minutes. After washing three times with blocking buffer, incubation
with rhodamine red-X conjugated goat anti-mouse secondary antibody
(1:250 dilution) or FITC-conjugated donkey anti-goat secondary
antibody (1:500 dilution; Jackson ImmunoResearch Laboratories, West
Grove, Pa.) was performed for 60 minutes, respectively. Three
washes were performed with blocking buffer, incubating with 5
.mu.g/mL of 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes,
Leiden, Netherlands) for 5 minutes during the first wash. Samples
without primary antibody were used as negative controls.
Fluorescent and phase-contrast microscopy were performed on a Zeiss
AXIOVERT 200 M Inverted Microscope.
[0183] All cells stained positively for both desmin and .alpha.-SMA
following 10 days of culture, demonstrating successful isolation
and purification of SC cells.
[0184] 3B--MSC and SC Culture Systems
[0185] Isolated SCs were co-cultured with MSCs, isolated and
characterized as described in Example 1 and Example 2, using direct
or indirect systems, as follows.
[0186] Direct Co-culture System: SCs and MSCs were seeded together
at a ratio of 1:1 in each well of a six-well plate (Corning Costar,
Acton, Mass.)
[0187] Indirect co-culture System: SCs and MSCs were co-cultured
using a Transwell configuration. Approximately 1.0.times.10.sup.5
SCs were seeded in the lower chamber with 0-1.0.times.10.sup.5 MSCs
seeded on the membrane inserts. MSCs were replaced with human
umbilical vein endothelial cells for controls. Co-cultures were
maintained in SC medium for 4 days.
[0188] 3C--MSCs Inhibit Collagen Synthesis in Activated Stellate
Cells
[0189] SC secretion of procollagen type-I C-peptide (PIP), a
peptide fragment cleaved from precursor collagen upon extracellular
secretion, was measured after four days of indirect co-culture as a
function of MSC to SC ratio, with the number of SCs remaining
constant Collagen synthesis was quantified using an ELISA for
procollagen type-I C-peptide (Takara-Bio Inc., Shiga, Japan). After
the co-culture period, the Transwell insert containing MSCs was
removed and the medium on the SCs was replaced with fresh medium.
Twenty-four hours later, medium was collected and procollagen
type-I C-peptide concentration was measured by ELISA.
[0190] PIP levels secreted by activated SCs (101.+-.11 pg/106
cells/day) were significantly reduced at a MSC:SC co-culture ratio
of 1:10 (41.+-.18 pg/106 cells/day; p=0.0491), with a 66% reduction
at a 1:1 co-culture ratio (34.+-.5 pg/106 cells/day; p=0.004).
[0191] Reduced PIP secretion was not observed in co-cultures of SCs
with human umbilical vein endothelial cells (MVEC), suggesting a
MSC-specific effect. These results suggest that soluble factors
released by MSCs inhibit the synthesis of procollagen in activated
SCs.
[0192] 3D--MSCs Induce Apoptosis in Activated SCs
[0193] The decrease in hepatic fibrosis observed after
transplantation of MSCs is accompanied by a reduction in the number
.alpha.-SMA+ (activated stellate) cells observed by
immunohistological staining (Sakaida et al., Hepatology,
40:1304-1311, 2004; Fang et al., Transplantation, 78:83-88, 2004;
Zhao et al., World J. Gastroenterol., 26:6167-6175, 2005). The
mechanism by which the reduction in the number of activated
stellate cells occurs is unclear; there is evidence for decreased
proliferative capacity, a reversion to a quiescent phenotype and
for apoptotic cell death. Therefore, we examined the fate of
activated SCs indirectly co-cultured with MSCs by measuring the
extent of SC dedifferentiation, proliferation and death.
[0194] SC dedifferentiation or reversion back to a quiescent state
was determined by analysis .alpha.-SMA expression, as described
above. The expression of .alpha.-SMA was not decreased after
co-culture. This observation suggests that SCs do not revert to a
quiescent phenotype in the presence of MSCs.
[0195] SC proliferation was assessed using flow cytometry to
quantify the population of SCs entering the S-phase of the cell
cycle, as measured by BrdU incorporation, after co-culture with
MSCs.
[0196] Briefly, twenty-four hours prior to analysis, the Transwell
insert containing MSCs was removed and SC cultures were treated
with 10 .mu.M BrdU. Cells were then washed with PBS three times,
trypsinized and centrifuged at 1200 rpm for 5 minutes. For
fixation, the pellet was resuspended in 70% ethanol for 45 minutes
at room temperature, centrifuged and washed twice with PBS. Cells
were incubated with 4 M HCl for 15 minutes at room temperature,
centrifuged, washed twice with PBS and incubated with a blocking
buffer composed of PBS and 10% FBS for 10 minutes. Cells were then
incubated with anti-BrdU antibody conjugated to Alexa-Fluor 488 dye
for 60 minutes at 37.degree. C., centrifuged and washed with PBS.
Negative controls consisted of cells incubated without the
antibody. Fluorescently labeled cells were analyzed by flow
cytometry.
[0197] As shown in FIG. 1, a decline, from 30.+-.9% to 15.+-.4%, in
BrdU.sup.+ SCs was observed at a 1:1 co-culture ratio
(p=0.043).
[0198] Microscopic observation of indirect co-cultures indicated a
decrease in the number of SCs adhering to the polystyrene plate. To
determine if apoptosis accounted for the observed changes in SC
number after co-culture, Annexin-V-FITC staining and flow cytometry
were used.
[0199] Briefly, quantification of cell apoptosis/necrosis was
determined using the Annexin-V FLUOS kit (Roche, Indianapolis,
Ind.) as per vendor instructions. After co-culture, SCs were
recovered and stained with Annexin V for 20 minutes and analyzed
using flow cytometry. Serum deprived SCs served as a positive
control for apoptosis. Results were gated based on fluorescent
signals greater than SC autofluorescence.
[0200] In SCs cultured alone, there was a basal level of apoptosis
(25%). Using the indirect co-culture system with a ratio of 1:1,
there was an approximate 2.5 fold increase in apoptosis (55%).
Co-culture with fibroblasts resulted in a level of apoptosis that
was similar to SCs alone (32%), demonstrating that the
pro-apoptotic effect was MSC-specific.
TABLE-US-00001 TABLE 1 Apoptosis of Activated SCs as a Function of
MSC Number MSC:SC Trial Trial Trial Mean St. Dev. ratio 1 2 3 Trial
4 (%) (%) p-value 0 12.24 18.47 16.23 15.87 15.68 2.54 --
1:10.sup.3 18.37 34.16 22.24 14.95 22.43 8.37 0.1008 1:10.sup.2
25.54 26.53 26.00 25.96 26.01 0.41 8.63 .times. 10.sup.-3 1:10
34.22 23.2 36.31 38.08 32.95 6.69 3.56 .times. 10.sup.-3 1:1 53.53
44.71 45.85 36.92 42.97 11.64 6.98 .times. 10.sup.-4
[0201] As shown in Table 1, significant SC death also occurred at
MSC:SC co-culture ratios of 1:100 or greater, suggesting that small
numbers of MSCs can release potent pro-apoptotic molecules that
induce SC death.
[0202] Taken together, these in vitro results imply that
transplantation of MSCs in vivo can ameliorate or resolve hepatic
fibrosis through a mechanism involving a highly selective
pro-apoptotic effect on activated SCs.
[0203] 3E--MSCs Secrete IL-10 and TNF-.alpha.
[0204] MSCs were exposed to IL-1, IL-6, or tumor necrosis
factor-.alpha. (TNF-.alpha.), all of which are cytokines known to
be involved in liver fibrosis, for 24 hours. IL-10 protein
secretion and mRNA levels were the determined by
enzyme-linked-immunosorbent-assay (ELISA) and RT-PCR,
respectively.
[0205] Briefly, human MSCs were treated with 2.5 ng/ml IL-6
(R&D Systems, Minneapolis, Minn.), 5 ng/ml IL-1 (R&D
Systems, Minneapolis, Minn.), or 25 ng/ml TNF-.alpha. (R&D
Systems, Minneapolis, Minn.) supplemented MSC expansion medium for
24 hours. MSCs cultured in expansion medium served as a negative
control. After treatment, cells were harvested and analyzed for
changes in gene expression.
[0206] Quantification of human IL-10 was determined using an ELISA
as per vendor instructions (Endogen, Rockford, Ill.). Supernatants
were sampled after 48 hours of co-culture and stored at -20.degree.
C. until analysis.
[0207] RNA was extracted from 0.1-1.0.times.10.sup.6 MSCs using the
NUCLEOSPIN RNA purification kit (BD Biosciences, Palo Alto, Calif.)
per the manufacturer's instructions. Approximately 1 .mu.g of total
mRNA was reverse transcribed to cDNA using the ONESTEP RT-PCR Kit
(Qiagen, Valencia, Calif.) per manufacturer's instructions and
amplified in a Perkin Elmer-Cetus Thermal Cycler 480. Cycling
conditions were: 1) 50.degree. C. for 30 minutes; 2) 95.degree. C.
for 15 minutes ; 3) 30 cycles at 94.degree. C. for 30 seconds,
55.degree. C. for 30 seconds, and 72.degree. C. for 1 minute; and
4) a final extension step at 72.degree. C. for 10 minutes. IL-10
was amplified using a combination of the sense and antisense
oligonucleotides SEQ ID NO:1 and SEQ ID NO:2, respectively, to
yield a 364 base pair (bp) PCR product. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was amplified using a combination of the
sense and antisense oligonucleotides SEQ ID NO:3 and SEQ ID NO:4,
respectively, to yield a 238 by PCR product.
TABLE-US-00002 5'-AAGCCTGACCACGCTTTCTA-3' SEQ ID NO: 1
5'-GTAGAGCGGGGTTTCACCA-3' SEQ ID NO: 2 5'-GAGTCAACGGATTTGGTCGT-3'
SEQ ID NO: 3 5'-TTGATTTTGGAGGGATCTCG-3' SEQ ID NO: 4
[0208] As shown in FIGS. 2-3, exposure to IL-6 (2.5 ng/ml) or
TNF-.alpha. (25 ng/ml), cytokines which are significantly elevated
in liver injury models, led to the upregulation of both IL-10 mRNA
(FIG. 2) and protein secretion into the culture medium (FIG.
3).
[0209] As shown in FIG. 4, IL-6 and TNF-.alpha. were both found to
be present in the SC/MSC direct co-culture system, as determined by
ELISA, as described above. IL-6 was secreted by activated SCs
(65.+-.1 pg/10.sup.6 cells/day), while TNF-.alpha. was secreted by
MSCs (23.+-.1 pg/10.sup.6 cells/day).
[0210] As shown in FIG. 5, activated SCs are capable of inducing
MSCs to secrete IL-10 when the cells are directly co-cultured at a
ratio of 1:1 (54 pg/10.sup.6 cells/day). These levels were similar
to the levels observed in FIG. 6.
[0211] As shown in FIG. 6, at a MSC:SC ratio of at least 1:10, SCs
were capable of inducing elevated IL-10 mRNA expression in MSCs
when cultured indirectly using the above described Transwell
configuration. Using the same system, IL-10 protein secretion
increased to 32.+-.7 pg/10.sup.6 cells/day (p<0.01).
[0212] Since both IL-6 and TNF-.alpha. were detected in MSC-SC
co-cultures, neutralization by monoclonal antibodies was performed
to determine the role of IL-6 and TNF-.alpha. on the release of
IL-10 by MSCs.
[0213] Neutralization of specific cytokines was performed during
indirect co-cultures. For all neutralization experiments, the ratio
of MSCs to SCs was 1:1. Anti-human IL-10 (BioLegend, San Diego,
Calif.), TNF-.alpha., (BioLegend, San Diego, Calif.), or HGF and
anti-rat IL-6 (Cell Sciences, Canton, Mass.) were diluted in SC
medium based on the half maximal inhibition concentrations given by
the manufacturer. Fresh medium with neutralizing antibodies was
added after 48 hours of co-culture.
[0214] As shown in FIG. 7, a significant decrease in IL-10 release
(48.+-.4 to 18.+-.1 pg/10.sup.6 cells/day; p<0.01) was observed
when the indirect co-cultures were treated with 1250 ng/ml of
anti-IL-6 neutralizing antibody. No significant difference was
observed after treatment with anti-TNF-.alpha. neutralizing
antibody. Taken together, these data imply that activated SCs
secrete IL-6, which induces MSCs to secrete IL-10.
[0215] 3F--MSCs Inhibit Activated SC Collagen Synthesis and
Proliferation Via IL-10 and TNF-.alpha. Paracrine Signaling
[0216] To determine if the previously observed suppression of
collagen synthesis and proliferation in activated SCs, after
indirect co-culture with MSCs, was mediated by MSC-derived IL-10
and TNF-.alpha., levels of PIP secretion by activated SCs was
measured after four days of indirect co-culture with MSCs in the
presence of neutralizing antibodies to IL-10, TNF-.alpha., or
both.
[0217] Partial normalization of PIP secretion was observed after
neutralization of IL-10 or TNF-.alpha. at antibody concentrations
of 1000 ng/ml and 500 ug/ml or greater, respectively.
Neutralization of both IL-10 and TNF-.alpha. in co-culture led to a
synergistic rise in PIP levels from 79.+-.9 pg/10.sup.6 cells/day
to 215.+-.20 pg/10.sup.6 cells/day (p=0.002) at maximal antibody
concentrations.
[0218] The effects of MSC-derived TNF-.alpha. and IL-10 on the
proliferation of activated SCs was analyzed. Neutralizing the
effects of IL-10 in a 1:1 co-culture led to a marginal rise in the
number of BrdU-positive cells, from 12% to 15% at the maximum
antibody concentration. Neutralization of TNF-.alpha. resulted in a
more significant effect on proliferation with an increase in
BrdU-positive cells from 13% to 33% (p=0.0197). Neutralization of
both cytokines led to the most significant increase proliferation
population (13% to 44%).
[0219] These data suggest a synergy between TNF-.alpha. and IL-10
signaling pathways (p=0.009).
[0220] 3G--MSC-Derived Hepatocyte Growth Factor (HGF) Induces
Apoptosis in Activated SCs
[0221] Given the observation that a relatively small number of MSCs
could cause SC apoptosis, indirect co-culture supernatants were
analyzed for potent pro-apoptotic signals.
[0222] As shown in FIG. 8, a considerable amount of hepatocyte
growth factor (HGF) in mono- and co-cultures was detected and was
produced at approximately equivalent levels by both SCs and
MSCs.
TABLE-US-00003 TABLE 2 Apoptosis of Activated SCs as a Function of
HGF Neutralization Anti-HGF Trial Mean St. Dev. (.mu.g ml.sup.-1) 1
Trial 2 Trial 3 Trial 4 (%) (%) p-value 0 53.53 44.71 45.85 36.92
42.97 11.64 -- 0.05 43.66 22.11 25.67 21.5 28.24 10.45 0.2056 0.5
39.32 11.87 19.26 23.94 23.59 11.60 0.1136 1.5 18.47 21.17 15.9
17.25 18.20 2.24 0.0390 5.0 13.81 17.76 14.38 -- 15.32 2.14
0.0413
[0223] As shown in Table 2, a decrease in apoptosis was observed as
a function of neutralizing antibody to HGF. This decrease in
apoptosis did not occur when the cultures were incubated with a
control antibody with no HGF specificity. These data support a role
for MSC-derived HGF in accelerating the rate of SC apoptosis.
[0224] In summary, the data presented in Example 3 demonstrates
that MSCs are capable of inhibiting the proliferative and
fibrogenic function of activated SCs in a paracrine manner and as a
function of MSC number. This inhibition is caused by MSC-derived
IL-10 and TNF-.alpha., which act synergistically. The secretion of
IL-10 by MSCs is a dynamic response to IL-6 secretion by activated
SCs. Secretion of IL-10 by MSCs in response to TNF-.alpha. was
observed after exogenous stimulation, but not during mono- or
co-culture. This result likely reflects the high levels of
stimulation used in vitro (e.g., approximately 25 ng ml.sup.-1)
compared to the low levels measured in co-culture (e.g.,
approximately 2.5 ng ml.sup.-1). This observation strongly implies
that a threshold concentration of TNF-.alpha. is necessary to
induce IL-10 expression in MSCs. IL-6 and TNF-.alpha. also increase
nuclear factor (NF)-kappaB signaling in various cells types. Thus,
NF-kappaB may also play a role in MSC cytokine expression during
inflammation.
[0225] MSCs also induced apoptosis in activated SCs, which is
mediated by HGF. The effect of MSC-derived HGF and IL-10 is likely
supplementary to the autocrine signaling of these proteins in SCs.
The above described data are summarized in FIG. 9.
[0226] These studies demonstrate for the first time that MSCs act
through multiple mechanisms to coordinate a dynamic, integrated
response to inflammation, particularly fibrosis. Similar
immunoprotective mechanisms may also influence the phenotype of
hepatocytes, kupffer cells, sinusoidal endothelial cells, and
immune cells that infiltrate the liver during inflammation.
Example 4
Acellular MSC Based Therapy--Generation of Acellular MSC
Conditioned Medium (MSC-CM)
[0227] This example demonstrates the production of an MSC-CM
composition.
[0228] MSCs were isolated and characterized as described in Example
1 and Example 2. In some embodiments, MSCs were characterized based
on the detection of cell surface expressed markers (e.g., CD14-,
CD105+, CD34-, CD45-, CD106+ and CD44+). Cells that did not satisfy
the characterization criterion described in Example 2 were
discarded. In some embodiments, human MSCs were provided by the
Tulane Center for Gene Therapy.
[0229] An MSC-CM composition was generated by culturing MSCs up to
a maximum cell density of 70-80% confluency. Cells were not
permitted to undergo differentiation. In other words, MSCs were
maintained in an undifferentiated state. In some embodiments, MSC
differentiation was monitored using the characterization criterion
described in Example 2. In some embodiments, a 70-80% confluence
corresponds to 1.times.10.sup.6 MSCs per 175 cm.sup.2 tissue
culture grade flask. In some embodiments, an MSC-CM composition was
generated using 2.times.10.sup.6 MSCs, which were obtained using
two 175 cm.sup.2 tissue culture grade flasks with each flask
containing 1.times.10.sup.6 cells.
[0230] MSC-CM was prepared as follows;
[0231] (1) 70-80% confluent MSCs were washed thoroughly with
phosphate buffered saline (PBS);
[0232] (2) MSCs from (1) were cultured in 15 ml serum free DMEM
supplemented with 0.05% bovine serum albumin to prevent protein
aggregation;
[0233] (3) MSCs were cultured for 24 hours;
[0234] (4) culture media was collected from (3); and
[0235] (5) collected culture media was concentrated 25-fold (e.g.,
25 times) using ultrafiltration with a 3 kD cutoff.
[0236] The MSC-CM composition was concentrated using
ultrafiltration units (Amicon Ultra-PL 3, Millipore, Bedford,
Mass., USA).
[0237] The MSC-CM composition was fractionated into heparin binding
and non-heparin binding fractions. For fractionation experiments,
concentrated an MSC-CM composition was passed over a
heparin-agarose column per vendor's instructions. Briefly, columns
were primed with 10 equivalents of binding buffer (10 mM sodium
phosphate, pH 7.0). The sample was applied, followed by 10
equivalent volumes of binding buffer, which was considered as the
heparin unbound fraction. The bound fraction was eluted with 10
volumes of binding buffer supplemented with 1 M NaCl. Flow-through
and eluted fractions were collected separately. Flow-through and
eluted fractions were collected and reconcentrated, as described
above.
Example 5
Experimental FHF Induction and Treatment Regimens
[0238] This example demonstrates the induction of FHF in an
experimental animal model.
[0239] The induction of fulminant hepatic failure (FHF) is
previously reported (Shinoda et al., J. Surg. Res., 137:130-140,
2007). Briefly, male Sprague-Dawley rats weighing 250-300 g were
used for FHF experiments. Hepatocytes were isolated from 150-200 g
female Lewis. All animals (Charles River Laboratories, Boston,
Mass.) were handled in accordance with the guidelines set forth by
the Committee on Laboratory Resources, National Institutes of
Health.
[0240] FHF was induced using two injections of Gal-N (Sigma
Aldrich, St Louis, Mo.), freshly dissolved in 0.9% NaCl solution
and administered i.p. with a 12-hour interval. Different dosages of
Gal-N were chosen for tissue analysis (0.6 g/kg) and survival
studies (1.2 g/kg), based on our previous studies. When
appropriate, twenty-four hours after FHF induction 0.9 ml of MSC-CM
or 0.9% NaCl solution (vehicle control) was injected through the
penile vein under ketamine/xylazine anesthesia (110 mg/kg and 0.4
mg/kg i.p. respectively). Animals receiving 0.6 g/kg were
sacrificed 36 hours later for tissue collection. Survival was
monitored every 12 hours for 28 days.
Example 6
MSC-CM Therapy Inhibits Gross Liver Change
[0241] This example demonstrated an hepatoprotective effect of
MSC-CM.
[0242] Gal-N induced FHF is typically accompanied by characteristic
changes in gross appearance of the liver consisting of increased
pallor and a soft and shrunken consistency.
[0243] FHF was induced in Sprague-Dawley rats using Gal-N, as
described in Example 5. Animals were treated with systemic
infusions of concentrated MSC-CM or with vehicle (control). A
sublethal dose of Gal-N (0.6 g/kg) was used for tissue analysis 36
hours after treatment and 1.2 g/kg Gal-N was administered for
survival studies. Liver histologies were analyzed as follows.
[0244] Formalin-fixed, paraffin-embedded liver specimens were
sectioned at 4 .mu.m and stained with hematoxylin & eosin
(H&E). Histological assessment was performed by a blinded
observer, who scored the liver sections using the following
criteria: "0" for normal histology, "1" for minor hepatocellular
death and inflammation, "2" for widely distributed patchy necrosis
with inflammation, "3" for complete lobular disruption and diffuse
hepatocyte necrosis with panlobular inflammation, and "4" for
mortality.
[0245] In this trial, one of four control animals died before
animals were sacrificed, confirming that the extent of injury in
the used model can be rapidly fatal. Necropsy was not performed on
this animal, but based on our prior experience with this model we
expect that gross appearance was abnormal.
[0246] Two of the 3 remaining control livers, which were soft and
shrunken with an abnormal pale appearance and a rough textured
surface. The liver of one vehicle treated rat appeared normal. In
contrast, none of the 4 MSC-CM treated livers demonstrated gross
pathological changes. In contrast to the affected control livers,
the livers from MSC-CM treated animals were larger with a dark
coloration and a glossy surface typical of a healthy liver.
[0247] As shown in FIG. 10, two of 16 control animals (12.5%)
survived the entire observation period. All other vehicle treated
rats (87.5%) died within 60 hours. In the MSC-CM group, 2 of 8
animals (25%) died in the first 60 hours. Four (50%) survived the
28-day study period. Overall, MSC-CM treatment significantly
improved survival of Gal-N induced FHF (p=0.017).
Example 7
CM Treatment Downregulates Systemic Inflammation
[0248] Severe liver injury (e.g., following Gal-N treatment) can
result in a local and systemic inflammatory response that can
ultimately lead to multi-organ failure and death. To investigate
the systemic inflammatory response in animals exposed to Gal-N,
serum samples were collected from Gal-N animals 36 hours after
treatment with a systemic injection of MSC-CM (n=4) or vehicle
(n=3) and analyzed by ELISA, as follows.
[0249] Blood samples were centrifuged at 12,000 rpm for 15 min in a
microcentrifuge and serum was collected for analysis.
Quantification of rat IL-1.beta., TNF-.alpha., IL-6, IL-2,
interleukin-1 receptor antagonist (IL-1ra) and IL-10 was determined
using ELISA per manufacturer's instructions (R&D Systems,
Minneapolis, Minn.).
[0250] As shown in FIGS. 11A-F, analysis of systemic cytokine
levels revealed a non-significant decrease for IL-1.beta.
(p=0.054), but significantly lowered levels of TNF-.alpha. (64%)
(p=0.0002) and IL-6 (54%) (p=0.0002), all of which are
pro-inflammatory cytokines known to be upregulated after liver
injury. Levels of IL-2 did not change (p=0.43). In contrast, the
concentration of soluble IL-1ra was 87% lower in MSC-CM treated
animals (p=0.0002). Levels of the anti-inflammatory cytokine IL-10
were increased 4-fold in MSC-CM treated animals (p=0.032).
[0251] These studies show that infusion of supernatants from MSC
downregulates the systemic inflammation typically associated with
FHF.
Example 8
MSC-CM Improves Liver Pathology
[0252] Thirty-six hours after systemic treatment with concentrated
MSC-CM or vehicle, livers samples of Gal-N rats were analyzed by
hematoxylin & eosin (H&R) staining of paraffin embedded
sections.
[0253] Microscopic evaluation of H&E stained liver sections
revealed profound hepatocellular death with cytoplasmic
vacuolization, panlobular mononuclear leukocyte infiltration and
severe distortion of tissue architecture in vehicle-treated
animals. In contrast, livers of MSC-CM treated animals demonstrated
only minor periportal immune cell infiltration with edema and
fibrin deposition, characteristic of tissue repair.
[0254] FIG. 12A shows semi-quantitative histological examination
confirmed significant differences between the groups. The average
score in the MSC-CM group was 1.5.+-.0.6 and 3.0.+-.0.8 for vehicle
treated animals (p=0.024).
[0255] Quantification of infiltrating immune cells was performed
using freely available ImageJ software (rsb.info.nih.gov/ij/). As
shown in FIG. 12B, a 58% decrease in the number of infiltrating
immune cells was observed after MSC-CM infusion (33.+-.9.3 compared
to 84.+-.37 in controls) (p=0.004).
[0256] These results demonstrate that MSC-CM therapy inhibits liver
damage and immune cell infiltration in Gal-N induced FHF.
Example 9
MSC-CM Inhibits In Vivo Hepatocellular Apoptosis
[0257] Hepatocellular apoptosis was analyzed according to the
following procedures.
[0258] Four-micron thick sections of formalin fixed liver tissue
were deparaffinized and rehydrated after baking at 60.degree. C.
for 1 hour. Peroxidase activity was blocked using 3% hydrogen
peroxide in ethanol for 15 minutes.
[0259] Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End
Labeling (TUNEL) was performed using the APOPTAG Peroxidase In Situ
Apoptosis Detection Kit (Chemicon International, Temecula, Calif.)
according to the vendor's instructions. The sections were developed
using 3,3'-diaminobenzidine and counter-stained with Gill's
Hematoxylin.
[0260] Quantification of TUNEL-reactivity and infiltrating immune
cells was performed using freely available ImageJ software
(rsb.info.nih.gov/ij/). Ten random 40.times. images were analyzed
per animal. Particles were quantified using appropriate criteria
for corresponding sizes of the nuclei. Particles of area greater
than 700 .mu.m2 were analyzed to specifically identify hepatocytes
from non-parenchymal and inflammatory cells.
[0261] To determine whether MSC-CM infusion decreases apoptotic
cell-death, the number of TUNEL-reactive nuclei in liver sections
was determined. In sections from vehicle-treated rats, many
apoptotic nuclei were observed. In contrast, few TUNEL-positive
nuclei were present after MSC-CM treatment. Quantification revealed
a 90% reduction in TUNEL-positive nuclei (8.3.+-.12/field of view)
when compared to control animals (81.+-.52) (p=0.009). These
observations confirm that MSC-CM therapy effectively reduces
hepatocellular death in this model of acute liver injury.
Example 10
MSC-CM Inhibits Hepatocyte Apoptosis In Vitro
[0262] Inhibition of hepatocellular death by MSC-CM therapy in vivo
can either be a direct effect of trophic molecules preserving liver
cells, or an indirect effect, for example through inhibition of the
immune response. Therefore, the ability of MSC-CM to directly
inhibit apoptosis was assayed in cultured hepatocytes, as
follows.
[0263] Primary rat hepatocytes were isolated using a two-step
collagenase perfusion procedure as described previously (Dunn et
al., FASEB J., 3:174-177, 1989). The yield was routinely 200-300
million hepatocytes with viability greater than 90% as determined
by trypan blue exclusion. Hepatocyte culture medium consisted of
DMEM supplemented with 10% FBS 14 ng/ml glucagon, 0.5 U/ml insulin,
20 ng/ml epidermal growth factor (EGF), 7.5 .mu.g/ml
hydrocortisone, 200 .mu.g/ml streptomycin and 200 U/ml penicillin.
Culture conditions were hepatocyte medium for control experiments;
hepatocyte medium mixed at a 50:1 ratio with the 25-fold
concentrated MSC-CM (2% MSC-CM); and hepatocyte medium mixed at a
12.5:1 ratio for 8% MSC-CM.
[0264] Isolated primary rat hepatocytes were cultured as described
above for a total of 7 days in 12-well plates at a density of
1.times.10.sup.5 cells/cm.sup.2 in a collagen gel sandwich
configuration. Apoptosis was induced using Actinomycin D (1 hour)
and TNF-.alpha. (8 hours). During exposure to TNF-.alpha.,
hepatocytes were cultured in hepatocyte medium only or hepatocyte
medium supplemented with 2% or 8% of 25.times. concentrated MSC-CM.
Hepatocytes were stained using a fluorescent Live Dead Assay
(Molecular Probes). Cell death was quantified using digital image
analysis of 4 images per well. Experiments were performed in
triplicate.
[0265] As shown in FIG. 14, a 22% increase in the fraction of
viable cells was observed when hepatocyte medium was supplemented
with 2% MSC-CM (46% viable compared to 38% viable in control
cultures) (p=0.005). No significant increase in hepatocytes
viability was seen with 8% MSC-CM (43%) (p=0.15). These experiments
demonstrate that low level MSC-CM has a direct anti-apoptotic
effect on hepatocytes. Therefore, MSC-CM is directly
hepatoprotective. In other words, MSC-CM has a direct preserving
effect on hepatocytes.
[0266] Rescue/protection from hepatocellular apoptosis was more
prominent in vivo than in vitro. This observation is likely due to
local and systemic inhibition of the apoptotic response.
Example 11
MSC-CM Enhances Liver Regeneration
[0267] Stimulation of endogenous repair programs is also a
potential mechanism of the above described MSC-CM-induced
therapeutic effect.
[0268] Liver samples of Gal-N rats were analyzed 36 hours after FHF
induction with MSC-CM or vehicle. Proliferating cell nuclear
antigen (PCNA) staining was performed by treating sections with 10
mM Citrate Buffer at pH 6.0 using a digital pressure cooker.
Subsequently, sections were blocked with 1.5% horse serum for 15
minutes and incubated with mouse monoclonal anti-PCNA (Clone 24, BD
Transduction Laboratories, San Jose, Calif.) at a 1:500 dilution
for 1 hour at room temperature. Primary antibody was detected using
Vectastain Elite ABC kit (Vector laboratories, Burlington, Calif.).
Sections were developed using 3,3'-diaminobenzidine and
counter-stained with Gill's Hematoxylin.
[0269] PCNA reactive cells were quantified and compared to
vehicle-treated animals. As shown in FIG. 15, 3-fold more
PCNA-reactive cells were observed in MSC-CM treated livers than
control livers.
[0270] The mRNA expression profiles of 10 genes known to be
upregulated during liver regeneration was performed using RT-PCR,
as described in Example 3E. Forward and reverse oligonucleotide
combinations are shown in Table 3.
TABLE-US-00004 TABLE 3 RT-PCR Oligonucleotides SEQ SEQ ID ID
Amplicon Gene NO: Forward (5'-3') NO: Reverse (5'-3') (bp) OSM 5
caactgggtgctttcagaca 6 aacccatgaagcgatggtag 253 AR 7
gtctttgtctccgccgtaag 8 ctgaacttctggagccttcg 244 TGF-.alpha. 9
gcaagttctgcctgttcctc 10 gcactgaaccaacccacttt 161 HGF 11
cgagctatcgcggtaaagac 12 tgtagctttcaccgttgcag 165 TNF 13
actcccagaaaagcaagcaa 14 cgagcaggaatgagaagagg 211 EGF 15
acaccgaaggtggctatgtc 16 tagagtcagggcaaggcagt 195 IL-6 17
ccggagaggagacttcacag 18 cagaattgccattgcacaac 134 SCF 19
caaaactggtggcgaatctt 20 gccacgaggtcatccactat 217 HG-EBF 21
gcctcctgtaattgctctgc 22 gccaaaaatcctggagcata 207 TIMP3 23
tgtacaccccagcctctttc 24 cttctcgccaagacctcaac 182 18s 25
atgacatcaagaaggtggtg 26 cataccaggaaatgagcttg 177
[0271] Visibly stronger bands were observed for each of the genes
analyzed. As shown in FIG. 16, this observation was confirmed using
quantitative analysis. Increases ranged from 4-fold to 27-fold.
[0272] These results demonstrate that administration of MSC-derived
soluble factors enhances liver regeneration programs during
FHF.
Example 12
MSC-CM Stimulates Hepatocyte Proliferation In Vitro
[0273] Hepatocyte duplication, a major component of liver
regeneration, is regulated by a complex interaction of paracrine
and endocrine signals involving non-parenchymal liver cell types as
well as extra-hepatic organs. To determine whether MSC-derived
factors can directly enhance hepatocyte replication, the effect of
MSC-CM on the in vitro proliferation of isolated primary
hepatocytes was explored.
[0274] Primary rat hepatocytes were isolated as described in
Example 9. Hepatocytes were subsequently seeded at a low density
(1.25.times.10.sup.3 cells/cm.sup.2) on a feeder layer of 3T3 J2
fibroblasts (8.times.10.sup.4 cells/cm.sup.2) previously exposed to
12 .mu.g/ml mitomycin-C for 2.5 hours to arrest growth. Hepatocytes
were allowed to proliferate with daily medium changes. Hepatocyte
culture medium is described in Example 9.
[0275] Cells were cultured with 10 .mu.M bromodeoxyuridine (BrdU;
Sigma). After 48 hours, cultures were fixed in 70% ethanol for 45
minutes and treated with 4N HCl and 0.2% TRITONX-10. Cells were
then incubated in blocking buffer for 30 minutes and incubated for
60 minutes with anti-BrdU-Alex594 (Invitrogen, Carlsbad, Calif.)
and rabbit anti-rat albumin (ICN Pharmaceuticals, Aurora, Ohio) at
37.degree. C., followed by FITC conjugated anti-rabbit IgG (ICN
Pharmaceuticals) at room temperature. BrdU positive cells in each
hepatocyte colony were counted in fluorescence microscopy images.
Albumin content in supernatant samples was determined by an
enzyme-linked immunosorbent assay (ELISA) using purified rat
albumin and a peroxidase-conjugated anti-albumin antibody (MP
Biomedicals, Aurora, Ohio). Urea content was determined with a
commercially available kit (StanBio Laboratory, Boerne, Tex.)
according to the vendor's instructions.
[0276] Proliferation of rat hepatocyte colonies on a feeder layer
of growth-inhibited 3T3-fibroblasts as visualized by double
immunofluorescence staining for BrdU and albumin. As shown in FIG.
17A, with 2% MSC-CM supplementation (represented in graph as 2), a
79% increase in BrdU-positive hepatocytes was observed (9 3.+-.12
per field of view with MSC-CM vs. 52.+-.14 in control cultures)
(p=0.001). When medium was supplemented with 8% MSC-CM (shown in
graph as "8"), no significant increase was measured (59.+-.14 BrdU)
(p=0.37).
[0277] As shown in FIG. 17B, in parallel to these findings, the
total amount of albumin secreted and urea synthesized per well was
increased in 2% MSC-CM supplemented cultures. Albumin levels were
29.+-.2.4 .mu.g/ml/day, compared to 20.+-.1.2 .mu.g/ml/d under
control conditions (p=0.006). No significant difference compared to
control was observed in 8% MSC-CM conditions (23.+-.2.2 .mu.g/ml/d)
(p=0.14).
[0278] As shown in FIG. 17C, urea synthesis shifted from 69.+-.8.1
.mu.g/ml/d in control cultures to 90.+-.10 .mu.g/ml/d in 2% MSC-CM
conditions (p=0.019), but was not significantly altered in the
presence of 8% MSC-CM (53.1 .mu.g/ml/d) (p=0.063).
[0279] In general, therefore, markers of hepatocyte proliferation
and function were significantly higher in the presence of 2% MSC-CM
in vitro. Although 8% MSC-CM also had an effect on hepatocyte
proliferation and function in vitro, it was not as pronounced as
those levels observed with 2% MSC-CM.
[0280] The data presented herein clearly demonstrates that MSC-CM
is capable of increasing hepatocyte proliferation. MSC-CM increased
the number of proliferating cells at least 3-fold in the
regenerating, injured liver. MSC cells are not required for the
observations described above. Secreted factors contained in MSC-CM
are sufficient in protecting hepatocytes from apoptosis and
promoting hepatocyte proliferation. Thus, systemic infusion of
MSC-CM represents an effective strategy for MSC therapy.
Example 13
MSC-Derived Factors Reverse Fulminant Hepatic Failure
[0281] This example demonstrates that MSC-derived molecules provide
survival benefits against parenchymal cell loss, wherein cell loss
is integrated with a local and systemic immune response, following
intravenous bolus administration of MSC-CM and/or extracorporeal
perfusion with a bioreactor containing MSCs (e.g., undifferentiated
MSCs).
[0282] Sprague-Dawley rats were intraperitoneally administered a
total of two injections of 1.2 g/kg of a hepatotoxin,
D-galactosamine (Gal-N), each separated by 12 hours, as described
in Example 5.
[0283] Animals treated 24 hours later with intravenous injections
into the penile vein of (1) whole MSCs or (2) MSC lysates. MSCs
were isolated and characterized as described in Examples 1 and
2.
[0284] A total of 2.times.10.sup.6 cells were administered to each
subject for whole cell MSC therapy. The volume of whole MSCs was
500 .mu.l.
[0285] Cell lysates were prepared by sonication. The dose of
sonicated cells administered was 2.times.10.sup.6 cells per
subject. The volume of MSC lysate was 500 .mu.l. Unlike MSC-CM, MSC
lysate is not concentrated.
[0286] Vehicle (PBS) and NIH 3T3-J2 fibroblast cell lysate were
administered as controls. The volume of each control was 500
.mu.l.
[0287] As shown in FIG. 18, no significant benefit was seen after
the intravenous infusion of 2.times.10.sup.6 human MSCs. This
observation is most likely due to poor engraftment, entrapment in
the alveolar capillary bed, and/or immune rejection of the cells.
In contrast, treatment with cellular lysates, derived from the same
cell mass used for transplantation, showed an increased survival
trend compared to vehicle (P<0.47) and fibroblast lysate
(P<0.36) controls.
Example 14
MSC-Derived Components Reverse FHF
[0288] The experiments described in this example demonstrate that
MSC-derived components promote survival in FHF-models and that this
effect is not species specific, e.g., the therapeutic potential of
MSC-CM is not species specific.
[0289] As shown in FIG. 19A, a longitudinal study using MSC-CM from
2.times.10.sup.6 human MSCs revealed a distinct survival benefit
compared to vehicle (P<0.032) and fibroblast (P<0.026)
concentrated medium.
[0290] 72 hour survival of FHF-induced rats was monitored as a
function of MSC mass from which MSC-CM was collected. As shown in
FIG. 19B, MSC-CM was most effective when derived from a MSC mass of
2.times.10.sup.6 cells.
[0291] The observation that xenogeneic MSC lysates and supernatants
decreased animal mortality suggests that these factors can cross
species barriers. Thus, the therapeutic potential of MSC-CM is not
species-specific.
Example 15
Combined Metabolic and Secretory Function in MSC-EB Provide
Hepatoprotection and Survival Benefit
[0292] A MSC-extracorporeal bioreactor (MSC-EB) was developed to
combine the effectiveness of MSC whole cells and MSC-CM in a single
device.
[0293] Extracorporeal device operation was previously reported
(Shinoda et al., J. Surg. Res., 137:130-140, 2007). Briefly, male
Sprague-Dawley rats weighing between 280 and 370 grams were
anaesthetized using intraperitoneal injections of ketamine and
xylazine at 110 and 0.4 mg/kg, respectively. The left carotid
artery and right jugular vein were cannulated and the animal was
placed in a metabolic cage. Twenty-four hours later, 1.2 g/kg Gal-N
freshly dissolved in physiological saline and adjusted to pH 7.3
with 1 N NaOH was injected i.p., followed by a second equal
injection 12 hours later, as described in Example 5. Twenty-four
hours after the first injection of Gal-N, the arterial and venous
lines were connected to an extracorporeal circuit. Plasma was
separated using a plasma separator (MicroKros, pore size 0.2
micron). Plasma was perfused through the polycarbonate, flat-plate
bioreactor and subsequently reunited with the cellular components
of the blood and returned to the animal. The extracorporeal
bioreactor was operated for 10 hours. Animals that died during
reactor operation and failed to receive adequate treatment (MSC-EB,
N=3 and Fibroblast-EB, N=2) were censored from analysis. Animal
survival was monitored every 12 hours. Plasma or whole blood was
analyzed for liver injury biomarkers (e.g., serum alanine
aminotransferase (ALT), serum aspartate aminotransferase (AST))
using a microfluidic metabolic assay (Picollo, Abaxis, Union City,
Calif.). An exemplary schematic representation of an extracorporeal
circuit is shown in FIG. 20.
[0294] Animals were treated 24 hours after FHF induction with a
human MSC-EB connected to the systemic circulation of the animal.
Bioreactors seeded with fibroblasts (fibroblast-EB) and acellular
(acellular-EB) bioreactors served as controls. After 10 hours of
extracorporeal perfusion, animals were taken off assist support and
monitored for survival. Plasma was obtained at the start of, and 24
hours after, bioreactor treatment and analyzed for hepatocyte
enzyme release. As shown in FIGS. 21A-B, liver serologies,
including aspartate aminotransferase (AST; P<0.02) and alanine
aminotransferase (ALT; P<0.001) were improved in animals treated
with the MSC-EB. These data demonstrate a hepatoprotective effect
of device therapy as shown by the reduction in biochemical markers
of hepatocyte death. As shown in FIG. 21C, 71% of animals treated
with the MSC-EB survived, compared to 14% in both acellular
(P<0.037) and fibroblast controls (P<0.05). Table 5 shows
liver serologies after MSC-EB treatment.
TABLE-US-00005 TABLE 5 Liver Serologies are Improved after MSC-EB
Treatment Post Para- MSC-EB Post MSC-EB % meter MSC Pre MSC-EB (24
hr) (48 hr) Change TB - 0.73 .+-. 0.75 0.9 N/A +23 (mg/dl) + 0.76
.+-. 0.26 1.16 .+-. 0.83 1.2 .+-. 0.84 +58 AST - 2007 .+-. 837.4
1999 N/A 0 (U/l) + 1513.2 .+-. 513.2 888 .+-. 272.6 940.8 .+-.
330.53 -41 ALT - 1222.33 .+-. 710.4 1233 N/A 0 (U/l) + 859.2 .+-.
125.7 168 .+-. 61.9 358.8 .+-. 198.4 -80 ALP - 216 .+-. 39.1 106
N/A -51 (U/l) + 192.8 .+-. 41 91.2 .+-. 23.2 98.4 .+-. 24.6 -53
Data are expressed as mean .+-. standard error of the mean (SEM).
Percent change refers to post MSC-EB (24 hours) relative to pre
MSC-EB. (-) is EB without MSCs. N = 5 for (-). N = 3 for (+). No
data acquired due to mortality (N/A).
[0295] As shown in Table 5, liver serologies were improved after
MSC-EB treatment.
Example 16
MSC-CM Therapy Inhibits Panlobular Leukocyte Invasion, Bile Duct
Duplication, and Hepatocellular Death
[0296] Post MSC-CM histopathological changes were evaluated using a
sub-lethal Gal-N regimen (0.6 g/kg) to induce acute liver injury,
while ensuring survival in our control-treated group for
comparison. It should be noted that even at this Gal-N dose,
mortality occurred in a vehicle-treated group (N=1). This confirms
that the extent of injury in this model can still be fatal. Gal-N
injured rats were treated with vehicle (N=4) or MSC-CM (N=4) 24
hours after injury and their livers were harvested 36 hours
thereafter for pathological analysis.
[0297] Liver tissue was harvested from rats induced with a
sub-lethal regimen of Gal-N (0.6 g/kg), 36 hours after treatment
with MSC-CM. Tissue was fixed in 10% buffered formalin, embedded in
paraffin, sectioned to 6-.mu.m thickness, and stained with
hematoxylin and eosin.
[0298] Microscopic evaluation of liver tissue from vehicle treated
rats revealed profound hepatocellular apoptosis, bile duct
duplication and panlobular mononuclear leukocyte infiltration with
cytoplasmic vacuolization and severe distortion of tissue
architecture. MSC-CM treated rats showed no signs of disseminated
inflammation, although minor periportal infiltration with edema and
fibrin deposition consistent with tissue repair was observed.
[0299] Histopathology was scored using the criteria described in
Example 6 ("0" for normal histology, "1" for minor hepatocellular
death and inflammation, "2" for widely distributed patchy necrosis
with inflammation, "3" for complete lobular disruption and diffuse
hepatocyte necrosis with panlobular inflammation, and "4" for
mortality). Clearly, MSC-CM treated livers presented a lower score
than vehicle treated livers.
[0300] Lower numbers of infiltrating leukocytes were observed in
MSC-CM treated livers.
Example 17
MSC-CM Alters Immune Cell Migration to the Liver
[0301] To investigate whether the lack of panlobular leukocyte
infiltration observed in MSC-CM treated livers may be due to the
MSC-CM-dependent diversion of immune cell migration away from an
inflamed, target organ, radiolabeled leukocytes were adoptively
transferred directly after MSC-CM or vehicle treatment, into Gal-N
(0.6 g/kg) injured rats. Briefly, leukocytes were isolated from
whole rat blood by NH4Cl erythrocyte lysis. Cells were pelleted,
washed once with PBS and resuspended in 0.9% saline containing the
In111 oxine isotope (GE Healthcare Biosciences Corp., Piscataway,
N.J.). Cells were labeled at 92% efficiency with high viability.
Approximately 15.times.10.sup.6 cells were infused into the penile
vein of Gal-N injured (0.6 g/kg) directly after treatment with
vehicle or MSC-CM.
[0302] Leukocyte trafficking was then monitored in these animals
using single photon emission computed tomography (SPECT) over time.
SPECT images were captured using a M.CAM gamma camera setup
(Siemens Medical Systems, Malvern, Pa.) at 0, 3 and 24 hours after
leukocyte infusion. An illustration of this protocol is provided in
FIG. 22.
[0303] Qualitatively, more leukocytes were seen migrating to the
liver in vehicle treated animals over time. In contrast, there was
a distinct decrease in signal intensity in the liver of MSC-CM
treated animals over time. These results suggest that there was a
selective pressure upon leukocytes to emigrate from the liver due
to MSC-CM, unlike control conditions where leukocytes eventually
migrated to the injured organ.
[0304] As shown in FIG. 22B, in rats administered a sub-lethal dose
of Gal-N (0.6 mg/kg), dramatic changes in leukocyte counts and
differentials were observed in peripheral blood cell populations in
MSC-CM treated animals.
[0305] Leukocyte distribution was also evaluated at the organ
level. Animals were sacrificed at 0.5 hour, 8 hour, and 24 hour
time points. Leukocyte levels were determined for indicated organs
using scintillation counts.
[0306] As shown in FIG. 22C, lymphoid organs and the liver are the
primary sites of MSC-CM activity (50% of total solid organs at 24
hours). Considerable MSC-CM activity was, however, observed in
every organ analyzed.
[0307] These data support the notion that altered leukocyte
migration may be a potential target of MSC-CM therapy. These data
also support the systemic use of MSC-CM therapy, e.g., for the
treatment of multi-organ failure in a subject. Thus, MSC-CM therapy
may provide a beneficial effect in the following organs, the lung,
the heart, the pancreas, the GI, the thymus, the lymph node, bone
marrow, the spleen, the liver, and the blood.
Example 18
MSC-CM Characterization
[0308] In an effort to understand the molecular mediators of the
observed effects of MSC therapy, we examined MSC-CM using a
high-density protein array.
[0309] Briefly, MSC supernatants were prepared by collecting
serum-free medium after 24 hour culture of approximately
2.times.10.sup.6 MSCs. Supernatants were analyzed for a panel of
specified proteins using an antibody array (RAYBIO Human Cytokine
Antibody Array C Series 2000, RayBiotech Inc., Norcross, Ga.) as
specified by the vendor.
[0310] As shown in FIG. 23A, MSC-CM contained 69 of the 174
proteins assayed, which included a broad spectrum of molecules
involved in immunomodulation and liver regeneration. As shown in
FIG. 23B. cluster analysis revealed that a large fraction (30%) of
MSC-CM was composed of chemokines, many of which were expressed at
high levels.
[0311] MSC-CM was then fractionated based on functionality using
affinity-based methods rather than other arbitrary molecular
criteria such as size or hydrophobicity, as follows. MSC-CM was
passed over an affinity column impregnated with heparin sulfate, a
known ligand for all chemokines and separated into bound and
unbound fractions. Each fraction was infused into FHF-induced rats
with overall survival as the study endpoint.
[0312] As shown in FIG. 23C, the therapeutic activity of MSC-CM was
restricted to the heparin bound fraction, providing a strong
correlation between chemokines and the survival benefit after
MSC-CM infusion in FHF-induced rats.
[0313] MSC-CM did not increase mRNA levels of the transcription
factor Foxp3 in peripheral blood mononuclear cells. Foxp3
expression is restricted to regulatory T cells, an endogenous
suppressor lymphocyte population.
[0314] MSC-CM is chemotactically active, while Fibroblast-CM is
inert. This was evaluated using a microfluidic chemotaxis chamber
previously described (Jeon et al., Nat. Biotech., 20:826-830,
2002). Neutrophils exposed to Fibroblast-CM do not show
morphological changes involved with chemotaxis, whereas neutrophils
exposed to MSC-CM have prominent filopodial extensions and are
chemotactically primed.
[0315] One component of MSC-CM that may be responsible for
increased hepatocyte replication is SDF-1a. Hepatocytes were
proliferated using aforementioned techniques in standard culture
medium alone or supplemented with 5 ng/ml SDF-1a (FIG. 29B) or the
SDF-1a receptor antagonist, AMD3100 at 1 uM. Increased SDF-1a
stimulation led to larger colonies, while blockade of SDF-1a
signaling led to smaller colonies. FIG. 24 shows urea synthesis in
SDF-1a and AMD3100 treated cells. Urea synthesis is a surrogate
biomarker for hepatocyte mass; the results show significant
differences between control conditions and modulation of the SDF-1a
signaling pathway.
Other Embodiments
[0316] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
26120DNAArtificial Sequencesynthetic polynucleotide 1aagcctgacc
acgctttcta 20219DNAArtificial Sequencesynthetic polynucleotide
2gtagagcggg gtttcacca 19320DNAArtificial Sequencesynthetic
polynucleotide 3gagtcaacgg atttggtcgt 20420DNAArtificial
Sequencesynthetic polynucleotide 4ttgattttgg agggatctcg
20520DNAArtificial Sequencesynthetic polynucleotide 5caactgggtg
ctttcagaca 20620DNAArtificial Sequencesynthetic polynucleotide
6aacccatgaa gcgatggtag 20720DNAArtificial Sequencesynthetic
polynucleotide 7gtctttgtct ccgccgtaag 20820DNAArtificial
Sequencesynthetic polynucleotide 8ctgaacttct ggagccttcg
20920DNAArtificial Sequencesynthetic polynucleotide 9gcaagttctg
cctgttcctc 201020DNAArtificial Sequencesynthetic polynucleotide
10gcactgaacc aacccacttt 201120DNAArtificial Sequencesynthetic
polynucleotide 11cgagctatcg cggtaaagac 201220DNAArtificial
Sequencesynthetic polynucleotide 12tgtagctttc accgttgcag
201320DNAArtificial Sequencesynthetic polynucleotide 13actcccagaa
aagcaagcaa 201420DNAArtificial Sequencesynthetic polynucleotide
14cgagcaggaa tgagaagagg 201520DNAArtificial Sequencesynthetic
polynucleotide 15acaccgaagg tggctatgtc 201620DNAArtificial
Sequencesynthetic polynucleotide 16tagagtcagg gcaaggcagt
201720DNAArtificial Sequencesynthetic polynucleotide 17ccggagagga
gacttcacag 201820DNAArtificial Sequencesynthetic polynucleotide
18cagaattgcc attgcacaac 201920DNAArtificial Sequencesynthetic
polynucleotide 19caaaactggt ggcgaatctt 202020DNAArtificial
Sequencesynthetic polynucleotide 20gccacgaggt catccactat
202120DNAArtificial Sequencesynthetic polynucleotide 21gcctcctgta
attgctctgc 202220DNAArtificial Sequencesynthetic polynucleotide
22gccaaaaatc ctggagcata 202320DNAArtificial Sequencesynthetic
polynucleotide 23tgtacacccc agcctctttc 202420DNAArtificial
Sequencesynthetic polynucleotide 24cttctcgcca agacctcaac
202520DNAArtificial Sequencesynthetic polynucleotide 25atgacatcaa
gaaggtggtg 202620DNAArtificial Sequencesynthetic polynucleotide
26cataccagga aatgagcttg 20
* * * * *