U.S. patent application number 12/709379 was filed with the patent office on 2010-09-23 for methods and platforms for drug discovery.
This patent application is currently assigned to iZumi Bio, Inc.. Invention is credited to Tetsuya Ishikawa, Hideki Masaki, Kazuhiro Sakurada, Shunichi Takahashi.
Application Number | 20100240090 12/709379 |
Document ID | / |
Family ID | 39253880 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240090 |
Kind Code |
A1 |
Sakurada; Kazuhiro ; et
al. |
September 23, 2010 |
METHODS AND PLATFORMS FOR DRUG DISCOVERY
Abstract
The present invention involves methods for identifying an agent
that corrects a phenotype associated with a health condition or a
predisposition for a health condition. The invention also involves
methods for identifying a diagnostic cellular phenotype,
determining the risk of a health condition in a subject, methods
for reducing the risk of drug toxicity in a human subject, and
methods for identifying a candidate gene that contributes to a
human disease. The invention also discloses human induced
pluripotent stem cell lines.
Inventors: |
Sakurada; Kazuhiro;
(Yokohama, JP) ; Masaki; Hideki; (Akita, JP)
; Ishikawa; Tetsuya; (Meguro-ku, JP) ; Takahashi;
Shunichi; (Kobe, JP) |
Correspondence
Address: |
WILSON, SONSINI, GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Assignee: |
iZumi Bio, Inc.
South San Francisco
CA
|
Family ID: |
39253880 |
Appl. No.: |
12/709379 |
Filed: |
February 19, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12484163 |
Jun 12, 2009 |
|
|
|
12709379 |
|
|
|
|
12157967 |
Jun 13, 2008 |
|
|
|
12484163 |
|
|
|
|
61040646 |
Mar 28, 2008 |
|
|
|
61061592 |
Jun 13, 2008 |
|
|
|
61061594 |
Jun 13, 2008 |
|
|
|
Current U.S.
Class: |
435/29 |
Current CPC
Class: |
C12N 5/0696 20130101;
C12N 2501/604 20130101; C12N 2501/603 20130101; C12N 2799/027
20130101; C12N 2501/606 20130101; C12N 2510/00 20130101; A61P 25/28
20180101; C12N 15/85 20130101; C12N 2501/602 20130101; A61P 3/10
20180101 |
Class at
Publication: |
435/29 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 15, 2007 |
JP |
JPO-2007-159382 |
Nov 20, 2007 |
EP |
PCT/EP2007/010019 |
Jun 13, 2008 |
EP |
PCT/EP2008/005047 |
Jun 13, 2008 |
IB |
PCT/IB2008/002540 |
Claims
1. A method for identifying an agent that corrects a phenotype
associated with a health condition or a predisposition for the
health condition, comprising: (i) contacting a first population of
cells from a human induced pluripotent stem cell line, or cells
differentiated from the human induced pluripotent stem cell line,
with a candidate agent; (ii) contacting a second population of
cells from the human induced pluripotent stem cell line, or cells
differentiated from the human induced pluripotent stem cell line,
with a negative control agent; (iii) assaying the phenotype in the
first population and second population after the contacting steps;
and (iv) identifying the candidate agent as correcting the
phenotype if the assayed phenotype of the first population after
the contacting step is closer to a normal phenotype than the
phenotype of the second population after the contacting step;
wherein the cells in the first and second populations of human
induced pluripotent stem cells: (a) comprise at least one
endogenous allele associated with the health condition or the
predisposition for the health condition; or (b) are generated from
a subject suffering from the health condition or the predisposition
for the health condition.
2-46. (canceled)
47. A method for evaluating a physiological function of a compound
comprising treating cells obtained by inducing differentiation of
an induced pluripotent stem (iPS) cell with the compound, wherein
the iPS cell is obtained by nuclear reprogramming of a somatic
cell, which comprises contacting a nuclear reprogramming factor
with the somatic cell to obtain an induced pluripotent stem
cell.
48. A method for evaluating the toxicity of a compound comprising
treating cells obtained by inducing differentiation of an induced
pluripotent stem (iPS) cell with the compound, wherein the iPS cell
is obtained by nuclear reprogramming of a somatic cell, which
comprises contacting a nuclear reprogramming factor with the
somatic cell to obtain an induced pluripotent stem cell.
49. A method for evaluating the effect of a compound on a cellular
phenotype comprising treating cells obtained by inducing
differentiation of an induced pluripotent stem (iPS) cell with the
compound, wherein the iPS cell is obtained by inducing pluripotency
of a somatic cell, which comprises contacting an induction factor
with the somatic cell to obtain an induced pluripotent stem
cell.
50. A method for evaluating the toxicity of a compound comprising
treating cells obtained by inducing differentiation of an induced
pluripotent stem (iPS) cell with the compound, wherein the iPS cell
is obtained by inducing pluripotency of a somatic cell, which
comprises contacting an induction factor with the somatic cell to
obtain an induced pluripotent stem cell.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 12/484,163, filed Jun. 12, 2009, which is a
continuation-in-part of U.S. application Ser. No. 12/157,967, filed
Jun. 13, 2008, which claims the benefit of U.S. Provisional
Application No. 61/040,646, filed Mar. 28, 2008, and which also
claims the benefit of International Application No.
PCT/EP2007/010019, filed Nov. 20, 2007, and which also claims the
benefit of Japanese Application No. JPO-2007-159382, filed Jun. 15,
2007; this application also claims the benefit of International
Application No. PCT/IB2008/002540, filed Jun. 13, 2008,
International Application No. PCT/EP2008/005047, filed Jun. 13,
2008, U.S. Provisional Application No. 61/061,592, filed Jun. 13,
2008, and U.S. Provisional Application No. 61/061,594, filed Jun.
13, 2008, all of which are herein incorporated by reference in
their entirety.
BACKGROUND OF THE INVENTION
[0002] The pharmaceutical industry has expended vast technical and
financial resources to develop novel therapeutic agents. Yet, the
failure rate (more than 90%) for lead compounds remains
persistently high. Often, lead drug compounds that meet
expectations in preclinical models, such as inbred animal models,
or a small number of cell lines, are toxic or ineffective when
administered to a human clinical trial patient population. A
fundamental deficiency in most current drug development efforts is
that they do not evaluate candidate drug efficacy and toxicity in
the context of the extreme genetic diversity of the human patient
population. In other words, in the present drug development
paradigm, drug efficacy and toxicity are not tested on many, if not
most, of the relevant genotype/phenotype combinations present in
the human population. Indeed, even after successful trials in a
relatively small human clinical trial population, unexpected
adverse effects can be revealed once these drugs are administered
to a broader human patient population.
SUMMARY OF THE INVENTION
[0003] The present invention involves methods for identifying an
agent that corrects a phenotype associated with a health condition
or a predisposition for a health condition comprising contacting a
first population of cells from a human induced pluripotent stem
cell line, or cells differentiated from the human induced
pluripotent stem cell line, with a candidate agent; contacting a
second population of cells from a human induced pluripotent stem
cell line, or cells differentiated from the human induced
pluripotent stem cell line, with a control agent; wherein the cells
in both populations comprise at least one endogenous allele
associated with the health condition or predisposition for the
health condition; assaying the two populations and identifying
candidate agents as correcting the phenotype if the first
population is closer to a normal phenotype following treatment than
the second population. The condition may be selected from health
conditions such as a neurodegenerative disorder, a neurological
disorder, a mood disorder, a cardiovascular disease, a metabolic
disorder, a respiratory disease, a drug sensitivity condition, an
eye disease, an immunological disorder, or a hematological disease.
The cells may be differentiated from induced stem cells to neural
stem cells, neurons, cardiomyocytes, hepatic stem cells, or
hepatocytes. The phenotype described may be apoptosis,
intracellular calcium level, calcium flux, protein kinase activity,
enzyme activity, cell morphology, receptor activation, protein
trafficking, intracellular protein aggregation, organellar
composition, motility, intercellular communication, protein
expression, or gene expression.
[0004] The invention also involves methods for identifying a
diagnostic cellular phenotype comprising comparing a set of cells
from a subject to cells from a subject free of the health condition
wherein both sets of cells were induced pluripotent stem cells, or
were cells differentiated from induced pluripotent stem cells, and
wherein the comparison is performed on a computer. The cells may be
differentiated from induced stem cells to neural stem cells,
neurons, cardiomyocytes, hepatic stem cells, or hepatocytes.
[0005] The invention also involves methods for determining the risk
of a health condition in a subject comprising comparing at least
one phenotype determined in a first set of cells derived from the
subject to the at least one phenotype determined in a second set of
cells derived from subjects free of the health condition and to the
at least one phenotype determined in a third set of cells derived
from subjects suffering from the health condition; and indicating
that the subject is at high risk for the health condition if the at
least one phenotype determined in the first set of cells is more
similar to the at least one phenotype determined in the third set
of cells than the at least one phenotype determined in the second
set of cells, wherein the first, second, and third sets of cells
were induced pluripotent stem cells, or were cells differentiated
from induced pluripotent stem cells, and wherein the comparison is
performed on a computer.
[0006] The invention also involves methods for reducing the risk of
drug toxicity in a human subject, comprising contacting one or more
cells differentiated from an induced pluripotent stem cell line
generated from the subject with a dose of a pharmacological agent,
assaying the contacted one or more differentiated cells for
toxicity, and prescribing or administering the pharmacological
agent to the subject if, and only if, the assay is negative for
toxicity in the contacted cells. The cells differentiated from the
induced pluripotent stem cell line may be hepatocytes,
cardiomyocytes, or neurons.
[0007] The invention also involves methods for identifying a
candidate gene that contributes to a human disease, comprising
comparing a global gene expression profile of cultured human cells
of a differentiated cell type from a plurality of healthy
individuals to a global gene expression profile of cultured human
cells of the differentiated cell type from a plurality of
individuals suffering from the human disease and identifying one or
more genes that have different expression levels as candidate genes
that contribute to the human disease, wherein the comparison is
performed on a computer.
[0008] The invention also discloses a human induced pluripotent
stem cell line generated from a subject diagnosed as suffering from
a health condition, or comprising at least one endogenous allele
associated with a health condition or a predisposition for the
health condition. The invention also discloses an isolated
population of human cells comprising neural stem cells or neurons
from a subject having at least one endogenous allele associated
with a neurodegenerative disorder, a neurological disorder, or a
mood disorder, or from a subject diagnosed with the
neurodegenerative disorder, neurological disorder, or mood
disorder. The invention also discloses an isolated population of
human cells comprising human cardiac progenitor cells or
cardiomyocytes from a subject having at least one endogenous allele
associated with a cardiovascular disease, or from a subject
diagnosed with the cardiovascular disease. The invention also
discloses an isolated population of human cells comprising hepatic
stem cells or hepatocytes from a subject having at least one
endogenous allele associated with a drug sensitivity condition, or
from a subject diagnosed with the drug sensitivity condition.
[0009] The invention further discloses a panel of genetically
diverse human induced pluripotent stem cell lines, comprising human
induced pluripotent stem cell lines generated from a plurality of
individuals each of which carry at least one polymorphic allele
that is unique among the plurality of individuals.
INCORPORATION BY REFERENCE
[0010] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, and patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0012] FIG. 1 is a schematic comparison of a traditional drug
discovery scheme (left) in which lead compounds are tested against
a disease target in heterologous systems (e.g., animal models)
prior to testing compound efficacy and safety in patients versus a
new drug discovery paradigm (right) in which lead compounds are
first identified based on their efficacy in correcting a
disease-relevant cellular phenotype in patient-derived,
disease-relevant cell types.
[0013] FIG. 2 is an overview of an exemplary, non-limiting, scheme
for patient iPSC-based disease modeling and drug discovery.
[0014] FIG. 3 is an overview of an exemplary, non-limiting, scheme
for patient iPSC-based testing of lead drug candidate efficacy and
safety in cells from a genetically diverse cohort of patient iPSC
lines.
[0015] FIG. 4 is an overview of an exemplary, non-limiting, scheme
for patient iPSC-based identification of predictive biomarkers for
drug efficacy and toxicity. Such biomarkers are used in, e.g.,
patient stratification for clinical trials of drug candidates, and
also for optimal dosing and safety of approved therapeutics in
specific patients or patient populations, which is sometimes
referred to as "personalized medicine."
[0016] FIG. 5 (Top Panel) shows photomicrographs of fibroblasts
from three SMN1.sup.-/- SMA patients and two SMN1.sup.-/+ healthy
control subjects; (Bottom Panel) shows photomicrographs of iPSC
colonies derived from the corresponding SMA case and control
subject fibroblasts illustrated in the top panel.
[0017] FIG. 6 shows photomicrographs of embryoid bodies obtained
from the SMA case and control iPSC lines shown in FIG. 5.
[0018] FIG. 7 shows immunofluorescence photomicrographs of staining
for ectodermal (TuJ1), mesodermal (Desmin), and endodermal (AFP)
lineage markers in cells differentiated from SM10d iPSCs.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0019] Genetic variations (e.g., polymorphic alleles) within and
among human patient populations underlie, to a large extent,
differences in individual disposition to diseases, disease
manifestation, disease severity, and response to treatment (e.g.,
to drug treatment). The prevalent animal and cellular models for
human disease and drug discovery provide a poor representation of
the genotypic/phenotypic spectrum extant in the patient populations
to be treated. For example, strains of mice and rats commonly used
in drug discovery are highly inbred, and thus only represent a very
narrow range of possible genotype/phenotype combinations in mice or
rats, let alone humans. Likewise, the relatively small number of
human cell lines used for drug screening may reflect the
genotypic/phenotypic scope of the individuals from which they were
derived, but not that of a genetically diverse population. Further,
most human cell lines are quite limited in their capacity to
generate or phenocopy specific differentiated cell types (e.g.,
neurons, cardiomyocytes, and hepatocytes) affected by a particular
health condition. Also, the cell lines are not representative of
cell populations in a subject, since cell lines have been altered
to indefinitely replicate. Importantly, in many cases animal models
or genetically modified cell models of disease simply fail to
adequately recapitulate the cellular disease phenotypes as they
actually occur in a human patient's cells. Thus, typical
preclinical drug discovery strategies miss many genotype/phenotypes
that are present in the human population and will have a direct
impact on the therapeutic efficacy and toxicity of a candidate drug
compound. A practical consequence of these facts is that more often
than not lead compounds fail in human clinical trials despite
successful preclinical testing in animal models and transformed
cell line models, as mentioned above. Ideally, drug screening and
drug target discovery would be performed in biological models that
recapitulate the genetic and phenotypic diversity present in a
human patient population and the appropriate disease state at the
cellular level, well before the clinical trial stage. These drug
discovery paradigms are illustrated schematically in FIG. 1. In the
traditional drug discovery model (left), candidate therapeutic
agents are selected for clinical trials in patients based on their
action on specific drug targets and their efficacy/lack of toxicity
in animal models. In an alternative drug discovery model (right)
the disease-relevant cells derived from patient iPSC lines, as
described herein, are the starting point for identification of lead
compounds based on their ability to ameliorate a disease-relevant
cellular phenotype in patient derived cells.
[0020] Accordingly, the present disclosure describes human induced
pluripotent stem cell lines from selected individuals (e.g.,
patients), genetically diverse panels of such cell lines,
differentiated cells derived from such cell lines, and methods for
their use in disease modeling, drug discovery, diagnostics, and
individualized therapy.
II. Definitions
[0021] "Candidate drug compound," as used herein, refers to any
test compound to be assayed for its ability to affect a functional
endpoint. Some examples of such functional endpoints are ligand
binding to a receptor, receptor antagonism, receptor agonism,
protein-protein interactions, enzymatic activities, transcriptional
responses, etc.
[0022] "Correcting" a phenotype, as used herein, refers to altering
a phenotype such that it more closely approximates a normal
phenotype.
[0023] "iPSC donor," as used herein, refers to a subject, e.g., a
human patient from which one or more induced stem cell lines have
been generated. Generally, the genome of an iPSC line corresponds
to that of its iPSC donor.
[0024] "Phenomic analysis," as used herein, refers to the analysis
of phenotypes (e.g., resting calcium level, gene expression
profiles, apoptotic index, electrophysiological properties,
sensitivity to free radicals, compound uptake and extrusion, kinase
activity, second messenger pathway responses) exhibited by a
particular type of cell (e.g., cardiomyocytes).
[0025] "Phenome," as used herein refers to the set of phenotypes
that is subject and cell-type specific. For example, the phenome of
hepatocytes and cardiomyocytes from the same individual will be
quite distinct even though they share the same genome.
[0026] An "endogenous allele," as used herein, refers to a
naturally occurring allele that is native to the genome of a cell,
i.e., an allele that is not introduced by recombinant
methodologies.
[0027] An "iPSC-derived cell," as used herein, refers to a cell
that is generated from an iPSC either by proliferation of the iPSC
to generate more iPSCs, or by differentiation of the iPSC into a
different cell type. iPSC-derived cells include cells not
differentiated directly from an iPSC, but from an intermediary cell
type, e.g., a glial progenitor cell, a neural stem cell, or a
cardiac progenitor cell.
[0028] A "normal" phenotype, as used herein, refers to a phenotype
(e.g., apoptotic rate, resting calcium level, kinase activity, gene
expression level) that falls within a range of phenotypes found in
healthy individuals or that are not associated with (e.g.,
predictive of) a health condition.
III. Induced Stem Cell Lines for Drug Screening and Drug Target
Discovery
A. Overview
[0029] The present disclosure provides human induced pluripotent
stem cell (iPSC) lines, panels of stem cell lines, and methods for
their use in drug discovery, diagnostic, and therapeutic methods as
described in detail below. The induced pluripotent stem cell lines
disclosed herein are characterized by long term self renewal, a
normal karyotype, and the developmental potential to differentiate
into a wide variety of cell types (e.g., neurons, cardiomyocytes,
and hepatocytes). Induced pluripotent stem cell lines can be
differentiated into cell lineages of all three germ layers, i.e.,
ectoderm, mesoderm, and endoderm.
[0030] An important nexus exists between a subject (e.g., a
patient) and iPSC lines generated from that subject. First, all of
the genotypes of iPSC lines and those of the corresponding subject
are identical. Thus, genotype-phenotype correlations, uncovered in
one are informative for the other, and vice versa. Second,
differentiated cells (e.g., neurons) derived ex vivo from an iPSC
line will exhibit a complete set of cellular phenotypes (referred
to herein as a "phenome") that are very similar, if not identical,
to those of differentiated cells in vivo in the corresponding
subject. This point is particularly relevant for developing
therapeutics targeted to cells that cannot be routinely obtained
from patients (e.g., neurons, cardiomyocytes, hepatocytes, or
pancreatic cells). For example, in the case of a patient suffering
from a neurodegenerative disease (e.g., parkinson's disease),
dopaminergic neurons, which are typically affected by this
condition, can be obtained non-invasively by differentiating an
iPSC line from the subject, and can then be screened in multiple
assays. Thus, iPSC lines provide a renewable source of
differentiated cells (e.g., inaccessible differentiated cells) in
which pathological cellular phenotypes that are associated with a
disease, cell type, and individual may be examined and screened
against test compounds. An exemplary, non-limiting embodiment of
this approach to disease modeling and drug discovery is
schematically illustrated in FIG. 2. iPSC lines and iPSC-derived
cells (e.g., motor neurons) are also useful for predicting the
efficacy and/or adverse side effects of a candidate drug compound
in specific individuals or groups of individuals, as schematically
illustrated in FIG. 3. For example, test compounds can be tested
for toxicity in hepatocytes differentiated from a genetically
diverse panel of induced pluripotent stem cells. Toxicity testing
in iPSC-derived hepatocytes can reveal both the overall likelihood
of toxicity of a test compound in a target patient population, and
the likelihood of toxicity in specific patients within that
population.
[0031] In effect, iPSC lines and iPSC-derived cells (e.g.,
pancreatic cells) can serve as "cellular avatars," that reveal
cellular phenotypes that are disease, cell-type, and
subject-specific to the extent the phenotypes are determined or
predisposed by the genome. Collectively, panels of patient induced
stem cell lines will represent a wide range of genotype/phenotype
combinations in a patient population. Thus, they are useful for
developing therapeutics that are effective and safe across a wide
range of the relevant target population, or for determining which
individuals can be treated effectively and safely with a given
therapeutic agent.
B. Screening and Selection of Subject Samples
[0032] Some of the methods described herein utilize induced stem
cell lines or panels of induced stem cell lines derived from
subjects that meet one or more pre-determined criteria. In some
cases subjects and cellular samples from such subjects may be
selected for the generation of induced stem cell lines and panels
of induced stem cell lines based on one or more of such
pre-determined criteria. These include, but are not limited to, the
presence or absence of a health condition in a subject (e.g.,
spinal muscular atrophy, Parkinson's disease, or amyotrophic
lateral sclerosis), one or more positive diagnostic criteria for a
health condition, a family medical history indicating a
predisposition or recurrence of a health condition, the presence or
absence of a genotype associated with a health condition, or the
presence of at least one polymorphic allele that is not already
represented in a panel of induced stem cell lines.
[0033] In some cases, a panel of induced stem cell lines is
generated specifically from individuals diagnosed with a health
condition, and from subjects that are free of the health condition.
Such health conditions include, without limitation,
neurodegenerative disorders; neurological disorders such as
cognitive impairment, and mood disorders; auditory disease such as
deafness; osteoporosis; cardiovascular diseases; diabetes;
metabolic disorders; respiratory diseases; drug sensitivity
conditions; eye diseases such as macular degeneration;
immunological disorders; hematological diseases; kidney diseases;
proliferative disorders; genetic disorders, traumatic injury,
stroke, organ failure, or loss of limb.
[0034] Examples of neurodegenerative disorders include, but are not
limited to, Alexander's disease, Alper's disease, Alzheimer's
disease, amyotrophic lateral sclerosis, ataxia telangiectasia,
Batten disease, bovine spongiform encephalopathy, Canavan disease,
Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob
disease, Huntington's disease, HIV-associated dementia, Kennedy's
disease, Krabbe's disease, lewy body dementia, Machado-Joseph
disease, multiple sclerosis, multiple system atrophy, narcolepsy,
neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher
Disease, Pick's disease, primary lateral sclerosis, prion diseases,
Refsum's disease, Sandhoffs disease, Schilder's disease, subacute
combined degeneration of spinal cord secondary to pernicious
anaemia, schizophrenia, spinocerebellar ataxia, spinal muscular
atrophy, Steele-Richardson-Olszewski disease, and tabes
dorsalis.
[0035] Examples of neurological disorders include, stroke,
cognitive impairment, and mood disorders.
[0036] Examples of immunological disorders include but are not
limited to acquired immune deficiency, leukemia, lymphoma,
hypersensitivities (allergy), autoimmune diseases, and severe
combined immune deficiency.
[0037] Examples of autoimmune diseases include but are not limited
to acute disseminated encephalomyelitis, addison's disease,
ankylosing spondylitis, antiphospholipid antibody syndrome,
autoimmune hemolytic anemia, autoimmune hepatitis, bullous
pemphigoid, coeliac disease, dermatomyositis, diabetes mellitus
type 1, Goodpasture's syndrome, Graves' disease, Guillain-Barre
syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura,
lupus erythematosus, multiple sclerosis, myasthenia gravis,
pemphigus, pernicious anaemia, polymyositis, primary biliary
cirrhosis, rheumatoid arthritis, Sjogren's syndrome, temporal
arthritis (also known as "giant cell arthritis"), vasculitis,
Wegener's granulomatosis.
[0038] Examples of cardiovascular diseases include but are not
limited to aneurysm, angina, arrhythmia, atherosclerosis,
cardiomyopathy, cerebrovascular accident (stroke), cerebrovascular
disease, congenital heart disease, congestive heart failure,
myocarditis, valve disease coronary, artery disease dilated,
cardiomyopathy, diastolic dysfunction, endocarditis, high blood
pressure (hypertension), hypertrophic cardiomyopathy, mitral valve
prolapse, myocardial infarction (heart attack), and venous
thromboembolism.
[0039] Examples of metabolic disorders include but are not limited
to acid lipase disease, amyloidosis, Barth Syndrome, biotinidase
deficiency, carnitine palmitoyl transferase deficiency type II,
central pontine myelinolysis, metabolic diseases of muscle
including muscular dystrophy, Farber's Disease, glucose-6-phosphate
dehydrogenase deficiency, gangliosidoses, trimethylaminuria,
Lesch-Nyhan syndrome, lipid storage diseases, metabolic myopathies,
methylmalonic aciduria, mitochondrial myopathies,
mucopolysaccharidoses, mucolipidoses, mucolipidoses,
mucopolysaccharidoses, multiple CoA carboxylase deficiency,
nonketotic hyperglycinemia, Pompe disease, propionic acidemia, type
I glycogen storage disease, urea cycle disorders, hyperoxaluria,
and oxalosis.
[0040] Examples of proliferative disorders include but are not
limited to one or more of the following: carcinomas, sarcomas,
lymphomas, leukemias, germ cell tumors, blastic tumors, prostate
cancer, lung cancer, colorectal cancer, bladder cancer, cutaneous
melanoma, breast cancer, endometrial cancer, and ovarian
cancer.
[0041] Further examples of diseases or disorders may be found in
U.S. application Ser. Nos. 12/157,967, WSGR Docket Number
36588-704.201; filed on Jun. 13, 2008; First Inventor Kazuhiro
Sakurada, 61/061,594, WSGR Docket Number 36588-707.101; filed on
Jun. 13, 2008; First Inventor Kazuhiro Sakurada, and WSGR Docket
Number 36588-704.502, filed Jun. 12, 2009; First Inventor Kazuhiro
Sakurada, which are hereby incorporated by reference. It is also
anticipated that the methods of the present invention include
marketing and selling products and services for the treatment of
diseases and disorders including, but not limited to, those
mentioned herein.
[0042] Such subjects may be identified in, e.g., gene association
studies, clinical studies, and hospitals, preferably after a final
diagnosis of a health condition has been made. Preferably, subjects
are identified in gene association studies that include
non-affected control individuals.
[0043] In other cases, iPSC lines are generated from subjects
screened for the presence or absence of at least one allele
associated with a health condition or a predisposition for a health
condition. Such alleles indicate that an individual, though not
exhibiting overt symptoms of a health condition, has a high risk of
developing the health condition. For example, BRCA1 have been used
to indicate a high likelihood of developing breast cancer.
Genotyping of subjects may be performed on samples from a number of
sources, e.g., blood banks, sperm banks, gene-association studies,
hospitals, clinical trials, or any other source as long as a living
cellular sample can be obtained from the individual that is
genotyped. While not wishing to be bound by theory, it is believed
that one or more that cellular phenotypes from individuals carrying
alleles associated with health conditions will exhibit
abnormalities that can serve as more reliable prognostic indicators
of a health condition in combination with a genotype than a
genotype alone. Further, identification of specific abnormal
cellular phenotypes associated with a health condition may indicate
a target pathway for screening of prophylactic and therapeutic
agents for the health condition.
[0044] There is an ongoing effort to identify associations between
polymorphic alleles present in the human population, e.g., single
polymorphisms (SNPs) and the occurrence of common health
conditions, e.g., neurodegenerative diseases, psychiatric
disorders, metabolic disorders, and cardiovascular diseases.
Various types of polymorphic alleles can be found in the human
genome as summarized in Table 1.
TABLE-US-00001 TABLE 1 Types of Interindividual Variation in the
Human Genome Genetic Frequency in change/variation Abbreviation
Description human genome Single nucleotide SNP Typically two
different nucleotides (biallelic 12,000,000 polymorphism SNPs) at
one defined position, but more rarely also triallelic variants
occur Deletions/Insertions InDel Deletions (or insertions,
depending on the allele >1,000,000 frequencies) of between 1 to
1000 nucleotides. More frequent are deletions of one or three
basepairs Varying number of VNTR Microsatellites also termed short
tandem repeat >500,000 tandem recaps (STR) polymorphisms are
typically tandem repeats of two, three or four nucleotides, but
repeats up to ten nucleotides in length may also classified in this
group. Minisatellites are VNTR polymorphisms in which 10-100
nucleotides are repeated in variable numbers. Repeated segments
often do not have exactly identical sequences. VNTRs with larger
repeat units (100-1000 bp) are termed satellites. Copy number CNV
Inheritable deletion of multiplication of DNA >1500 loci
variation segments larger than 1 kb. Currently, about 1500 covering
12% of CNVs distributed through all chromosomes are the genome
known; estimated to cover 12% of the human genome length.
[0045] A number of studies have identified alleles associated with
a health condition or a predisposition towards a health
condition.
[0046] Examples of alleles associated with health conditions are
known in the art. See, e.g., the databases listed in Table 2.
TABLE-US-00002 TABLE 2 List of Publicly Available Databases
Containing Alleles Associated with a Health Condition or
Predisposition to a Health Condition Name of Website Website URL
Brief Description Alzgene www.alzforum.org/res/com/gen/alzgene
Collection of published genetic association studies performed on
Alzheimer Disease phenotypes, from database searches and journals'
contents lists. Case and control data presented. Cytokine Gene
www.nanea.dk/cytokinesnps/ Regularly updated database Polymorphism
with Medline-based records in Human from a systematic review of
Disease cytokine gene polymorphisms associated with human disease.
Data extracted from two publications about the study. HuGE
Navigator hugenavigator.net HuGE Navigator provides access to a
continuously updated knowledge base in human genome epidemiology,
including information on population prevalence of genetic variants,
gene-disease associations, gene-gene and gene-environment
interactions, and evaluation of genetic tests. GenAtlas
www.genatlas.org Regularly updated database of genes, phenotypes
and references. Among numerous databases are brief sections on
disorders associated with genes, with lists of citations. May be
biased towards statistically significant results. GeneCanvas
genecanvas.idf.inserm.fr Database of cardiovascular candidate genes
and their polymorphisms investigated at INSERM (Paris, France).
Data include gene frequencies and linkage disequilibrium
statistics. Genetic geneticassociationdb.nih.gov Database of human
genetic Association association studies of Database complex
diseases and disorders, based on Medline records. Data extracted
from publications. Human Obesity obesitygene.pbrc.edu Database of
obesity-related Gene Map genes, including P values Database for
association and references. Biased in favour of statistically
significant results. Infevers fmf.igh.cnrs.fr/infevers Database of
genetic associations in hereditary inflammatory disorders, with
voluntarily submitted entries. Submissions are validated by an
editorial board member. MedGene medgene.med.harvard.edu/MEDGENE/
Automated database of gene disease association studies in Medline.
OMIM www.ncbi.nlm.nih.gov/omim/ Database of human genes and genetic
disorders, containing textual information with links to Medline and
sequence records in the Entrez system, and links to additional
related resources at NCBI and elsewhere. PharmGKB www.pharmgkb.org
Database of genomic data and clinical information from participants
in pharmacogenetics research studies. Welcomes submission of
primary data. T1DBase t1dbase.org/ Database of type 1 diabetes
data, including information from collaborating laboratories. Some
indication given of unpublished data.
[0047] Some examples of health condition-associated alleles and
their corresponding studies are provided in Table 3.
TABLE-US-00003 TABLE 3 Some Examples of Alleles Associated with a
Health Condition Polymorphism(s) Disease identified References
Bipolar rs420259 The Wellcome Trust Case Control disorder
Consortium (2007), Nature, 447: 661-678 Coronary rs1333049 The
Wellcome Trust Case Control artery Consortium (2007), Nature,
disease 447: 661-678 Crohn's rs17221417 The Wellcome Trust Case
Control disease rs11209026 Consortium (2007), Nature, rs10210302
447: 661-678 rs9858542 rs17234657 rs1000113 rs10761659 rs10883365
rs17221417 rs2542151 Hypertension The Wellcome Trust Case Control
Consortium (2007), Nature, 447: 661-678 Rheumatoid rs6679677 The
Wellcome Trust Case Control arthritis rs6457617 Consortium (2007),
Nature, 447: 661-678 Type 1 rs11761231 The Wellcome Trust Case
Control Diabetes rs6679677 Consortium (2007), Nature, rs9272346
447: 661-678 rs11171739 rs17696736 rs12708716 Type 2 rs4506565 The
Wellcome Trust Case Control Diabetes rs9465871 Consortium (2007),
Nature, rs9939609 447: 661-678 Gallstone rs1187534 Bush, et al.,
(2007), Nat Genet, disease (D19H) 39: 995-999 Myocardial rs10757278
Helgadottir, et al., (2007), Science, Infarction 316: 1491-1493
Atrial rs2200733 Gudbjartsson, et al., (2007), Nature, fibrillation
448: 353-357 Type 2 rs1801282 Warren, et al., (2007) diabetes
rs13266634 Pharmacogenomics, 7: 180-189 rs1111875 rs7903146 rs5219
rs4402960 rs7754840 rs10811661 rs9300039 rs8050136 Type 2
rs13266634 Saxena, et al., (2007), Science, diabetes rs1111875 316:
1331-1336 rs7903146 rs5219 rs1801282 rs10811661 rs4402960 rs7754840
Rheumatoid rs3761847 Plenge, et al., (2007), N Engl J Med,
arthritis 357: 1199-209 Exfoliation rs1048661 + Thorleifsson, et
al., (2007), Science, Glaucoma rs3825942 317: 1397-1400 Breast
rs2981582 Easton, et al., (2007), Nature, Cancer rs12443620 447:
1087-1093 rs8051542 rs889312 rs3817198 rs2107425 rs13281615
Colorectal rs6983267 Tomlinson, et al., (2007), Nat Genet, cancer
39: 984-988
[0048] The sequence and other information for any rs-identified SNP
can be accessed on the world wide web through the SNP database of
the National Center for Biotechnology Information
(www.ncbi.nlm.nih.gov/); pulldown menu="SNP."
[0049] Subjects may be screened for alleles in genes that affect
response to a therapeutic agent or to a class of therapeutic
agents. Examples of such alleles include, but are not limited to,
alleles of drug metabolizing enzymes, such as Glucose 6 phosphate
dehydrogenase (G6PDH), Butyrlcholine esterase, N-acetyltransferase,
Cytochrome P450 isoforms (e.g., 2B6, 2D6, C19, 2C9), Thiopurine
S-methyltransferase, Dihydropyrimidine dehydrogenase, and Uridin
diphospho-glucuronic acid transferase type 1A1. Alleles of
Cytochrome P450 enzyme isoforms can be found, e.g., in a database
provided under the "Home Page of the Human Cytochrome P450 (CYP)
Allele Nomenclature Committee," at "www.cypalleles.ki.se/." Some
alleles occur in genes that affect drug transport, including, e.g.,
multiple drug resistance conferring transporters (MDRs), breast
cancer resistance protein (BRCP), multidrug
resistance-associated-associated proteins (MRPs), and organic
anion-transporting polypeptide (OATP1B1). Other alleles occur in
genes that encode drug targets, including, but not limited to,
Vitamin K epoxide reductase, Factor V, G-protein coupled receptors
(GPCRs). Of note, GPCRs are one of the most common drug targets.
Examples of polymorphic alleles in GPCRs can be found in, e.g., the
GPCR Natural Variants ("NaVa") Database, which is accessible on the
internet at "nava.liacs.nl/" The GPCR NaVa database describes
sequence variants within the family of human G Protein-Coupled
Receptors (GPCRs). It includes: rare mutations (frequency <1%);
polymorphisms (frequency >1%), including Single Nucleotide
Polymorphisms (SNPs); variants without estimates of allele
frequency.
[0050] Polymorphic alleles of interest may be detected and scored
in a nucleic acid sample from a subject by any of a number of
methods known in the art. For example, detection of multiple
alleles may be performed by conducting a nucleic acid array-based
assay on a nucleic acid sample from a subject, where the nucleic
acid array comprises allele-specific probes (e.g., SNP-specific
probes), which, under high stringency hybridization conditions,
selectively hybridize with and discriminate between the nucleic
acid sequences of two or more polymorphic alleles of interest,
e.g., alleles of G-protein coupled receptors.
[0051] The nucleic acid arrays used to detect polymorphisms may be
commercially available nucleic acid arrays. For example, the
Affymetrix.RTM. Genome-Wide SNP Array 6.0 includes probes for more
than 906,000 SNPs and more than 946,000 probes for the detection of
copy number variation. Alternatively, the nucleic acid arrays may
be custom-made to include to a limited subset of alleles of
interest. The design of suitable probe arrays for analysis of
predetermined polymorphisms and interpretation of the hybridization
patterns is described in detail in WO 95/11995; EP 717,113; and WO
97/29212. Such arrays typically contain first and second groups of
probes which are designed to be complementary to different allelic
forms of the polymorphism. Each group contains a first set of
probes, which is subdivided into subsets, one subset for each
polymorphism. Each subset contains probes that span a polymorphism
and proximate bases and are complementary to one allelic form of
the polymorphism. Thus, within the first and second probe groups
there are corresponding subsets of probes for each polymorphism.
The hybridization patterns of these probes to target samples can be
analyzed by footprinting or cluster analysis, as described above.
For example, if the first and second probes groups contain subsets
of probes respectively complementarity to first and second allelic
forms of a polymorphic site spanned by the probes, then on
hybridization of the array to a sample that is homozygous for the
first allelic form all probes in the subset from the first group
show specific hybridization, whereas probes in the subset from the
second group that span the polymorphism show only mismatch
hybridization. The mismatch hybridization is manifested as a
footprint of probe intensities in a plot of normalized probe
intensity (i.e., target/reference intensity ratio) for the subset
of probes in the second group. Conversely, if the target sample is
homozygous for the second allelic form, a footprint is observed in
the normalized hybridization intensities of probes in the subset
from the first probe group. If the target sample is heterozygous
for both allelic forms then a footprint is seen in normalized probe
intensities from subsets in both probe groups although the
depression of intensity ratio within the footprint is less marked
than in footprints observed with homozygous alleles. Analysis of
the hybridization pattern of a nucleic acid array to a nucleic acid
sample indicates which allelic form is present at some or all of
the SNP sequences represented on the array. Thus, an individual or
an iPSC line generated from an individual can be characterized with
a polymorphic profile representing allelic variants of interest,
e.g., alleles associated with a health condition.
[0052] In other embodiments, an allele is detected using a primer
extension reaction or amplification reaction. For example, a
nucleic acid sample containing (or suspected of containing) a
target nucleic acid molecule can be contacted with an
oligonucleotide primer that, upon further contact with a
polymerase, can be extended up to and, if desired, beyond the
position of the SNP. In addition, the nucleic acid sample can be
contacted with an amplification primer pair, comprising a first
primer and a second primer, which selectively hybridize to
complementary strands of a target nucleic acid molecule and, in the
presence of polymerase, allow for generation of an amplification
product. For convenience, the primers of an amplification primer
pair are referred to as a "first primer" and a "second primer";
however, reference herein to a "first primer" or a "second primer"
is not intended to indicate any importance, order of addition, or
the like. It will be further recognized that an amplification
primer pair requires that the first and second primer comprise what
are commonly referred to as a forward primer and a reverse
primer.
[0053] A primer extension or PCR amplification reaction can be
designed such that the presence of a particular nucleotide at an
SNP position can be determined by the presence or size of the
extension and/or amplification product, in which case the SNP can
be determined using a method such as gel electrophoresis, capillary
gel electrophoresis, or mass spectrometry; or the amplification
product can be sequenced to determine the nucleotide at the SNP
position. In addition, the SNP can be detected indirectly, for
example, by further contacting the sample with a detector
oligonucleotide, which can selectively hybridize to a nucleotide
sequence of the first amplification product comprising the SNP
position; and detecting selective hybridization of the detector
oligonucleotide, as above.
[0054] Various other methods useful for genotyping are known to the
art and can be applied to the present methods. For example, PCR can
be performed using TaqMan.RTM. reagents, followed by reading the
plates at this endpoint. Molecular beacons, Amplifluor.RTM. or
TriStar.RTM. reagents and methods similarly can be used
(Stratagene; Intergen). Amplification products also can be detected
using an ELISA format, for example, using a design in which one
primer is biotinylated and the other contains digoxygenin. The
amplification products are then bound to a streptavidin plate,
washed, reacted with an enzyme-conjugated antibody to digoxygenin,
and developed with a chromogenic, fluorogenic, or chemiluminescent
substrate for the enzyme. Alternatively, a radioactive method can
be used to detect generated amplification products, for example, by
including a radiolabeled deoxynucleoside triphosphate into the
amplification reaction, then blotting the amplification products
onto DEAE paper for detection. In addition, if one primer is
biotinylated, then streptavidin-coated scintillation proximity
assay plates can be used to measure the PCR products. Additional
methods of detection can use a chemiluminescent label, for example,
a lanthanide chelate such as used in the DELFIA.RTM. assay (Pall
Corp.), an electrochemiluminescent label such as ruthenium
tris-bipyridy (ORI-GEN), or a fluorescent label, for example, using
fluorescence correlation spectroscopy.
[0055] An assay system that is commercially available and can be
used to identify a nucleotide occurrence of one or more SNPs is the
SNP-IT.RTM. assay system (Orchid BioSciences, Inc.; Princeton
N.J.). In general, the SNP-IT.RTM. method is a three step primer
extension reaction. In the first step a target nucleic acid
molecule is isolated from a sample by hybridization to a capture
primer, which provides a first level of specificity. In a second
step the capture primer is extended from a terminating nucleotide
triphosphate at the target SNP site, which provides a second level
of specificity. In a third step, the extended nucleotide
triphosphate can be detected using a variety of known formats,
including, for example, by direct fluorescence, indirect
fluorescence, an indirect colorimetric assay, mass spectrometry, or
fluorescence polarization. Reactions conveniently can be processed
in 384 well format in an automated format using a SNP stream.RTM.
instrument (Orchid BioSciences, Inc.).
[0056] Various methods for genotyping SNP alleles, selected as
described herein, are readily adaptable to high throughput assays.
For example, an amplification reaction such as PCR can be performed
using inexpensive robotic thermocyclers for a specified number of
cycles, then the amplification product generated can be determined
at the endpoint of the reaction. Furthermore, the methods can be
performed in a multiplex format, for example, using differentially
labeled oligonucleotide probes, or performing oligonucleotide
ligation assays that result in different sized ligation products,
or amplification reactions that result in different sized
amplification products. In another example, high-throughput mass
spectrometry is used to detect SNP alleles in a target nucleic acid
sample. Mass spectrometric methods for SNP genotyping are described
in, e.g., U.S. Pat. Nos. 7,132,519, 6,994,998; and U.S. Patent
Application No 20060275789.
[0057] Where hybridization-based methods are used, high stringency
conditions are those that result in perfect matches remaining in
hybridization complexes, while imperfect matches melt off.
Similarly, low stringency conditions are those that allow the
formation of hybridization complexes with both perfect and
imperfect matches. High stringency conditions are known in the art;
see for example Maniatis et al. (1989), Molecular Cloning: A
Laboratory Manual, 2d Edition; and Short Protocols in Molecular
Biology, ed. Ausubel, et al. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen (1993), Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes,
"Overview of principles of hybridization and the strategy of
nucleic acid assays." Generally, stringent conditions are selected
to be about 5-10 C lower than the thermal melting point (Tm) for
the specific sequence at a defined ionic strength pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions will be those in
which the salt concentration is less than about 1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
3.degree. C. for short probes (e.g. 10 to 50 nucleotides) and at
least about 6.degree. C. for long probes (e.g. greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. In another
embodiment, less stringent hybridization conditions are used; for
example, moderate or low stringency conditions may be used, as are
known in the art. See, e.g., Maniatis and Ausubel, supra, and
Tijssen, supra.
C. Methods for Inducing Pluripotent Stem Cell Lines
[0058] iPSC lines may be induced from a wide variety of mammalian
cells, e.g., human somatic cells, such as fibroblasts, bone
marrow-derived mononuclear cells, skeletal muscle cells, adipose
cells, peripheral blood mononuclear cells, macrophages,
hepatocytes, keratinocytes, oral keratinocytes, hair follicle
dermal cells, gastric epithelial cells, lung epithelial cells,
synovial cells, kidney cells, skin epithelial cells or osteoblasts.
Methods for inducing multipotent and pluripotent stem cell lines
are further disclosed in U.S. application Ser. Nos. 12/157,967,
WSGR docket number 36588-704.201; filed Jun. 13, 2008; first
inventor Kazuhiro Sakurada, 61/061,594, WSGR Docket Number
36588-707.101; filed on Jun. 13, 2008; First Inventor Kazuhiro
Sakurada, and 61/061,565, WSGR Docket Number 36588-702.101; filed
on Jun. 13, 2008; First Inventor Kazuhiro Sakurada, which are
hereby incorporated by reference in their entirety.
[0059] The cells to be induced can originate from many different
types of tissue, e.g., bone marrow, skin (e.g., dermis, epidermis),
muscle, adipose tissue, peripheral blood, foreskin, skeletal
muscle, or smooth muscle. The cells can also be derived from
neonatal tissue, including, but not limited to: umbilical cord
tissues (e.g., the umbilical cord, cord blood, cord blood vessels),
the amnion, the placenta, or other various neonatal tissues (e.g.,
bone marrow fluid, muscle, adipose tissue, peripheral blood, skin,
skeletal muscle etc.).
[0060] The cells can be derived from neonatal or post-natal tissue
collected from a mammal within the period from birth, including
cesarean birth, to death. For example, the tissue may be from a
mammal who is >10 minutes old, >1 hour old, >1 day old,
>1 month old, >2 months old, >6 months old, >1 year
old, >2 years old, >5 years old, >10 years old, >15
years old, >18 years old, >25 years old, >35 years old,
>45 years old, >55 years old, >65 years old, <80 years
old, <70 years old, <60 years old, <50 years old, <40
years old, <30 years old, <20 years old or <10 years old.
In some examples, the tissue is from a human age 18, 20, 21, 23,
24, 25, 28, 29, 31, 33, 34, 35, 37, 38, 40, 41, 42, 43, 44, 47, 51,
55, 61, 63, 65, 70, 77, or 85 years old.
[0061] The cells may be from non-embryonic tissue, e.g., at a stage
of development later than the embryonic stage. In some cases, the
cells may be derived from a fetus. In some cases, the cells are not
from a fetus. In some cases, the cells are from an embryo. In some
cases, the cells are not from an embryo.
[0062] The cells can be obtained from a single cell or a population
of cells. The population may be homogenous or heterogeneous. The
cells may be a population of cells found in a human cellular
sample, e.g., a biopsy or blood sample. In some cases, the cells
are a cell line. In some cases, the cells are somatic cells. In
some cases, the cells are derived from cells fused to other cells.
In some cases, the cells are not derived from cells fused to other
cells. In some cases, the cells are not derived from cells
artificially fused to other cells. In some cases, the cells are
not: a cell that has been fused with an embryonic stem cell, or a
cell that has undergone the procedure known as somatic cell nuclear
transfer.
[0063] The cellular population may include both differentiated and
undifferentiated cells. In some cases, the population primarily
contains differentiated cells. In other cases, the population
primarily contains undifferentiated cells, e.g., undifferentiated
stem cells. The undifferentiated cells within the population may be
induced to become pluripotent or multipotent. In some cases,
differentiated cells within the cellular population are induced to
become pluripotent or multipotent.
[0064] The cellular population may include undifferentiated cells
such as mesenchymal stem cells (MSCs), see, e.g., Pittenger et al.
(1999), Science 284 (5411): 143-7, multipotent adult progenitor
cells (MAPCs), see, e.g., Jahagirdar et al. (2005), Stem Cell Rev.
1(1): 53-9, and/or marrow-isolated adult multilineage inducible
(MIAMI) cells (D'Ippolioto et al., (2004), J. Cell Sci. 117 (Pt
14): 2971-81. MSCs are multipotent cells that arise from the
mesenchyme during development. In some cases, the undifferentiated
stem cells (e.g., mesenchymal stem cells, MAPCs and MIAMI cells)
are stem cells that have not undergone epigenetic inactivating
modification by heterochromatin formation due to DNA methylation or
histone modification of at least four genes, at least three genes,
at least two genes, at least one gene, or none of the following:
Nanog, Oct3/4, Sox2 and Tert. Activation, or expression of such
genes, e.g., Tert, Nanog, Oct3/4 or Sox2, may occur when human
pluripotent stem cells are induced from undifferentiated stem cells
present in a human postnatal tissue.
[0065] Methods for obtaining human somatic cells are well
established, as described in, e.g., Schantz and Ng (2004), A Manual
for Primary Human Cell Culture, World Scientific Publishing Co.,
Pte, Ltd. In some cases, the methods include obtaining a cellular
sample, e.g., by a biopsy, blood draw, or alveolar or other
pulmonary lavage. Other suitable methods for obtaining various
types of human somatic cells include, but are not limited to, the
following exemplary methods:
Bone Marrow
[0066] The donor is given a general anesthetic and placed in a
prone position. From the posterior border of the ilium, a
collection needle is inserted directly into the skin and through
the iliac surface to the bone marrow, and liquid from the bone
marrow is aspirated into a syringe. A mononuclear cell fraction is
then prepared from the aspirate by density gradient centrifugation.
The collected crude mononuclear cell fraction is then cultured
prior to use in the methods described herein for induction
pluripotency. For convenience, methods for induction of
pluripotency, as described herein, are collectively referred to as
"induction."
Postnatal Skin
[0067] Skin tissue containing the dermis is harvested, for example,
from the back of a knee or buttock. The skin tissue is then
incubated for 30 minutes at 37.degree. C. in 0.6% trypsin/DMEM
(Dulbecco's Modified Eagle's Medium)/F-12 with 1%
antibiotics/antimycotics, with the inner side of the skin facing
downward.
[0068] After the skin tissue is turned over to scrub slightly the
inner side with tweezers, the skin tissue is finely cut into 1 mm2
sections using scissors, which are then centrifuged at 1200 rpm and
room temperature for 10 minutes. The supernatant is removed, and to
the tissue precipitate is added 25 ml of 0.1% trypsin/DMEM/F-12/1%
antibiotics, antimycotics, and stirred using a stirrer at
37.degree. C. and 200-300 rpm for 40 minutes. After confirming that
the tissue precipitate is fully digested, 3 ml fetal bovine serum
(FBS) (manufactured by JRH) is added, and filtered sequentially
with gauze (Type I manufactured by PIP), a 100 .mu.m nylon filter
(manufactured by FALCON) and a 40 .mu.m nylon filter (manufactured
by FALCON). After centrifuging the resulting filtrate at 1200 rpm
and room temperature for 10 minutes to remove the supernatant,
DMEM/F-12/1% antibiotics, antimycotics is added to wash the
precipitate, and then centrifuged at 1200 rpm and room temperature
for 10 minutes. The cell fraction thus obtained is then cultured
prior to induction.
Postnatal Skeletal Muscle
[0069] After the epidermis of a connective tissue containing muscle
such as the lateral head of the biceps brachii muscle or the
sartorius muscle of the leg is cut and the muscle tissue is
excised, it is sutured. The whole muscle obtained is minced with
scissors or a scalpel, and then suspended in DMEM (high glucose)
containing 0.06% collagenase type IA and 10% FBS, and incubated at
37.degree. C. for 2 hours.
[0070] By centrifugation, cells are collected from the minced
muscle, and suspended in DMEM (high glucose) containing 10% FBS.
After passing the suspension through a microfilter with a pore size
of 40 .mu.m and then a microfilter with a pore size of 20 .mu.m,
the cell fraction obtained may be cultured according to the method
described in 6. below as crude purified cells containing
undifferentiated stem cells, and used for the induction of human
pluripotent stem cells of the present invention.
Postnatal Adipose Tissue
[0071] Cells derived from adipose tissue for use in the present
invention may be isolated by various methods known to a person
skilled in the art. For example, such a method is described in U.S.
Pat. No. 6,153,432, which is incorporated herein in its entirety. A
preferred source of adipose tissue is omental adipose tissue. In
humans, adipose cells are typically isolated by fat aspiration.
[0072] In one method of isolating cells derived from adipose cells,
adipose tissue is treated with 0.01% to 0.5%, preferably 0.04% to
0.2%, and most preferably about 0.1% collagenase, 0.01% to 0.5%,
preferably 0.04%, and most preferably about 0.2% trypsin and/or 0.5
ng/ml to 10 ng/ml dispase, or an effective amount of hyaluronidase
or DNase (DNA digesting enzyme), and about 0.01 to about 2.0 mM,
preferably about 0.1 to about 1.0 mM, most preferably 0.53 mM
concentration of ethylenediaminetetraacetic acid (EDTA) at 25 to
50.degree. C., preferably 33 to 40.degree. C., and most preferably
37.degree. C. for 10 minutes to 3 hours, preferably 30 minutes to 1
hour, and most preferably 45 minutes.
[0073] Cells are passed through nylon or a cheese cloth mesh filter
of 20 microns to 800 microns, more preferably 40 microns to 400
microns, and most preferably 70 microns. Then the cells in the
culture medium are subjected to differential centrifugation
directly or using Ficoll or Percoll or another particle gradient.
The cells are centrifuged at 100 to 3000.times.g, more preferably
200 to 1500.times.g, most preferably 500.times.g for 1 minute to 1
hours, more preferably 2 to 15 minutes and most preferably 5
minutes, at 4 to 50.degree. C., preferably 20 to 40.degree. C. and
more preferably about 25.degree. C.
[0074] The adipose tissue-derived cell fraction thus obtained may
be cultured according to the method described herein as crude
purified cells containing undifferentiated stem cells, and used for
the induction of human pluripotent or multipotent stem cells.
Blood
[0075] About 50 ml to about 500 ml vein blood or cord blood is
collected, and a mononuclear cell fraction is obtained by the
Ficoll-Hypaque method, as described in, e.g., Kanof et al. (1993),
Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D.
H. Margulies, E. M. Shevack, and W. Strober, eds.), ch.
7.1.1.-7.1.5, John Wiley & Sons, New York).
[0076] After isolation of the mononuclear cell fraction,
approximately 1.times.10.sup.7 to 1.times.10.sup.8 human peripheral
blood mononuclear cells are suspended in a RPMI 1640 medium
containing 10% fetal bovine serum, 100 .mu.g/ml streptomycin and
100 units/ml penicillin, and after washing twice, the cells are
recovered. The recovered cells are resuspended in RPMI 1640 medium
and then plated in a 100 mm plastic petri dish at a density of
about 1.times.10.sup.7 cells/dish, and incubated in a 37.degree. C.
incubator at 8% CO.sub.2. After 10 minutes, cells remaining in
suspension are removed and adherent cells are harvested by
pipetting. The resulting adherent mononuclear cell fraction is then
cultured prior to the induction period as described herein. In some
cases, the peripheral blood-derived or cord blood-derived adherent
cell fraction thus obtained may be cultured according to the method
described herein as crude purified cells containing
undifferentiated stem cells, and used for the induction of human
pluripotent stem cells of the present invention.
Induction
[0077] During the induction process, forced expression of certain
polypeptides is carried out in cultured cells for a period of time,
after which the iPSCs are screened for a number of morphological
and gene expression properties that characterize multipotent and
pluripotent stem cells. Induced cells that meet these screening
criteria may then be subcloned and expanded. In some cases, the
cells to be induced may be cultured for a period of time prior to
the induction procedure. Alternatively, the cells to be induced may
be used directly in the induction process without a prior culture
period. In some embodiments, the type of cell culture medium used
is the same or very similar before, during, and after the induction
process. In other cases, different cell culture media are used at
different points. For example, one type of culture medium may be
used directly before the induction process, while a second type of
media is used during the induction process. At times, a third type
of culture medium is used during the induction process.
[0078] Cells may be cultured in medium supplemented with a
particular serum. In some embodiments, the serum is fetal bovine
serum (FBS). The serum can also be fetal calf serum (FCS). In some
cases, the serum may be Human AB serum. Mixtures of serum may also
be used, e.g. mixture of FBS and Human AB, FBS and FCS, or FCS and
Human AB.
[0079] Culture of cells may be carried out under a low serum
culture conditions prior to, during, or following induction. A "low
serum culture condition" refers to the use of a cell culture medium
containing a concentration of serum ranging from 0% (v/v) (i.e.,
serum-free) to about 5% (v/v), e.g., 0% to 2%, 0% to 2.5%, 0% to
3%, 0% to 4%, 0% to 5%, 0.1% to 2%, 0.1% to 5%, 0.1%, 0.5%, 1%,
1.2%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4%. In some embodiments, the
serum concentration is from about 0% to about 2%. In some cases,
the serum concentration is about 2%. In some cases, the serum
concentration is preferably 2% or less. In other embodiments, cells
are cultured under a "high serum condition," i.e., greater than 5%
serum to about 20% serum, e.g., 6%, 7%, 8%, 10%, 12%, 15%, or 20%.
Culturing under high serum conditions may occur prior to, during,
and/or after induction.
[0080] Some representative media that the cells can be cultured in
include: MAPC, FBM, ES, MEF-conditioned ES (MC-ES), and mTeSR.TM.
(available, e.g., from StemCell Technologies, Vancouver, Canada),
See Ludwig et al (2006), Nat Biotechnol, 24(2):185-187. In other
cases, alternative culture conditions for growth of human ES cells
are used, as described in, e.g., Skottman et al (2006),
Reproduction, 132(5):691-698. In some embodiments, the cells are
cultured in MAPC, FBM, MC-ES, or mTeSR.TM. prior to and/or during
the introduction of induction factors to the cells; and the cells
are cultured in MC-ES or mTeSR.TM. medium later in the induction
process.
[0081] MAPC (2% FBS) Medium may comprise: 60% Dulbecco's Modified
Eagle's Medium-low glucose, 40% MCDB 201, Insulin Transferrin
Selenium supplement, (0.01 mg/ml insulin; 0.0055 mg/ml transferrin;
0.005 .mu.g/ml sodium selenite), 1.times. linolenic acid albumin (1
mg/mL albumin; 2 moles linoneic acid/mole albumin), 1 nM
dexamethasone, 2% fetal bovine serum, 1 nM dexamethasone, 10.sup.-4
M ascorbic acid, and 10 .mu.g/ml gentamycin.
[0082] FBM (2% FBS) Medium may comprise: MCDB202 modified medium,
2% fetal bovine serum, 5 .mu.g/ml insulin, 50 mg/ml gentamycin, and
50 ng/ml amphotericin-B.
[0083] ES Medium may comprise: 40% Dulbecco's Modified Eagle's
Medium (DMEM) 40% F12 medium, 2 mM L-glutamine, 1.times.
non-essential amino acids (Sigma, Inc., St. Louis, Mo.), 20%
Knockout Serum Replacement.TM. (Invitrogen, Inc., Carlsbad,
Calif.), and 10 .mu.g/ml gentamycin.
[0084] MC-ES medium may be prepared as follows. ES medium is
conditioned on mitomycin C-treated murine embryonic fibroblasts
(MEFs), harvested, filtered through a 0.45-.mu.M filter, and
supplemented with about 0.1 mM .beta. mercaptoethanol, about 10
ng/ml bFGF or FGF-2, and, optionally, about 10 ng/ml activin A. In
some cases, irradiated MEFs are used in place of the mitomycin
C-treated MEFs.
[0085] When either low or high serum conditions are used for
culturing the cells, one or more growth factors such as fibroblast
growth factor (FGF)-2; basic FGF (bFGF); platelet-derived growth
factor (PDGF), epidermal growth factor (EGF); insulin-like growth
factor (IGF); or insulin can be included in the culture medium.
Other growth factors that can be used to supplement cell culture
media include, but are not limited to one or more: Transforming
Growth Factor .beta.-1 (TGF .beta.-1), Activin A, Noggin,
Brain-derived Neurotrophic Factor (BDNF), Nerve Growth Factor
(NGF), Neurotrophin (NT)-1, NT-2, or NT 3. In some cases, one or
more of such factors is used in place of the bFGF or FGF-2 in the
MC-ES medium or other cell culture medium.
[0086] In some cases, the concentration of growth factors in the
culture media described herein (e.g., MAPC, FBM, MC-ES, mTeSR.TM.)
is from about 2 ng/ml to about 20 ng/ml, e.g., about 2 ng/ml, 3
ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 10 ng/ml, 12
ng/ml, 14 ng/ml, 15 ng/ml, 17 ng/ml, or 20 ng/ml. In some
embodiments, the concentration of bFGF or FGF2 is from about 2
ng/ml to about 5 ng/ml; from about 5 ng/ml to about 8 ng/ml; from
about 9 ng/ml to about 11 ng/ml; from about 11 ng/ml to about 15
ng/ml; or from about 15 ng/ml to about 20 ng/ml.
[0087] The growth factors may be used alone or in combination. For
example, FGF-2 may be added alone to the medium; in another
example, both PDGF and EGF are added to the culture medium.
[0088] In some examples, following initiation of the forced
expression of genes or polypeptides (e.g., immediately after a
retroviral infection period) in cells, the "iPSCs" are maintained
in MC-ES medium as described herein.
[0089] In some embodiments, cells are maintained in the presence of
a rho, or rho-associated, protein kinase (ROCK) inhibitor to reduce
apoptosis. In some cases, an inhibitor of Rho associated kinase is
added to the culture medium. For example, the addition of Y-27632
(Calbiochem; water soluble) or Fasudil (HA1077: Calbiochem), an
inhibitor of Rho associated kinase (Rho associated coiled
coil-containing protein kinase) may be used to culture the human
pluripotent stem cells of the present invention. In some cases the
concentration of Y-27632 or Fasudil, is from about 5 .mu.M to about
20 e.g., about 5 .mu.M, 10 .mu.M, 15 .mu.M, or 20 .mu.M.
[0090] The cells may be cultured for about 1 to about 12 days e.g.,
2 days, 3 days, 4.5 days, 5 days, 6.5 days, 7 days, 8 days, 9 days,
10 days, or any other number of days from about 1 day to about 12
days prior to undergoing the induction methods described
herein.
[0091] In some cases, the iPSCs are cultured in complete ES medium
in a 37.degree. C., 5% CO.sub.2 incubator, with medium changes
about every 1 to 2 days. In some embodiments, induced the iPSCs are
cultured and observed for about 14 days to about 40 days, e.g., 15,
16, 17, 18, 19, 20, 23, 24, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37,
38 days, or any other period from about 14 days to about 40 days
prior to identifying and selecting clones comprising "iPSCs" based
on morphological characteristics. Morphological characteristics for
identifying iPSC clones include, but are not limited to, a small
cell size with a high nucleus-to-cytoplasm ratio; formation of
small monolayer colonies within the space between parental cells
(e.g., between fibroblasts).
[0092] The cells may be plated at a cell density of about
1.times.10.sup.3 cells/cm.sup.2 to about 1.times.10.sup.4
cells/cm.sup.2, e.g., 2.times.10.sup.3 cells/cm.sup.2,
3.5.times.10.sup.3 cells/cm.sup.2, 6.times.10.sup.3 cells/cm.sup.2,
7.times.10.sup.3 cells/cm.sup.2, 9.times.10.sup.3 cells/cm.sup.2,
or any other cell density from about 1.times.10.sup.3
cells/cm.sup.2 to about 1.times.10.sup.4 cells/cm.sup.2.
[0093] The cells can be plated and cultured directly on tissue
culture-grade plastic. Alternatively, cells are plated and cultured
on a coated substrate, e.g., a substrate coated with fibronectin,
gelatin, matrigel.TM., collagen, or laminin. Suitable cell culture
vessels include, e.g., 35 mm, 60 mm, 100 mm, and 150 mm cell
culture dishes, 6-well cell culture plates, and other
size-equivalent cell culture vessels. In some cases, the cells are
cultured with feeder cells. For example, the cells may be cultured
on a layer, or carpet, of MEFs.
[0094] Media with low concentrations of serum may be particularly
useful to enrich for undifferentiated stem cells. The
undifferentiated cells cultured under low serum conditions may or
may not share certain properties with MSCs, MAPCs, and/or MIAMI
cells. Differences in phenotype may be due, in part, to culture
methods used to obtain MSCs, MAPCs and MIAMI cells. For example,
MSCs are often obtained by isolating the non-hematopoeitic cells
(e.g., interstitial cells) adhering to a plastic culture dish when
tissue, e.g., bone marrow, fat, muscle, or skin etc., is cultured
in a culture medium containing a high-concentration serum (5% or
more). However, even under these culture conditions, a very small
number of undifferentiated cells can be maintained, especially if
the cells were passaged under certain culture conditions (e.g., low
passage number or low-density culturing).
[0095] In some embodiments, in order to culture and grow human
pluripotent stem cells induced from the undifferentiated stem cells
of the present invention present in a human postnatal tissue, it is
preferred that the cells are subcultured every 5 to 7 days in a
culture medium containing the additives described herein on a
MEF-covered plastic culture dish or a matrigel-coated plastic
culture dish. In some cases, the cells may be cultured at a low
density, which may be accomplished by splitting the cells from
about 1:6 to 1:3 or by plating the cells at 10.sup.3 cells/cm.sup.2
to 3.times.10.sup.4 cells/cm.sup.2.
[0096] Primary culture ordinarily occurs immediately after the
cells are isolated from a donor, e.g., human. The primary cells can
be subjected to a second subculture, a third subculture, a fourth
subculture, and greater than four subcultures. A "second"
subculture describes primary culture cells subcultured once, a
"third" subculture describes primary cultures subcultured twice, a
"fourth" subculture describes primary cells subcultured three
times, etc. The culture techniques described herein may generally
include culturing from the period between the primary culture and
the fourth subculture, but other culture periods may also be
employed. Preferably, cells are cultured from primary culture to
second subculture.
[0097] Inducing a cell to become pluripotent can be accomplished in
numerous ways. In some embodiments, the methods for induction of
pluripotency in one or more cells include forcing expression of a
set of induction factors (Ws). In some cases, the set of IFs
includes one or more: an Oct3/4 polypeptide, a Sox2 polypeptide, a
Klf4 polypeptide, or a c-Myc polypeptide. In some cases, the set
does not include a c-Myc polypeptide. For example, the set of IFs
can include: an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4
polypeptide, but not a c-Myc polypeptide. In some cases, the set of
IFs does not include polypeptides that might increase the risk of
cell transformation.
[0098] In some cases, the set may include a c-Myc polypeptide. In
certain cases, the c-Myc polypeptide is a constitutively active
variant of c-Myc. In some instances, the set includes a c-Myc
polypeptide capable of inducible activity, e.g., a c-Myc-ER
polypeptide, see, e.g., Littlewood, et al. (1995) Nucleic Acid Res.
23(10):1686-90.
[0099] In other cases, the set of IFs may include: an Oct3/4
polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, but not a
TERT polypeptide, a SV40 Large T antigen polypeptide, HPV16 E6
polypeptide, a HPV16 E7 polypeptide, or a Bmi1 polypeptide. In some
cases, the set of IFs does not include a TERT polypeptide. In some
cases, the set of IFs does not include a SV40 Large T antigen. In
other cases, the set of IFS does not include a HPV16 E6 polypeptide
or a HPV16 E7 polypeptide.
[0100] In some cases, the set of Ws includes three Ws, wherein two
of the three IFs are an Oct3/4 polypeptide and a Sox2 polypeptide.
In other cases, the set of IFs includes two Ws, wherein the two
polypeptides are a c-Myc polypeptide and a Sox2 polypeptide In some
cases, the set of induction factors is limited to Oct 3/4, Sox2,
and Klf4 polypeptides. In other cases, the set of induction factors
may be limited to a set of four Ws: an Oct3/4 polypeptide, a Sox2
polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide.
[0101] A set of Ws may include Ws in addition to an Oct 3/4, a
Sox2, and a Klf4 polypeptide. Such additional Ws include, but are
not limited to Nanog, TERT, LIN28, CYP26A1, GDF3, FoxD3, Zfp42,
Dnmt3b, Ecat1, and Tel1 polypeptides. In some cases, the set of
additional Ws does not include a c Myc polypeptide. In some cases,
the set of additional IFs does not include polypeptides that might
increase the risk of cell transformation.
[0102] Forced expression of Ws may be maintained for a period of at
least about 7 days to at least about 40 days, e.g., 8 days, 9 days,
10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17
days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 33
days, or 37 days.
[0103] The efficiency of inducing pluripotency in cells of a human
population of cells is from at least about 0.001% to at least about
0.01% of the total number of cells to be induced, e.g., 0.002%,
0.0034%, 0.004%, 0.005%, 0.0065%, 0.007%, 0.008%, or 0.0085%.
D. HDAC Inhibitor
[0104] Induction of the cells may be accomplished by combining
histone deacetylase (HDAC) inhibitor treatment with forced
expression of sets of Ws. The cells to be induced may be
undifferentiated stem cells present in a human postnatal tissue. In
other cases, the cells to be induced are differentiated cells or
are a mixture of differentiated or undifferentiated cells.
[0105] The HDAC may be combined with the forced expression of a
specific set of Ws, e.g., Oct 3/4, a Sox2, and a Klf4. For example,
a human somatic cell is induced to become pluripotent after HDAC
inhibitor treatment is combined with forced expression of Oct3/4,
Sox2 and Klf4 or forced expression of Oct3/4, Sox2, Klf4, and
c-Myc. In some cases, human pluripotent stem cells can be induced
by introducing three genes of Oct3/4, Sox2 and Klf4 or three genes
of Oct3/4, Sox2 and Klf4 plus the c-Myc gene or a HDAC inhibitor
into undifferentiated stem cells present in a human postnatal
tissue in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not
undergone epigenetic inactivation. In still other cases, human
pluripotent stem cells are induced by introducing three genes of
Oct3/4, Sox2 and Klf4 or three genes of Oct3/4, Sox2 and Klf4 plus
the c-Myc gene or a histone deacetylase inhibitor into
undifferentiated stem cells after the undifferentiated stem cells
were amplified by a primary culture or a second subculture, or a
subculture in a low density and subculturing in a culture medium
comprising a low-concentration serum.
[0106] Cells may be treated with one or more HDACs for about 2
hours to about 5 days, e.g., 3 hours, 6 hours, 12 hours, 14 hours,
18 hours, 1 day, 2 days, 3 days, or 4 days. Treatment with HDAC
inhibitor may be initiated prior to beginning forced expression of
IFs in the cells. In some cases, HDAC inhibitor treatment begins
during or after forced expression of IFs in the cells. In other
cases, HDAC inhibitor treatment begins prior to forced expression
and is maintained during forced expression.
[0107] Suitable concentrations of an HDAC inhibitor range from
about 0.001 nM to about 10 mM, depending on the particular HDAC
inhibitor to be used, but are selected so as to not significantly
decrease cell survival in the treated cells. The HDAC concentration
may range from 0.01 nM, to 1000 nM. In some embodiments, the HDAC
concentration ranges from about 0.01 nM to about 1000 nM, e.g.,
about 0.05 nM, 0.1 nM, 0.5 nM, 0.75 nM, 1.0 nM, 1.5 nM, 10 nM, 20
nM, 40 nM, 50 nM, 100 nM, 200 nM, 300 nM, 500 nM, 600 nM, 700 nM,
800 nM, or other concentration from about 0.01 nM to about 1000 nM.
Cells are exposed for 1 to 5 days or 1 to 3 days. For example,
cells are exposed 1 day, 2 days, 3 days, 4 days or 5 days.
[0108] Multiple varieties of HDAC inhibitors can be used for the
induction experiments. In a preferred embodiment, the HDAC
inhibitor MS-275 is used. Examples of suitable HDAC inhibitors
include, but are not limited to, any the following:
[0109] A. Trichostatin A and its analogs, for example: trichostatin
A (TSA); and trichostatin C (Koghe et al. 1998, Biochem. Pharmacol.
56: 1359-1364).
[0110] B. Peptides, for example: oxamflatin
[(2E)-5-[3-[(phenylsulfonyl)aminophenyl]-pent-2-ene-4-inohydroxamic
acid (Kim et al., Oncogene 18: 2461-2470 (1999)); Trapoxin A
(cylco-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10--
epoxy-decanoyl) (Kijima et al., J. Biol. Chem. 268: 22429-22435
(1993)); FR901228, depsipeptide (Nakajima et al., Ex. Cell RES.
241: 126-133 (1998)); FR225497, cyclic tetrapeptide (H. Mori et
al., PCT International Patent Publication WO 00/08048 (Feb. 17,
2000)); apicidin, cyclic tetrapeptide
[cyclo-(N--O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino--
8-oxodecanoyl)] (Darkin-Rattray et al., Proc. Natl. Acad. Sci.
U.S.A. 93: 13143-13147 (1996); apicidin Ia, apicidin Ib, apicidin
Ic, apicidin IIa, and apicidin IIb (P. Dulski et al., PCT
International Patent Publication WO 97/11366); HC-toxin, cyclic
tetrapeptide (Bosch et al., Plant Cell 7: 1941-1950 (1995));
WF27082, cyclic tetrapeptide (PCT International Patent Publication
WO 98/48825); and chlamydocin (Bosch et al., supra).
[0111] C. Hybrid polar compounds (HPC) based on hydroxamic acid,
for example: salicyl hydroxamic acid (SBHA) (Andrews et al.,
International J. Parasitology 30: 761-8 (2000)); suberoylanilide
hydroxamic acid (SAHA) (Richon et al., Proc. Natl. Acad. Sci.
U.S.A. 95: 3003-7 (1998)); azelaic bishydroxamic acid (ABHA)
(Andrews et al., supra); azelaic-1-hydroxamate-9-anilide (AAHA)
(Qiu et al., Mol. Biol. Cell 11: 2069-83 (2000)); M-carboxy
cinnamic acid bishydroxamide (CBHA) (Ricon et al., supra);
6-(3-chlorophenylureido) carpoic hydroxamic acid, 3-Cl-UCHA)
(Richon et al., supra); MW2796 (Andrews et al., supra); and MW2996
(Andrews et al., supra).
[0112] D. Short chain fatty acid (SCFA) compounds, for example:
sodium butyrate (Cousens et al., J. Biol. Chem. 254: 1716-23
(1979)); isovalerate (McBain et al., Biochem. Pharm. 53: 1357-68
(1997)); valproic acid; valerate (McBain et al., supra); 4-phenyl
butyric acid (4-PBA) (Lea and Tulsyan, Anticancer RESearch 15:
879-3 (1995)); phenyl butyric acid (PB) (Wang et al., Cancer
RESearch 59: 2766-99 (1999)); propinate (McBain et al., supra);
butylamide (Lea and Tulsyan, supra); isobutylamide (Lea and
Tulsyan, supra); phenyl acetate (Lea and Tulsyan, supra);
3-bromopropionate (Lea and Tulsyan, supra); tributyrin (Guan et
al., Cancer RESearch 60: 749-55 (2000)); arginine butyrate;
isobutyl amide; and valproate.
[0113] E. Benzamide derivatives, for example: MS-275
[N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzam-
ide] (Saito et al., Proc. Natl. Acad. Sci. U.S.A. 96: 4592-7
(1999)); and a 3'-amino derivative of MS-275 (Saito et al., supra);
and CI-994.
[0114] A histone deacetylase inhibitor treatment may be carried
out, for example, as follows. The concentration of the HDAC
inhibitor may depend on a particular inhibitor, but is preferably
0.001 nM to about 10 mM, and more preferably about 0.01 nM to about
1000 nM. The effective amount or the dosage of a histone
deacetylase inhibitor is defined as the amount of the histone
deacetylase inhibitor that does not significantly decrease the
survival rate of cells, specifically undifferentiated stem cells.
Cells are exposed for 1 to 5 days or 1 to 3 days. The exposure
period may be less than one day. In a specific embodiment, cells
are cultured for about 1 to 5 days, and then exposed to an
effective amount of a histone deacetylase inhibitor. However, the
histone deacetylase inhibitor may be added at the start of
culturing. Within such a time frame, a gene-carrying vehicle such
as a vector containing a nucleic acid encoding three genes (Oct3/4,
Sox2 and Klf4) is introduced into cultured cells by a known
method.
E. IF Expression Vectors
[0115] Forced expression of the IFs may comprise introducing one or
more mammalian expression vectors encoding an Oct3/4, a Sox2, and a
Klf4 polypeptide to a population of cells. The IFs may be
introduced into the cells as exogenous genes. In some cases, the
exogenous genes are integrated into the genome of a host cell and
its progeny. In other cases, the exogenous genes persist in an
episomal state in the host cell and its progeny. Exogenous genes
are genes that are introduced to the cell from an external source.
A gene as used herein is a nucleic acid that includes an open
reading frame encoding a polypeptide of interest, e.g., an IF. The
gene preferably includes a promoter operably linked to an open
reading frame. In some cases, a natural version of the gene may
already exist in the cell but an additional "exogenous gene" is
added to the cell to induce polypeptide expression.
[0116] The one or more mammalian expression vectors may be
introduced into greater than 20% of the total population of cells,
e.g., 25%, 30%, 35%, 40%, 44%, 50%, 57%, 62%, 70%, 74%, 75%, 80%,
90%, or other percent of cells greater than 20%. A single mammalian
expression vector may contain two or more of the just-mentioned
IFs. In other cases, one or more expression vectors encoding an Oct
3/4, Sox2, Klf4, and c Myc polypeptide are used. In some
embodiments, each of the IFs to be expressed is encoded on a
separate mammalian expression vector.
[0117] In some cases, the IFs are genetically fused in frame with a
transport protein amino acid sequence, e.g., that of a VP22
polypeptide as described in, e.g., U.S. Pat. Nos. 6,521,455,
6,251,398, and 6,017,735. Such VP22 sequences confer intercellular
transport of VP22 fusion polypeptides from cells that have been
transfected with a VP22 fusion polypeptide expression vector to
neighboring cells that have not been transfected or transduced.
See, e.g., Lemken et al (2007), Mol Ther, 15(2):310-319.
Accordingly, the use of IF-VP22 fusion polypeptides can
significantly increase the functional efficiency of transfected
mammalian expression vectors in the induction methods described
herein.
[0118] Examples of suitable mammalian expression vectors include,
but are not limited to: recombinant viruses, nucleic acid vectors,
such as plasmids, bacterial artificial chromosomes, yeast
artificial chromosomes, human artificial chromosomes, cDNA, cRNA,
and PCR product expression cassettes. Examples of suitable
promoters for driving expression of IFs in include retroviral LTR
elements; constitutive promoters such as CMV, HSV1-TK, SV40, EF-1a,
13 actin; PGK, and inducible promoters, such as those containing
Tet-operator elements. In some cases, one or more of the mammalian
expression vectors encodes, in addition to an IF, a marker gene
that facilitates identification or selection of cells that have
been transfected or infected. Examples of marker genes include, but
are not limited to, fluorescent protein genes, e.g., for EGFP,
DS-Red, YFP, and CFP; proteins conferring resistance to a selection
agent, e.g., the neoR gene, and the blasticidin resistance
gene.
1. Recombinant Viruses
[0119] Forced expression of an IF may be accomplished by
introducing a recombinant virus carrying DNA or RNA encoding an IF
to one or more cells. Additionally, the recombinant virus may carry
DNA or RNA encoding more than 1 IF. This includes multiple copies
of a single IF or multiple Ws contained within a single virus. For
ease of reference, at times a virus will be referred to herein by
the IF it is encoding. For example, a virus encoding an Oct3/4
polypeptide, may be described as an "Oct3/4 virus." In certain
cases, a virus may encode more than one copy of an IF or may encode
more than one IF, e.g., two IFs, at a time.
[0120] Different combinations or sets of recombinant viruses may be
introduced to the cells. The set of recombinant viruses may include
combinations included in any set of IFs described herein. The set
of recombinant viruses may include at least: an Oct3/4 virus, a
Sox2 virus, and a Klf4 virus. The set of recombinant viruses may be
limited to a set of four recombinant viruses: an Oct3/4 virus, a
Sox2 virus, a Klf4 virus, and a c-Myc virus. In some cases, the set
of recombinant viruses is limited to a set of at least: an Oct3/4
virus, a Sox2 virus, a Klf4 virus, and a c-Myc virus. In some
cases, the set of recombinant viruses is limited to Oct 3/4, Sox2,
and Klf4 viruses. The set of recombinant viruses may be limited a
set of at least: an Oct3/4 virus, a Sox2 virus, and a Klf4 virus.
In some cases, the set of recombinant viruses includes three
recombinant viruses, wherein two of the three recombinant viruses
are an Oct3/4 virus and a Sox2 virus. In still other cases, the set
of recombinant viruses may be limited to a Sox2 virus and a c-Myc
virus.
[0121] In some cases, the set of recombinant viruses does not
include a recombinant virus that encodes a polypeptide that might
increase the risk of cell transformation, e.g., a c-Myc
polypeptide. For example, the set of recombinant viruses can
include: an Oct3/4 virus, a Sox2 virus, and a Klf4 virus but not a
c-Myc virus.
[0122] In other cases, the set of recombinant viruses includes a
c-Myc virus. The c-Myc polypeptide encoded by the c-Myc virus may
be wild-type c-Myc or a constitutively active variant of c-Myc. In
some instances, the set includes a virus encoding c-Myc polypeptide
capable of inducible activity, e.g., a c-Myc-ER polypeptide, see,
e.g., Littlewood, et al. (1995) Nucleic Acid Res.
23(10):1686-90.
[0123] The set of recombinant viruses may include: an Oct3/4 virus,
a Sox2 virus, and a Klf4 virus, but not a TERT virus, a SV40 Large
T antigen virus, HPV16 E6 virus, a HPV16 E7 virus, or a Bmi1 virus.
At times, the set of recombinant viruses does not include a TERT
virus. In some cases, the set of recombinant viruses does not
include a SV40 virus. In other cases, the set of recombinant
viruses does not include a HPV16 E6 virus or a HPV16 E7 virus.
[0124] A set of recombinant viruses may include viruses in addition
to an Oct 3/4, a Sox2, and a Klf4 virus. Such additional
recombinant viruses include, but are not limited to Nanog, TERT,
CYP26A1, GDF3, FoxD3, Zfp42, Dnmt3b, Ecat1, and Tel1 viruses. In
some cases, the set of recombinant viruses includes any IF variant
described herein.
[0125] Individual viruses may be added to the cells sequentially in
time or simultaneously. In some cases, at least one virus, e.g., an
Oct3/4 virus, a Sox2 virus, a Klf4 virus, or a c-Myc virus, is
added to the cells at a time different from the time when one or
more other viruses are added. In some examples, the Oct3/4 virus,
Sox2 virus and KlF4 virus are added to the cells simultaneously, or
very close in time, and the c-Myc virus is added at a time
different from the time when the other viruses are added.
[0126] At least two recombinant viruses may be added to the cells
simultaneously or very close in time. In some examples, Oct3/4
virus and Sox2 virus are added simultaneously, or very close in
time, and the Klf4 virus or c-Myc virus is added at a different
time. In some examples, Oct3/4 virus and Sox2 virus; Oct3/4 virus
and Klf4 virus; Oct3/4 virus and c-Myc virus; Sox2 virus and Klf4
virus; Sox2 virus and c-Myc virus; or Klf4 and c-Myc virus are
added simultaneously or very close in time.
[0127] In some cases, at least three viruses, e.g., an Oct3/4
virus, a Sox2 virus, and a Klf4 virus, are added to the cells
simultaneously or very close in time. In other instances, at least
four viruses, e.g., Oct3/4 virus, Sox2 virus, Klf4 virus, and c-Myc
virus are added to the cells simultaneously or very close in
time.
[0128] At times, the efficiency of viral infection can be improved
by repetitive treatment with the same virus. In some cases, one or
more Oct3/4 virus, Sox2 virus, Klf4 virus, or c-Myc virus is added
to the cells at least two, at least three, or at least four
separate times.
[0129] Examples of recombinant viruses include, but are not
limited, to retroviruses (including lentiviruses); adenoviruses;
and adeno-associated viruses. Often, the recombinant retrovirus is
murine moloney leukemia virus (MMLV), but other recombinant
retroviruses may also be used, e.g., Avian Leukosis Virus, Bovine
Leukemia Virus, Murine Leukemia Virus (MLV), Mink-Cell
focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis
virus, Gibbon Abe Leukemia Virus, Mason Pfizer Monkey Virus, or
Rous Sarcoma Virus, see, e.g., U.S. Pat. No. 6,333,195.
[0130] In other cases, the recombinant retrovirus is a lentivirus
(e.g., Human Immunodeficiency Virus-1 (HIV-1); Simian
Immunodeficiency Virus (SW); or Feline Immunodeficiency Virus
(FIV)), See, e.g., Johnston et al (1999), Journal of Virology
73(6)" 4991-5000 (FIV); Negre D et al (2002) Current Topics in
Microbiology and Immunology 261:53-74 (SIV); Naldini et al (1996)
Science. 272:263-267 (HIV).
[0131] The recombinant retrovirus may comprise a viral polypeptide
(e.g., retroviral env) to aid entry into the target cell. Such
viral polypeptides are well-established in the art, see, e.g., U.S.
Pat. No. 5,449,614. The viral polypeptide may be an amphotropic
viral polypeptide, e.g., amphotropic env, that aids entry into
cells derived from multiple species, including cells outside of the
original host species. See, e.g., id. The viral polypeptide may be
a xenotropic viral polypeptide that aids entry into cells outside
of the original host species. See, e.g., id. In some embodiments,
the viral polypeptide is an ecotropic viral polypeptide, e.g.,
ecotropic env, that aids entry into cells of the original host
species. See, e.g., id.
[0132] Examples of viral polypeptides capable of aiding entry of
retroviruses into cells include but are not limited to: MMLV
amphotropic env, MMLV ecotropic env, MMLV xenotropic env, vesicular
stomatitis virus-g protein (VSV-g), HIV-1 env, Gibbon Ape Leukemia
Virus (GALV) env, RD114, FeLV-C, FeLV-B, MLV 10A1 env gene, and
variants thereof, including chimeras. See e.g., Yee et al (1994),
Methods Cell Biol. Pt A:99-112 (VSV-G); U.S. Pat. No. 5,449,614. In
some cases, the viral polypeptide is genetically modified to
promote expression or enhanced binding to a receptor.
[0133] In general, a recombinant virus is produced by introducing a
viral DNA or RNA construct into a producer cell. In some cases, the
producer cell does not express exogenous genes. In other cases, the
producer cell is a "packaging cell" comprising one or more
exogenous genes, e.g., genes encoding one or more gag, pol, or env
polypeptides and/or one or more retroviral gag, pol, or env
polypeptides. The retroviral packaging cell may comprise a gene
encoding a viral polypeptide, e.g., VSV-g that aids entry into
target cells. In some cases, the packaging cell comprises genes
encoding one or more lentiviral proteins, e.g., gag, pol, env, vpr,
vpu, vpx, vif, tat, rev, or nef. In some cases, the packaging cell
comprises genes encoding adenovirus proteins such as E1A or E1B or
other adenoviral proteins. For example, proteins supplied by
packaging cells may be retrovirus-derived proteins such as gag,
pol, and env, lentivirus-derived proteins such as gag, pol, env,
vpr, vpu, vpx, vif, tat, rev, and nef; and adenovirus-derived
proteins such as E1A and E1B. In many examples, the packaging cells
supply proteins derived from a virus that differs from the virus
from which the viral vector derives.
[0134] Packaging cell lines include but are not limited to any
easily-transfectable cell line. Packaging cell lines can be based
on 293T cells, NIH3T3, COS or HeLa cell lines. As packaging cells,
any cells may be used that can supply a lacking protein of a
recombinant virus vector plasmid deficient in at least one gene
encoding a protein required for virus packaging. Examples of
packaging cell lines include but are not limited to: Platinum-E
(Plat-E); Platinum-A (Plat-A); BOSC 23 (ATCC CRL 11554); and Bing
(ATCC CRL 11270), see, e.g., Morita et al (2000) Gene Therapy
7:1063-1066; Onishi et al (1996) Experimental Hematology
24:324-329; U.S. Pat. No. 6,995,009. Commercial packaging lines are
also useful, e.g., Ampho-Pak 293 cell line, Eco-Pak 2-293 cell
line, RetroPack PT67 cell line, and Retro-X Universal Packaging
System (all available from Clontech).
[0135] The retroviral construct may be derived from a range of
retroviruses, e.g., MMLV, HIV-1, SIV, Fly, or other retrovirus
described herein. The retroviral construct may encode all viral
polypeptides necessary for more than one cycle of replication of a
specific virus. In some cases, the efficiency of viral entry is
improved by the addition of other factors or other viral
polypeptides. In other cases, the viral polypeptides encoded by the
retroviral construct do not support more than one cycle of
replication, e.g., U.S. Pat. No. 6,872,528. In such circumstances,
the addition of other factors or other viral polypeptides can help
facilitate viral entry. In an exemplary embodiment, the recombinant
retrovirus is HIV-1 virus comprising a VSV-g polypeptide but not
comprising a HIV-1 env polypeptide.
[0136] The retroviral construct may comprise: a promoter, a
multi-cloning site, and/or a resistance gene. Examples of promoters
include but are not limited to CMV, SV40, EF1.alpha., .beta. actin;
retroviral LTR promoters, and inducible promoters. The retroviral
construct may also comprise a packaging signal (e.g., a packaging
signal derived from the MFG vector; a psi packaging signal).
Examples of retroviral constructs known in the art include but are
not limited to: pMX, pBabeX or derivatives thereof. See e.g.,
Onishi et al (1996) Experimental Hematology 24:324-329. In some
cases, the retroviral construct is a self-inactivating lentiviral
vector (SIN) vector, see, e.g., Miyoshi et al., (1998) J Virol.
72(10): 8150-8157. In some cases, the retroviral construct is
LL-CG, LS-CG, CL-CG, CS-CG, CLG or MFG. Miyoshi et al., (1998) J
Virol. 72(10): 8150-8157; Onishi et al (1996) Experimental
Hematology 24:324-329; Riviere et al. (1995) PNAS 92: 6733-6737.
Virus vector plasmids (or constructs), include: pMXs, pMXs-IB,
pMXs-puro, pMXs-neo (pMXs-IB is a vector carrying the
blasticidin-resistant gene in stead of the puromycin-resistant gene
of pMXs-puro) [Experimental Hematology, 2003, 31 (11): 1007-14],
MFG [Proc. Natl. Acad. Sci. U.S.A. 92, 6733-6737 (1995)], pBabePuro
[Nucleic Acids Research 18, 3587-3596 (1990)], LL-CG, CL-CG, CS-CG,
CLG [Journal of Virology 72: 8150-8157 (1998)] and the like as the
retrovirus system, and pAdex1 [Nucleic Acids Res. 23: 3816-3821
(1995)] and the like as the adenovirus system. In exemplary
embodiments, the retroviral construct comprises blasticidin (e.g.,
pMXs-IB), puromycin (e.g., pMXs-puro, pBabePuro); or neomycin
(e.g., pMXs-neo). See, e.g., Morgenstern et al. (1990) Nucleic
Acids Research 18: 3587-3596.
[0137] The retroviral construct may encode one or more IFs. In an
exemplary embodiment, pMX vectors encoding Oct3/4, Sox2, Klf4, or
c-Myc polypeptides, or variants thereof, are generated or obtained.
For example, Oct3/4 is inserted into pMXs-puro to create
pMX-Oct3/4; Sox2 is inserted into pMXs-neo to create pMX-Sox2; Klf4
is inserted into pMXs-IB to create pMX-Klf4; and c-Myc is inserted
into pMXs-IB to create pMX-c-Myc.
[0138] Methods of producing recombinant viruses from packaging
cells and their uses are well-established, see, e.g., U.S. Pat.
Nos. 5,834,256; 6,910,434; 5,591,624; 5,817,491; 7,070,994; and
6,995,009, incorporated herein by reference. Many methods begin
with the introduction of a viral construct into a packaging cell
line. The viral construct may be introduced by any method known in
the art, including but not limited to: the calcium phosphate method
[Kokai (Japanese Unexamined Patent Publication) No. 2-227075], the
lipofection method [Proc. Natl. Acad. Sci. U.S.A. 84: 7413 (1987)],
the electroporation method, microinjection, Fugene transfection,
and the like, and any method described herein.
[0139] In one example, pMX-Oct3/4, pMX-Sox2, pMX-Klf4 or pMX-c-Myc
is introduced into PlatE cells by Fugene HD (Roche) transfection.
The cell culture medium may be replaced with fresh medium
comprising FBM (Lonza) supplemented with FGM-2 Single Quots
(Lonza). In some embodiments, the medium is replaced from about 12
to about 60 hours following the introduction of the viral
construct, e.g., from about 12 to about 18 hours; about 18 to about
24; about 24 to about 30; about 30 to about 36; about 36 to about
42; about 42 to about 48; about 48 to about 54; or about 54 to
about 60 hours following introduction of the viral construct to the
producer cells. The medium may be replaced from about 24 to about
48 hours after introduction of the viral construct to the producer
cells. The supernatant can be recovered from about 4 to about 24
hours following the addition of fresh media, e.g., about 4 hours.
In some cases, the supernatant may be recovered about every 4 hours
following the addition of fresh media. The recovered supernatant
may be passed through a 0.45 uM filter (Millipore). In some cases,
the recovered supernatant comprises retrovirus derived from one or
more: pMX-Oct3/4, pMX-Sox2, pMX-Klf4 or pMX-c-Myc.
[0140] Adenoviral transduction may be used to force expression of
the sets of IFs. Methods for generating adenoviruses and their use
are well established as described in, e.g., Straus, The Adenovirus,
Plenum Press (NY 1984), 451 496; Rosenfeld, et al, Science,
252:431-434 (1991); U.S. Pat. Nos. 6,203,975, 5,707,618, and
5,637,456. In other cases, adenoviral-associated viral transduction
is used to force expression of the sets of Ws. Methods for
preparing adeno-associated viruses and their use are well
established as described in, e.g., U.S. Pat. Nos. 6,660,514 and
6,146,874.
[0141] In an exemplary embodiment, an adenoviral construct is
obtained or generated, wherein the adenoviral construct, e.g.,
Adeno-X, comprises DNA encoding Oct3/4, Sox2, Klf4, or c-Myc. An
adenoviral construct may be introduced by any method known in the
art, e.g., Lipofectamine 2000 (Invitrogen) or Fugene HD (Roche),
into HEK 293 cells. In some cases, the method further comprises (1)
collecting the cells when they exhibit a cytopathic effect (CPE),
such effect occurring from about 10 to about 20 days, e.g., about
11, 13, 14, 15, 18, or 20 days after transfection (2) subjecting
the cells to from about 2 to about 5 freeze-thaw cycles, e.g.,
about 3, (3) collecting the resulting virus-containing liquid; (4)
purifying the virus using an adenovirus purification kit (Clontech)
and (5) storing the virus at -80.degree. C. In some cases, the
titer, or plaque-forming unit (PFU), of the adenoviral stocks is
determined using an Adeno-X rapid titer kit (Clontech), as
described herein.
[0142] The cells may be infected with a recombinant retrovirus that
naturally targets a different cell type or cells originating from a
different host. To aid infection efficiency, an exogenous receptor
may be first introduced into the human cells. For example, an
exogenous mouse receptor may be added to human cells, e.g.,
postnatal dermal fibroblasts, in order help entry of murine moloney
leukemia virus (MMLV). The exogenous receptor may improve infection
efficiency by facilitating viral entry, especially if the receptor
recognizes a viral polypeptide, e.g., MMLV env, or HIV env.
Examples of exogenous receptors include but are not limited to any
receptor recognized by a specific retrovirus or lentivirus known in
the art. For example, a murine receptor, mCAT1, GenBank Accession
No NM.sub.--007513 protein is used in order to aid MMLV infection
of a human target cell. In another example, a CXCR4 or CCR5
receptor is used to aid HIV-1 infection of a target cell.
[0143] The exogenous receptor may be introduced by methods
described herein. Methods of introducing the exogenous receptor
include but are not limited to: calcium phosphate transfection,
Lipofectamine transfection, Fugene transfection, microinjection, or
electroporation. In exemplary embodiments, a virus, e.g.,
recombinant adenovirus or retrovirus (including lentivirus), is
used to introduce the exogenous receptor to the target cell. In a
further exemplary embodiment, a recombinant adenovirus is used to
introduce MCAT1 to human cells and then a recombinant retrovirus,
e.g., MMLV, is used to introduce the IF genes, e.g., Oct 3/4, a
Sox2, a Klf4, or c-Myc, to the cells.
[0144] In some cases, a solution of adenovirus comprising DNA
encoding the mCAT1 protein, e.g., an adenovirus generated by using
a pADEX-mCAT1 construct, is generated or obtained. The adenovirus
solution can comprise Hanks' balanced salt solution. In exemplary
embodiments, infection of cells is accomplished by: (1) contacting
the p-ADEX-mCAT1 adenovirus solution with cells, e.g., human,
non-embryonic fibroblasts, at a multiplicity of infection (m.o.i.)
from about 1:5 to about 1:50, e.g., about 1:5, about 1:7; about
1:10; about 1:15, about 1:20, about 1:25; about 1:30, about 1:35;
about 1:40; about 1:45, or about 1:50; (2) incubating the cells
with the adenovirus solution at room temperature from about 15
minutes to about 2 hours, e.g., about 15 minutes, about 30 minutes,
about 45 minutes, about 1 hour, about 1.25 hours, about 1.5 hours,
about 1.75 hours, or about 2 hours; and (3) culturing the somatic
cell population in culture medium from about 24 hours to about 60
hours, e.g., about 24 hours, about 30 hours, about 36 hours, about
42 hours, about 48 hours, about 54 hours, or about 60 hours.
[0145] The cells can be infected using a wide variety of methods.
In some cases, the infection of cells occurs by (1) combining one
or more, two or more, three or more, or all four: pMX-Oct3/4
retrovirus, pMX-Sox2 retrovirus, pMX-Klf4, or pMX-c-Myc to obtain a
retrovirus solution (2) supplementing the retrovirus solution with
from about 2 ug/ml to about 15 ug/ml Polybrene, e.g., about 2
ug/ml, about 3 ug/ml, about 5 ug/ml, about 7 ug/ml, about 10 ug/ml,
about 12 ug/ml, or about 15 ug/ml Polybrene; (3) contacting the
retroviral solution with the somatic cells, at a m.o.i. of from
about 1:100 to about 1:500, e.g., about 1:100, about 1:150, about
1:200, about 1:250, about 1:300, about 1:350, about 1:400, about
1:450, or about 1:500 m.o.i.; (4) allowing the contacting of step
(3) to continue at 37.degree. C. from about 2 hours to about 24
hours, e.g., about 2 hours, about 3 hours, about 4 hours, about 5
hours, about 6 hours, about 7 hours, about 9 hours, about 10 hours,
about 11 hours, about 12 hours, about 14 hours, about 15 hours,
about 16 hours, about 17 hours, about 18 hours, about 19 hours,
about 20 hours, about 21 hours, about 22 hours, about 23 hours, or
about 24 hours; (5) soon after the contacting of step (4), changing
the medium to MC-ES medium, as described herein; and (6) changing
the MC-ES medium with fresh medium every 1 to 2 days. In some
cases, infection of somatic cells occurs by following steps (1)
through (6) described herein, with the added step of pre-incubating
the somatic cells for a length of time, e.g., about 48 hours, prior
to contacting the cells with the retroviral solution. Such
pre-incubation may be necessary when the somatic cell expresses an
exogenous receptor that was introduced by viral transduction,
transfection, or other method. Thus, in some embodiments, if an
adenovirus or lentivirus is used to introduce an exogenous
receptor, e.g., mCAT1, to the somatic cell; such cells may need to
be cultured for a length of time from at least about 30 hours to at
least about 60 hours, e.g., about 30, about 35, about 40, about 48,
about 52, about 55, or about 60 hours.
[0146] The infection of cells may be accomplished by any method
known in the art. e.g., Palsson, B., et al. WO95/10619. Apr. 20,
1995; Morling, F. J. et al. (1995). Gene Therapy. 2: 504-508; Gopp
et al. (2006) Methods Enzymol. 420:64-81. For example, the
infection may be accomplished by spin-infection or "spinoculation"
methods that involve subjecting the cells to centrifugation during
the period closely following the addition of virus to the cells. In
some cases, virus may be concentrated prior to the infection, e.g.,
by ultracentrifugation. In some cases, other technologies may be
used to aid or improve entry of retroviruses into the target cell.
For example, the retrovirus may be contacted with a liposome or
immunoliposome to aid or direct entry into a specific cell type.
See, e.g., Tan et al. (2007) Mol. Med. 13(3-4): 216-226.
[0147] The methods of infecting cells described herein may be used
to infect cells expressing an exogenous receptor, e.g., MCAT1 or
other exogenous receptor described herein. Depending on how the
exogenous receptor was introduced, the preincubation period of the
cells prior to infection may need to be varied. In some cases,
cells that do not express an exogenous receptor are used. Some
recombinant retroviruses, e.g., VSV-G pseudotyped recombinant
retroviruses, may not need the aid of an exogenous receptor in
order to efficiently enter cells. In some examples, VSV-G
pseudotyped recombinant retrovirus is introduced to cells following
the method described herein, except that the timing of the
preculturing of the cells may vary.
2. Nucleic Acid Vectors
[0148] Nucleic acid vector transfection (e.g., transient
transfection) methods may be used to introduce IFs into human
cells. Methods for preparation of transfection-grade nucleic acid
expression vectors are well established. See, e.g., Sambrook and
Russell (2001), "Molecular Cloning: A Laboratory Manual," 3rd ed,
(CSHL Press). Examples of high efficiency transfection efficiency
methods include "nucleofection," as described in, e.g., Trompeter
(2003), J Immunol Methods, 274(1-2):245-256, and in international
patent application publications WO2002086134, WO200200871, and
WO2002086129, transfection with lipid-based transfection reagents
such as Fugene.RTM. 6 and Fugene.RTM. HD (Roche), DOTAP, and
lipofectamine.TM. LTX in combination with the PLUS.TM. (Invitrogen,
Carlsbad, Calif.), Dreamfect.TM. (OZ Biosciences, Marseille,
France), GeneJuice.TM. (Novagen, Madison, Wis.), polyethylenimine
(see, e.g., Lungwitz et al (2005), Eur J Pharm Biopharm,
60(2):247-266), and GeneJammer.TM. (Stratagene, La Jolla, Calif.),
and nanoparticle transfection reagents as described in, e.g., U.S.
patent application Ser. No. 11/195,066.
3. Protein Transduction
[0149] The induction methods may use protein transduction to
introduce at least one of the IFs directly into cells. In some
cases, protein transduction method includes contacting cells with a
composition containing a carrier agent and at least one purified
polypeptide comprising the amino acid sequence of one of the
above-mentioned IFs. Examples of suitable carrier agents and
methods for their use include, but are not limited to, commercially
available reagents such as Chariot.TM. (Active Motif, Inc.,
Carlsbad, Calif.) described in U.S. Pat. No. 6,841,535;
Bioport.RTM. (Gene Therapy Systems, Inc., San Diego, Calif.),
GenomeONE (Cosmo Bio Co., Ltd., Tokyo, Japan), and ProteoJuice.TM.
(Novagen, Madison, Wis.), or nanoparticle protein transduction
reagents as described in, e.g., in U.S. patent application Ser. No.
138,593.
[0150] The protein transduction method may comprise contacting a
cells with at least one purified polypeptide comprising the amino
acid sequence of one of the above-mentioned TAs fused to a protein
transduction domain (PTD) sequence (IF-PTD fusion polypeptide). The
PTD domain may be fused to the amino terminal of an IF sequence;
or, the PTD domain may be fused to the carboxy terminal of an IF
sequence. In some cases, the IF-PTD fusion polypeptide is added to
cells as a denatured polypeptide, which may facilitate its
transport into cells where it is then renatured. Generation of PTD
fusion proteins and methods for their use are established in the
art as described in, e.g., U.S. Pat. Nos. 5,674,980, 5,652,122, and
6,881,825. See also, Becker-Hapak et al (2003), Curr Protocols in
Cell Biol, John Wiley & Sons, Inc. Exemplary PTD domain amino
acid sequences include, but are not limited to, any of the
following: YGRKKRRQRRR (SEQ ID NO:1); RKKRRQRR (SEQ ID NO:2);
YARAAARQARA (SEQ ID NO:3); THRLPRRRRRR (SEQ ID NO:4); and
GGRRARRRRRR (SEQ ID NO:5).
[0151] In some cases, individual purified IF polypeptides are added
to cells sequentially at different times. In other embodiments, a
set of at least three purified IF polypeptides, but not a purified
c-Myc polypeptide, e.g., an Oct3/4 polypeptide, a Sox2 polypeptide,
and a Klf4 polypeptide are added to cells. In some embodiments, a
set of four purified IF polypeptides, e.g., purified Oct3/4, Sox2,
Klf4, and c-Myc polypeptides are added to cells. In some
embodiments, the purified IF polypeptides are added to cells as one
composition (i.e., a composition containing a mixture of the IF
polypeptides). In some embodiments, cells are incubated in the
presence of a purified IF polypeptide for about 30 minutes to about
24 hours, e.g., 1 hours, 1.5 hours, 2 hours, 2.5 hours, 3 hours,
3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16
hours, 18 hours, 20 hours, or any other period from about 30
minutes to about 24 hours. In some embodiments, protein
transduction of cells is repeated with a frequency of about every
day to about every 4 days, e.g., every 1.5 days, every 2 days,
every 3 days, or any other frequency from about every day to about
every four days
[0152] Forced expression of IFs may also be achieved by using
nucleic acid-free IF-containing protein transducing nanoparticles
(PTN). Details of methods for generating and using PTNs are found
in, e.g., Link et al (2006), Nuc Acids Res, 34(2):e16.
[0153] In some cases, the methods described herein utilize protein
transduction and expression vector transduction/transfection in any
combination to force expression of a set of IFs as described
herein. In some embodiments, retroviral expression vectors are used
to force expression of Oct 3/4, a Sox2, and a Klf4 polypeptides in
cells, and purified c-Myc purified polypeptide is introduced into
cells by protein transduction as described herein. HDAC inhibitor
treatment can be used in addition to the purified IF polypeptide.
In some cases, a set of at least three purified IF polypeptides,
but not a purified c-Myc polypeptide, e.g., an Oct3/4 polypeptide,
a Sox2 polypeptide, and a Klf4 polypeptide are added to cells which
are also subjected to HDAC inhibitor treatment.
F. Subcloning Induced Cell Colonies
[0154] Cell colonies may be subcloned by any method known in the
art. In some cases, the iPSCs are cultured and observed for about
14 days to about 40 days, e.g., 15, 16, 17, 18, 19, 20, 23, 24, 27,
28, 29, 30, 31, 33, 34, 35, 36, 37, 38 days, or any other period
from about 14 days to about 40 days prior to identifying and
selecting clones comprising "iPSCs" based on morphological
characteristics. Morphological characteristics for identifying iPSC
clones include, but are not limited to, a small cell size with a
high nucleus-to-cytoplasm ratio; formation of small monolayer
colonies within the space between parental cells (e.g., between
fibroblasts).
[0155] After washing cell cultures with a physiological buffer,
e.g., Hank's balanced salt solution, colonies displaying the
morphological characteristics of interest are surrounded by a
cloning ring to the bottom of which silicone grease has been
applied. About 100 .mu.l (or 50 .mu.l to 150 .mu.l) of "Detachment
Medium For Primate ES Cells" (manufactured by ReproCELL, Tokyo
Japan) is then added to the cloning ring and incubated at
37.degree. C. for about 20 minutes to form a cell suspension. The
cell suspension in the ring containing the detached colonies is
then added to about 2 ml of MC ES medium (or other medium described
herein), and plated in one well of a MEF-coated 24-well plate or
other cell culture vessel of equivalent surface area. After
culturing the colony-derived cells in a 5% CO.sub.2 cell culture
incubator at 37.degree. C. for about 14 hours, the medium is
replaced. Subsequently, the medium is replaced about every two days
until about 8 days later when a second subculture is carried
out.
[0156] In some embodiments, in the first subculture, the medium is
removed, the cells are washed with Hank's balanced salt solution,
and Detachment Medium For Primate ES Cells (ReproCell, Tokyo,
Japan) is then added to the cells and incubated at 37.degree. C.
for 10 minutes. After the incubation, MC-ES medium (2 ml) is added
to the resulting cell suspension to quench the activity of the
Detachment Medium. The cell suspension is then transferred to a
centrifuge tube, and centrifuged at 200.times.g at 4.degree. C. for
5 minutes. The supernatant is removed, the cell pellet is
resuspended in MC ES medium, and the resuspended cells are plated
on four wells of a MEF-coated 24-well plate and cultured for about
seven days until a second subculture is prepared.
[0157] In the second subculture, prepared by the method described
above, cells are plated on a 60 mm cell culture dish coated with
matrigel at a concentration of 20 .mu.g/cm.sup.2. About eight days
later (approximately 5 weeks after initiating forced expression of
IFs), a third subculture is prepared in which cells are plated on
two matrigel-coated 60 mm cell culture dishes, one of which can
subsequently be used for gene expression analysis and the other for
continued passaging as described below. One of the subcultures is
used for gene expression analysis, as described herein, and the
other is passaged as needed to maintain a cell line derived from
the iPSC clone.
G. Passaging and Maintaining Induced Cells
[0158] After subcloning, the iPSCs may be subcultured about every 5
to 7 days. In some cases, the cells are washed with Hank's balanced
salt solution, and dispase or Detachment Medium For Primate ES
Cells is added, and incubated at 37.degree. C. for 5 to 10 minutes.
When approximately more than half of the colonies are detached,
MC-ES medium is added to quench enzymatic activity of the
detachment medium, and the resulting cell/colony suspension is
transferred to a centrifuge tube. Colonies in the suspension are
allowed to settle on the bottom of the tube, the supernatant is
carefully removed, and MC-ES medium is then added to resuspend the
colonies. After examining the size of the colonies, any extremely
large ones are broken up into smaller sizes by slow up and down
pipetting. Appropriately sized colonies are plated on a
matrigel-coated plastic culture dish with a base area of about 3 to
6 times that before subculture.
[0159] Examples of culture media useful for culturing human
pluripotent stem cells induced from undifferentiated stem cells
present in a human postnatal tissue of the present invention
include, but are not limited to, the ES medium, and a culture
medium suitable for culturing human ES cells such as
MEF-conditioned ES medium (MC-ES) or other medium described herein,
e.g., mTeSRT.TM.. In some examples, the cells are maintained in the
presence of a ROCK inhibitor, as described herein.
IV. Analysis of Induced Cells
[0160] Cell colonies subcultured from those initially identified on
the basis of morphological characteristics may be assayed for any
of a number of properties associated with pluripotent stem cells,
including, but not limited to, expression of alkaline phosphatase
activity, expression of ES cell marker genes, expression of protein
markers, hypomethylation of Oct3/4 and Nanog promoters relative to
a parental cells, long term self-renewal, normal diploid karyotype,
and the ability to form a teratoma comprising ectodermal,
mesodermal, and endodermal tissues.
[0161] A number of assays and reagents for detecting alkaline
phosphatase activity in cells (e.g., in fixed cells or in living
cells) are known in the art. In an exemplary embodiment, colonies
to be analyzed are fixed with a 10% formalin neutral buffer
solution at room temperature for about 5 minutes, e.g., for 2 to 5
minutes, and then washed with PBS. A chromogenic substrate of
alkaline phosphatase, 1 step BCIP
(5-Bromo-4-Chloro-3'-Indolylphosphate p-Toluidine Salt) and NBT
(Nitro-Blue Tetrazolium Chloride) manufactured by Pierce (Rockford,
Ill.) is then added and reacted at room temperature for 20 to 30
minutes. Cells having alkaline phosphatase activity are stained
blue-violet.
[0162] Putative iPS cell colonies tested for alkaline phosphatase
activity may be then assayed for expression of a series of human
embryonic stem cell marker (ESCM) genes including, but not limited
to, Nanog, TDGF1, Dnmt3b, Zfp42, FoxD3, GDF3, CYP26A1, TERT, Oct
3/4, Sox2, Sa114, and HPRT. See, e.g., Assou et al (2007), Stem
Cells, 25:961-973. Many methods for gene expression analysis are
known in the art. See, e.g., Lorkowski et al (2003), Analysing Gene
Expression, A Handbook of Methods: Possibilities and Pitfalls,
Wiley-VCH. Examples of suitable nucleic acid-based gene expression
assays include, but are not limited to, quantitative RT-PCR
(qRT-PCR), microarray hybridization, dot blotting, RNA blotting,
RNAse protection, and SAGE.
[0163] In some embodiments, levels of ESCM gene mRNA expression
levels in putative iPS cell colonies are determined by qRT-PCR.
Putative iPS cell colonies are harvested, and total RNA is
extracted using the "Recoverall total nucleic acid isolation kit
for formaldehyde- or paraformaldehyde-fixed, paraffin-embedded
(FFPE) tissues" (manufactured by Ambion, Austin, Tex.). In some
instances, the colonies used for RNA extraction are fixed colonies,
e.g., colonies that have been tested for alkaline phosphatase
activity. The colonies can be used directly for RNA extraction,
i.e., without prior fixation. In an exemplary embodiment, after
synthesizing cDNA from the extracted RNA, the target gene is
amplified using the TaqMan.RTM. PreAmp mastermix (manufactured by
Applied Biosystems, Foster City, Calif.). Real-time quantitative
PCR is performed using an ABI Prism 7900HT using the following PCR
primer sets (from Applied Biosystems) for detecting mRNA of the
above-mentioned ESCM genes: Nanog, Hs02387400_g1, Dnmt3b,
Hs00171876_m1, FoxD3, Hs00255287_s1, Zfp42, Hs01938187_s1, TDGF1,
Hs02339499_g1, TERT, Hs00162669_m1, GDF3, Hs00220998_m1, CYP26A1,
Hs00175627_m1, GAPDH, Hs99999905_ml).
[0164] Putative iPS cell colonies may be assayed by an
immunocytochemistry method for expression of protein markers
including, but not limited to, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,
CD9, CD24, Thy-1, and Nanog. A wide range of immunocytochemistry
assays, e.g., fluorescence immunocytochemistry assays, are known as
described in, e.g., Harlow et al (1988), Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
353-355, and see also, The Handbook--A Guide to Fluorescent Probes
and Labeling Technologies, Molecular Probes, Inc., Eugene, Oreg.,
(2004).
[0165] In an exemplary embodiment, expression of one or more of the
above-mentioned protein markers in putative iPS cell colonies is
assayed as follows. Cultured cells are fixed with 10% formaldehyde
for 10 min and blocked with 0.1% gelatin/PBS at room temperature
for about an hour. The cells are incubated overnight at 4.degree.
C. with primary antibodies against SSEA-3 (MC-631; Chemicon),
SSEA-4 (MC813-70; Chemicon), TRA-1-60 (ab16288; abcam), TRA-1-81
(ab16289; abcam), CD9 (M-L13; R&D systems), CD24 (ALB9; abcam),
Thy1 (5E10; BD Bioscience), or Nanog (MAB1997; R&D Systems).
For Nanog staining, cells are permeabilized with 0.1% Triton
X-100/PBS before blocking. The cell colonies are washed with PBS
three times, then incubated with AlexaFluor 488-conjugated
secondary antibodies (Molecular Probes) and Hoechst 33258 (Nacalai)
at room temperature for 1 h. After further washing, fluorescence is
detected with a fluorescence microscope, e.g., Axiovert 200M
microscope (Carl Zeiss).
[0166] Expression of embryonic stem cell (ESC) marker genes in iPSC
colonies may be assayed in live cells, which increases the
efficiency of identifying iPSC colonies following an induction
method as described herein. Examples of ESC marker genes useful for
identifying induced stem cell colonies include, e.g., Oct3/4,
Nanog, Klf4, Lin28, Sox2, c-Myc, or TERT. In some embodiments, mRNA
for one or more of these genes is detected in live cells. In other
embodiments, mRNAs for two or more of the ESC marker genes is
detected. In one approach, cells are contacted with one or more
molecular beacon probes that hybridize to and signal the presence
of one or more stem cell marker genes. Molecular beacons (MBs) are
single-stranded oligonucleotide hybridization probes that form a
stem-and-loop structure. The loop contains a probe sequence that is
complementary to a target sequence, and the stem is formed by the
annealing of complementary arm sequences that are located on either
side of the probe sequence. A fluorophore is covalently linked to
the end of one arm and a quencher is covalently linked to the end
of the other arm. MBs do not fluoresce when they are free in
solution. However, when they hybridize to a target sequence they
undergo a conformational change that enables them to fluoresce
brightly. The probe sequence may range in length from about 15 to
about 30 nucleotides depending on the GC content of the target
probe sequence. Generally, the GC content of the target probe
sequence should be from about 40 to about 60%. The flanking stem
sequences may range from about 5 to about 7 nucleotides with a GC
content of about 75 to about 100 percent. The design of MBs and
their use to detect mRNA expression in living cells is known in the
art, as described in, e.g., Rhee et al (2008), Nuc Acid Res,
36(5):e30. Useful algorithms for determining melting temperatures
of an MB duplex and an MB/target duplex are known in the art. See,
e.g., the "Mfold" algorithm described in Zucker (2003), Nuc Acids
Res 31(13): 3406-3415, which is public available on a web server:
frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form1.cgi.
See also the Hyther Server at: ozone3.chem.wayne.edu/. Typical
parameters for use in these algorithms are 200 nM concentration for
beacons and nucleic acid target, a folding temperature of
37.degree. C., and ionic condition of 10 mM KCl and 5 mM MgCl2. The
iPSC colonies to be evaluated may be contacted about 14 days to
about 50 days after initiating induction, e.g., 14 days to 21 days,
14 days to 28 days, 20 days to 45 days, 25 days to 40 days, 30 days
to 35 days, 30 days to 50 days after induction. Preferably, cells
are contacted with as low a concentration of an MB and as short a
period as compatible with reliably detecting a signal. In some
embodiments, the concentration of an MB of about 0.1 .mu.M to about
5 .mu.M (for each MB), e.g., 0.1 .mu.M to 0.5 .mu.M, 0.2 .mu.M to 1
.mu.M, 0.5 .mu.M to 2 .mu.M, or 3 .mu.M to 5 .mu.M. Incubation
periods with a MB may range from about 5 minutes to about two
hours, e.g., 15 minutes to 30 minutes, 20 minutes to one hour, 30
minutes to 1.5 hours, 45 minutes to 2 hours, or any other time
period form about 5 minutes to two hours. In some cases, MBs are
introduced into the cells without the use of a transfection
reagent. In other cases, a transfection reagent optimized for
oligonucleotide transfection is utilized, e.g., TransIT.RTM. oligo
transfection reagent kit or any other transfection reagents known
in the art. In other cases, streptolysin-O is used to transiently
permealize cells to allow entry of the MBs into the cells. This
method is described in, e.g., Rhee et al supra and Santangelo et al
(2004), Nuc Acids Res, 32(6): e57.
[0167] In some cases, MBs are added to adherent cell cultures and
cell colonies found to be positive for expression of one or more
ESC marker genes are picked off the substrate as described above.
In other cases, MBs are added to iPSCs in suspension and
ESC-positive cells are selected by FACS or any other fluorescence
based sorting method. Alternatively, MBs are added to adherent
iPSCs, which are then dispersed prior to FACS selection. Use of
FACS for selection of iPSCs is particularly useful for high
throughput generation of iPSC lines and panels of iPSC lines.
A. Methylation Analysis
[0168] In some embodiments, a characteristic of the iPSCs is
reduced methylation of the genomic promoters of Oct3/4 and Nanog
relative to those of their parental cells. Suitable Oct3/4 promoter
regions to be analyzed include, but are not limited to, the Oct3/4
proximal promoter including conserved region 1 (CR1) and the Oct3/4
promoter distal enhancer including CR4. Suitable Nanog promoter
regions to be analyzed include, but are not limited to, the Nanog
proximal promoter including the Oct3/4 and Sox2 binding sites. See,
e.g., Rodda et al (2005), J Biol Chem, 280:24731-24737 and Yang et
al (2005), J Cell Biochem, 96:821-830. A number of methods for the
quantitative analysis of genomic DNA are known as described in,
e.g., Brena et al (2006), J Mol Med, 84(5):365-377. In an exemplary
embodiment, genomic DNA isolated from putative iPSCs and cells used
for a comparison is isolated and treated with bisulfate.
Bisulfite-treated genomic DNA is then PCR-amplified with primers
containing a T7 promoter sequence. Afterwards, RNA transcripts are
generated using T7 polymerase and then treated with RNAse A to
generate methylation-specific cleavage products. Methylation of
individual CpG sites is assessed by MALDI-TOF mass spectrometry of
the cleavage products. A detailed description of the method is
provided in, e.g., Ehich et al (2005), Proc Natl Acad Sci USA,
102:15785-15790.
B. Self-Renewal Assay
[0169] One of the characteristics of stem cells is their ability to
proliferate continuously without undergoing senescence.
Accordingly, iPSCs are assessed for their ability to be passaged
continuously in vitro. In some cases, the iPSCs are assayed for
their ability to be passaged for at least about 30 to at least
about 100 times in vitro, e.g., about 33, 35, 40, 45, 51, 56, 60,
68, 75, 80, 90, 93, 100, or any other number of passages from at
least about 30 to at least about 100 passages.
[0170] In another evaluation, iPSCs are assayed for their ability
to proliferate for a period of about 30 days to about 500 days from
initiation of forced expression of IFs in parental cells, e.g., 40
days, 50 days, 60 days, 70 days, 80 days, 100 days, 150 days, 180
days, 200 days, 250 days, 300 days, 400 days, 450 days or any other
period from about 30 days to about 500 days from initiation of
forced expression of IFs in the parental cells. In some
embodiments, long-term self-renewal of iPSCs is determined when the
cells are passaged in a defined medium (e.g., mTeSR1 medium) and in
the absence of feeder cells, e.g., mTeSR1 medium as described
herein. In other embodiments, cells are passaged in MC-ES medium as
described herein.
C. Karyotype Analysis
[0171] As another possible analysis, iPSCs are assessed for
diploidy and a normal, stable karyotype, e.g., stable after the
cells of have been passaged for at least one year in vitro. A
number of karotype analysis methods are known in the art. In some
embodiments, the karyotype analysis method is multicolor FISH as
described in, e.g., Bayani et al (2004), Curr Protoc Cell Biol,
Chapter 22: Unit 22.5. In other embodiments, the karyotype analysis
includes a molecular karyotype analysis as described in, e.g.,
Vermeesch et al (2007), Eur J Hum Genet, 15(11):1105-1114. In an
exemplary embodiment, iPSCs are pretreated with 0.02 .mu.g/ml
colecemid for about 2 to about 3 hours, incubated with about 0.06
to about 0.075M KCl for about 20 minutes, and then fixed with
Carnoy's fixative. Afterwards, for multicolor FISH analysis, cells
are hybridized with multicolor FISH probes, e.g., those in the
Star*FISH.COPYRGT. Human Multicolour FISH (M-FISH) Kit from Cambio,
Ltd (Cambridge, UK).
D. Teratoma Analysis
[0172] It is generally believed that pluripotent stem cells have
the ability to form a teratoma, comprising ectodermal, mesodermal,
and endodermal tissues, when injected into an immunocompromised
animal. Induced cells or induced pluripotent stem cells (iPS) or ES
cell-like pluripotent stem cells may refer to cells having an in
vitro long-term self-renewal ability and the pluripotency of
differentiating into three germ layers, and said pluripotent stem
cells may form a teratoma when transplanted into a test animal such
as mouse.
[0173] The iPSCs may be assessed for pluripotency in a teratoma
formation assay in an immunocompromised animal model. The
immunocompromised animal may be a rodent that is administered an
immunosuppressive agent, e.g., cyclosporin or FK-506. For example,
the immunocompromised animal model may be a SCID mouse. About
0.5.times.10.sup.6 to about 2.0.times.10.sup.6, e.g.,
0.6.times.10.sup.6, 0.8.times.10.sup.6, 1.0.times.10.sup.6,
1.2.times.10.sup.6, 1.5.times.10.sup.6, 1.7.times.10.sup.6, or
other number of iPSCs from about 0.5.times.10.sup.6 to about
2.0.times.10.sup.6 iPSCs/mouse may be injected into the medulla of
a testis of a 7- to 8-week-old immunocompromised animal. After
about 6 to about 8 weeks, the teratomas are excised after perfusing
the animal with PBS followed by 10% buffered formalin. The excised
teratomas are then subjected to immunohistological analysis. One
method of distinguishing human teratoma tissue from host (e.g.,
rodent) tissue includes immunostaining for the human-specific
nuclear marker HuNu. Immunohistological analysis includes
determining the presence of ectodermal (e.g., neuroectodermal),
mesodermal, and endodermal tissues. Protein markers for ectodermal
tissue include, but are not limited to, nestin, GFAP, and integrin
.beta.1. Protein markers for mesodermal tissue include, but are not
limited to, collagen II, Brachyury, and osteocalcin. Protein
markers for endodermal tissue include, but are not limited to,
.alpha.-fetoprotein (.alpha. FP) and HNF3beta.
E. Global Gene Expression
[0174] In some embodiments, global gene expression analysis is
performed on putative iPS cell colonies. Such global gene
expression analysis may include a comparison of gene expression
profiles from a putative iPS cell colony with those of one or more
cell types, including but not limited to, (i) parental cells, i.e.,
one or more cells from which the putative iPS cell colony was
induced; (ii) a human ES cell line; or (iii) an established iPS
cell line. As known in the art, gene expression data for human ES
cell lines are available through public sources, e.g., on the world
wide web in the NCBI "Gene Expression Omnibus" database. See, e.g.,
Barrett et al (2007), Nuc Acids Res, D760-D765. Thus, in some
embodiments, comparison of gene expression profiles from a putative
iPS colony to those of an ES cell line entails comparison
experimentally obtained data from a putative iPS cell colony with
gene expression data available through public databases. Examples
of human ES cell lines for which gene expression data are publicly
available include, but are not limited to, hE14 (GEO data set
accession numbers GSM151739 and GSM151741), Sheff4 (GEO Accession
Nos GSM194307, GSM194308, and GSM193409), h_ES 01 (GEO Accession
No. GSM194390), h_ES H9 (GEO Accession No. GSM194392), and h_ES
BG03 (GEO Accession No. GSM194391).
[0175] It is also possible to accomplish global gene expression by
analyzing the total RNA isolated from one or more iPS cell lines by
a nucleic acid microarray hybridization assay. Examples of suitable
microarray platforms for global gene expression analysis include,
but are not limited to, the Human Genome U133 plus 2.0 microarray
(Affymetrix) and the Whole Human Genome Oligo Micoarray (Agilent).
A number of analytical methods for comparison of gene expression
profiles are known as described in, e.g., Suarez-Farinas et al
(2007), Methods Mol Biol, 377:139-152, Hardin et al (2007), BMC
Bioinformatics, 8:220, Troyanskaya et al (2002), Bioinformatics,
18(11):1454-1461, and Knudsen (2002), A Biologist's Guide to
Analysis of DNA Microarray Data, John Wiley & Sons. In some
embodiments, gene expression data from cells produced by the
methods described herein are compared to those obtained from other
cell types including, but not limited to, human ES cell lines,
parental cells, and multipotent stem cell lines. Suitable
statistical analytical metrics and methods include, but are not
limited to, the Pearson Correlation, Euclidean Distance,
Hierarchical Clustering (See, e.g., Eisen et al (1998), Proc Natl
Acad Sci USA, 95(25): 14863-14868), and Self Organizing Maps (See,
e.g., Tamayo et al (1999), Proc Natl Acad Sci USA,
96(6):2907-2912.
F. Methods for Differentiating Induced Stem Cell Lines
[0176] iPSC lines may be differentiated into cell-types of various
lineages. Examples of differentiated cells include any
differentiated cells from ectodermal (e.g., neurons and
fibroblasts), mesodermal (e.g., cardiomyocytes), or endodermal
(e.g., pancreatic cells) lineages. The differentiated cells may be
one or more: pancreatic beta cells, neural stem cells, neurons
(e.g., dopaminergic neurons), oligodendrocytes, oligodendrocyte
progenitor cells, hepatocytes, hepatic stem cells, astrocytes,
myocytes, hematopoietic cells, or cardiomyocytes.
[0177] The differentiated cells derived from the iPSCs may be
terminally differentiated cells, or they may be capable of giving
rise to cells of a specific lineage. For example, iPSCs can be
differentiated into a variety of multipotent cell types, e.g.,
neural stem cells, cardiac stem cells, or hepatic stem cells. The
stem cells may then be further differentiated into new cell types,
e.g., neural stem cells may be differentiated into neurons; cardiac
stem cells may be differentiated into cardiomyocytes; and hepatic
stem cells may be differentiated into hepatocytes. Methods for
differentiating iPSCs are further disclosed in U.S. application
Ser. Nos. 12/157,967, WSGR docket number 36588-704.201; filed Jun.
13, 2008; first inventor Kazuhiro Sakurada, 61/061,594, WSGR Docket
Number 36588-707.101; filed on Jun. 13, 2008; First Inventor
Kazuhiro Sakurada, and 61/061,565, WSGR Docket Number
36588-702.101; filed on Jun. 13, 2008; First Inventor Kazuhiro
Sakurada, which are hereby incorporated by reference in their
entirety.
[0178] There are numerous methods of differentiating the iPSCs into
a more specialized cell type. Methods of differentiating iPSCs may
be similar to those used to differentiate other stem cells,
particularly ES cells, MSCs, MAPCs, MIAMI, hematopoietic stem cells
(HSCs). In some cases, the differentiation occurs ex vivo; in some
cases the differentiation occurs in vivo.
[0179] Any known method of generating neural stem cells from ES
cells may be used to generate neural stem cells from iPSCs, See,
e.g., Reubinoff et al. (2001) Nat. Biotechnol. 19(12):1134-40. For
example, neural stem cells may be generated by culturing the iPSCs
as floating aggregates in the presence of noggin, or other bone
morphogenetic protein antagonist, see e.g., Itsykson et al. (2005)
Mol Cell Neurosci. 30(1):24-36. In another example, neural stem
cells may be generated by culturing the iPSCs in suspension to form
aggregates in the presence of growth factors, e.g., FGF-2, Zhang et
al. (2001), Nat. Biotech. (19) 1129-1133. In some cases, the
aggregates are cultured in serum-free medium containing FGF-2. In
another example, the iPSCs are co-cultured with a mouse stromal
cell line, e.g., PA6 in the presence of serum-free medium
comprising FGF-2. In yet another example, the iPSCs are directly
transferred to serum-free medium containing FGF-2 to directly
induce differentiation.
[0180] Neural stems derived from the iPSCs may be differentiated
into neurons, oligodendrocytes, or astrocytes. Dopaminergic neurons
play a central role in Parkinson's Disease and are thus of
particular interest. In order to promote differentiation into
dopaminergic neurons, iPSCs may be co-cultured with a PA6 mouse
stromal cell line under serum-free conditions, see, e.g., Kawasaki
et al. (2000) Neuron 28(1):31-40. Other methods have also been
described, see, e.g., Pomp et al. (2005), Stem Cells 23(7):923-30;
U.S. Pat. No. 6,395,546.
[0181] Oligodendrocytes may also be generated from the iPSCs. For
example, oligodendrocytes may be generated by co-culturing iPSCs or
neural stem cells with stromal cells, e.g., Lee et al. (2000)
Nature Biotechnol 18:675-679. In another example, oligodendrocytes
may be generated by culturing the iPSCs or neural stem cells in the
presence of a fusion protein, in which the Interleukin (IL)-6
receptor, or derivative, is linked to the IL-6 cytokine, or
derivative thereof.
[0182] Astrocytes may also be produced from the iPSCs. Astrocytes
may be generated by culturing iPSCs or neural stem cells in the
presence of neurogenic medium with bFGF and EGF, see e.g., Brustle
et al. (1999) Science 285:754-756.
[0183] Induced cells may be differentiated into pancreatic beta
cells by methods known in the art, e.g., Lumelsky et al. (2001)
Science 292:1389-1394; Assady et al., (2001) Diabetes 50:1691-1697;
D'Amour et al (2006) Nat Biotechnol: 1392-1401' D'Amour et al.
(2005) Nat Biotechnol 23:1534-1541. The method may comprise
culturing the iPSCs in serum-free medium supplemented with Activin
A, followed by culturing in the presence of serum-free medium
supplemented with all-trans retinoic acid, followed by culturing in
the presence of serum-free medium supplemented with bFGF and
nicotinamide, e.g., Jiang et al. (2007) Cell Res 4:333-444. In
other examples, the method comprises culturing the iPSCs in the
presence of serum-free medium, activin A, and Wnt protein from
about 0.5 to about 6 days, e.g., about 0.5, 1, 2, 3, 4, 5, 6, days;
followed by culturing in the presence of from about 0.1% to about
2%, e.g., 0.2%, FBS and activin A from about 1 to about 4 days,
e.g., about 1, 2, 3, 4 days; followed by culturing in the presence
of 2% FBS, FGF-10, and KAAD-cyclopamine
(keto-N-aminoethylaminocaproyl dihydro cinnamoylcyclopamine and
retinoic acid from about 1 to about 5 days, e.g., 1, 2, 3, 4, or 5
days; followed by culturing with 1% B27, gamma secretase inhibitor
and extendin-4 from about 1 to about 4 days, e.g., 1, 2, 3, or 4
days; and finally culturing in the presence of 1% B27, extendin-4,
IGF-1, and HGF for from about 1 to about 4 days, e.g., 1, 2, 3, or
4 days.
[0184] Hepatic cells or hepatic stem cells may be differentiated
from the iPSCs. For example, culturing the iPSCs in the presence of
sodium butyrate may generate hepatocytes, see e.g., Rambhatla et
al. (2003) Cell Transplant 12:1-11. In another example, hepatocytes
may be produced by culturing the iPSCs in serum-free medium in the
presence of Activin A, followed by culturing the cells in
fibroblast growth factor-4 and bone morphogenetic protein-2, e.g.,
Cai et al. (2007) Hepatology 45(5):1229-39. In an exemplary
embodiment, the iPSCs are differentiated into hepatic cells or
hepatic stem cells by culturing the iPSCs in the presence of
Activin A from about 2 to about 6 days, e.g., about 2, about 3,
about 4, about 5, or about 6 days, and then culturing the iPSCs in
the presence of hepatocyte growth factor (HGF) for from about 5
days to about 10 days, e.g., about 5, about 6, about 7, about 8,
about 9, or about 10 days.
[0185] The method may also comprise differentiating iPSCs into
cardiac muscle cells. In an exemplary embodiment, the method
comprises culturing the iPSCs in the presence of noggin for from
about two to about six days, e.g., about 2, about 3, about 4, about
5, or about 6 days, prior to allowing formation of an embryoid
body, and culturing the embryoid body for from about 1 week to
about 4 weeks, e.g., about 1, about 2, about 3, or about 4
weeks.
[0186] In other examples, cardiomyocytes may be generated by
culturing the iPSCs may in the presence of LIF, or by subjecting
them to other methods in the art to generate cardiomyocytes from ES
cells, e.g., Bader et al. (2000) Circ Res 86:787-794, Kehat et al.
(2001) J Clin Invest 108:407-414; Mummery et al. (2003) Circulation
107:2733-2740.
[0187] Examples of methods to generate other cell-types from iPSCs
include: (1) culturing iPSCs in the presence of retinoic acid,
leukemia inhibitory factor (LIF), thyroid hormone (T3), and insulin
in order to generate adipoctyes, e.g., Dani et al. (1997) J. Cell
Sci 110:1279-1285; (2) culturing iPSCs in the presence of BMP-2 or
BMP-4 to generate chondrocytes, e.g., Kramer et al. (2000) Mech Dev
92:193-205; (3) culturing the iPSCs under conditions to generate
smooth muscle, e.g., Yamashita et al. (2000) Nature 408: 92-96; (4)
culturing the iPSCs in the presence of beta-mercaptoethanol to
generate keratinocytes, e.g., Bagutti et al. (1996) Dev Biol 179:
184-196; Green et al. (2003) Proc Natl Acad Sci USA
100:15625-15630; (5) culturing the iPSCs in the presence of
Interleukin-3(IL-3) and macrophage colony stimulating factor to
generate macrophages, e.g., Lieschke and Dunn (1995) Exp Hemat
23:328-334; (6) culturing the iPSCs in the presence of IL-3 and
stem cell factor to generate mast cells, e.g., Tsai et al. (2000)
Proc Natl Acad Sci USA 97:9186-9190; (7) culturing the iPSCs in the
presence of dexamethasone and stromal cell layer, steel factor to
generate melanocytes, e.g., Yamane et al. (1999) Dev Dyn
216:450-458; (8) co-culturing the iPSCs with fetal mouse
osteoblasts in the presence of dexamethasone, retinoic acid,
ascorbic acid, beta-glycerophosphate to generate osteoblasts, e.g.,
Buttery et al. (2001) Tissue Eng 7:89-99; (9) culturing the iPSCs
in the presence of osteogenic factors to generate osteoblasts,
e.g., Sottile et al. (2003) Cloning Stem Cells 5:149-155; (10)
overexpressing insulin-like growth factor-2 in the iPSCs and
culturing the cells in the presence of dimethyl sulfoxide to
generate skeletal muscle cells, e.g., Prelle et al. (2000) Biochem
Biophys Res Commun 277:631-638; (11) subjecting the iPSCs to
conditions for generating white blood cells, e.g., Rathjen et al.
(1998) Reprod Fertil Dev 10:31-47; or (12) culturing the iPSCs in
the presence of BMP4 and one or more: SCF, FLT3, IL-3, IL-6, and
GCSF to generate hematopoietic progenitor cells, e.g., Chadwick et
al. (2003) Blood 102:906-915.
[0188] In some cases, sub-populations of differentiated cells may
be purified or isolated. In some cases, one or more monoclonal
antibodies specific to the desired cell type are incubated with the
cell population and those bound cells are isolated. In other cases,
the desired subpopulation of cells expresses a reporter gene that
is under the control of a cell type specific promoter.
[0189] In a specific embodiment, the hygromycin B
phosphotransferase-EGFP fusion protein is expressed in a cell type
specific manner. The method of purifying comprises sorting the
cells to select green fluorescent cells and reiterating the sorting
as necessary, in order to obtain a population of cells enriched for
cells expressing the construct (e.g., hygromycin B
phosphotransferase-EGFP) in a cell-type-dependent manner. Selection
of desired sub-populations of cells may also be accomplished by
negative selection of proliferating cells with the herpes simplex
virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system
or by positive selection of cells expressing a bicistronic
reporter, e.g., Anderson et al. (2007) Mol. Ther.
(11):2027-2036.
G. Panels of Induced Stem Cell Lines
[0190] In some cases, the methods described herein utilize a panel
of iPSC lines or a panel of cells differentiated from iPSC lines. A
panel of iPSC lines comprises multiple iPSC lines, e.g., iPSC
lines, that meet certain selection criteria. Also provided herein
are panels of cells differentiated from iPSC lines as described
herein. Such panels of differentiated cells include, but are not
limited to, panels of neural stem cells, neurons, retinal cells,
glial progenitor cells, glial cells, cardiac progenitor cells,
cardiomyocytes, pancreatic progenitor cells, pancreatic beta cells,
hepatic stem cells, hepatocytes or lung progenitor cells. In some
cases, the selection criteria for inclusion of an iPSC line in a
panel of iPSC lines are determined prior to generating the iPSC
lines that will constitute the panel. In other cases, the selection
criteria are applied to iPSC lines generated before hand, e.g., a
bank of iPSC lines. Selection criteria include, but are not limited
to, the presence or absence of a particular health condition in an
iPSC donor, a positive drug response in an iPSC donor, negative,
positive, or adverse drug responses in an iPSC donor, the presence
or absence of a particular phenotype in an iPSC line or in cells
differentiated from the iPSC line, and the presence or absence of
one or more polymorphic alleles in the cell lines or their
corresponding donors.
[0191] In some embodiments, where selection criteria include the
presence or absence of one or more polymorphic alleles, the panel
includes genetically diverse human iPSC lines in which each iPSC
line carries at least one polymorphic allele that is unique among
the iPSCs to be included in the panel, e.g., 5 to 10, 20 to 50, 50
to 200, 200 to 500, 500 to 1000, 1000 to 5000, 5000 to 20000, or
20000 to 50000 polymorphic alleles that are unique within the panel
of iPSC lines. Such polymorphic alleles may include, e.g., a SNP
allele, a promoter allele, or a protein-encoding allele.
Polymorphic alleles can be screened and scored for by genotyping
using any of a number of known genotyping assays. In some cases,
the genotyping assay is a multiplexed genotyping assay, e.g., a
nucleic acid microarray assay platform such as a "SNP chip." In
some cases, the one or more polymorphic alleles are pre-selected.
In some embodiments, the one or more preselected alleles are
polymorphic alleles associated with a health condition or a
predisposition to a health condition. Examples of polymorphic
alleles associated with a health condition or a predisposition to a
health condition, include, but are not limited to, polymorphic
alleles associated with a neurodegenerative disorder, a
neurological disorder, an eye disease, a mood disorder, a
respiratory disease, a cardiovascular disease, an immunological
disorder, a hematological disease, a metabolic disorder, or a drug
sensitivity condition. Some examples of polymorphic alleles
associated with a health condition are provided in Table 3 above.
Polymorphic alleles may include polymorphic alleles in an encoded
protein or a regulatory sequence affecting the expression of the
encoded protein. In some cases, the encoded protein is a drug
target. Examples of drug target proteins include, but are not
limited to, GPCRs, ion channels, kinases, enzymes, and
transcription factors.
[0192] In other embodiments, the one or more polymorphic alleles
are pre-selected based on the presence of a high degree of
surrounding linkage disequilibrium in the genome, which has been
proposed as a signature of genomic loci that are likely to impact
many common health conditions. Methods for identifying SNPs having
a high surrounding linkage disequilibrium and genes near such SNPs
are described in, e.g., Wang et al (2006), Proc Natl Acad Sci USA,
103(1):135-140.
[0193] In some cases, a panel of iPSC lines includes iPSC lines
generated from subjects that are diagnosed as suffering from one or
more health conditions. The one or more health conditions may be
one or more health conditions that are common to all of the iPSC
donors, or they may be health conditions that are different between
the iPSC donors.
[0194] In certain cases, a panel of iPSC lines includes iPSC lines
generated from subjects that are both diagnosed as suffering from a
health condition and carry a polymorphic allele associated with a
health condition, e.g., a polymorphic allele associated with the
diagnosed health condition.
[0195] A panel of iPSC lines may include iPSC lines from at least
about 10 individuals to at least about 50,000 individuals, e.g., 10
to 50, 20 to 100, 50 to 250, 100 to 1000, 250, to 2000, 500 to
5000, 1000 to 10,000, 2500 to 20,000, 10,000, to 30,000, 20,000 to
40,000, or 30,000 to 50,000 individuals.
[0196] A panel of iPSC lines may include iPSC lines from at least
two ethnic groups, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25,
30, or 50 ethnic groups. Examples of ethnic groups include, but are
not limited to, Europeans, Japanese, Chinese, and the Yoruba of
Nigeria, and ethnic groups listed in Table 4.
TABLE-US-00004 TABLE 4 Exemplary Ethnic Groups Africa Bantu Biaka
Mandenka Mbuti pygmy Mozabite San Yoruba Native America Colombian
Karitiana Maya Pima Surui Asia Ctrl/South Balochi Brahui Burusho
Hazara Kalash Makrani Pathan Sindhi Uyghur Western Asia Bedouin
Druze Eastern Asia Cambodian Dai Daur Han (N. China) Han (S. China)
Hezhen Japanese Lahu Miao Mongola Naxi Oroqen She Tu Tujia Xibo
Yakut Yi Europe Adygei Basque French North Italian Orcadian Russian
Sardinian Tuscan Oceania Melanesian Papuan
IV. Methods for Use of Induced Stem Cell Lines and Panels of
Induced Stem Cell Lines
A. Overview
[0197] The iPSC lines and panels of iPSC lines described herein are
useful in a number of methods relating to drug discovery and
development. Typically, a drug candidate compound will be evaluated
in a biochemical assay (e.g., a receptor binding assay) that
evaluates only a single or very few sequence variants of the drug
target expressed in a patient population. Thus, such assays provide
little information as to how effective the drug candidate compound
is likely to be in patients that express a drug target allele that
differs from the particular drug target allele that was originally
screened. Along the same lines, drug candidate compounds often
undergo functional cellular screens in one or few cell lines
engineered to express a specific allele of the drug target, again
ignoring the genetic diversity of a human patient population not
only with respect to the drug target itself, but also to that of
the various downstream signal transduction proteins that play a
role in the response endpoint of cells to a drug. Likewise, adverse
effects of candidate drug compounds (e.g., liver toxicity) are
generally evaluated in inbred animal models, which are likely to be
uninformative for a variable fraction of a human patient
population. In contrast, drug screening in panels of genetically
diverse iPSC lines, as described herein, addresses the lack of
genetic diversity in the prevailing drug screening models.
[0198] The panels of genetically diverse iPSC lines described
herein (e.g., human iPSC lines) or cells differentiated from panels
of genetically diverse iPSC lines, as described herein, may be used
to identify test compounds that act on a drug target of interest.
In some embodiments, the panels of iPSCs cell lines include a
sufficient number of iPSC lines such that at least two, e.g., at
least 3, 5, 10, 20, 50, 100, or 200 polymorphic alleles of a drug
target (e.g., a GPCR, ion channel, or kinase) are represented in
the panel. In some embodiments, panels of iPSC lines are derived
from subjects diagnosed as suffering from a health condition or
identified as having a predisposition to the health condition. In
other embodiments, the iPSC line panels comprise iPSC lines each of
which that has at least one polymorphic allele associated with a
health condition or a predisposition to the health condition.
[0199] Drug targets for many health conditions are known. Such drug
targets may include, but are not limited to, receptors, GPCRs,
growth factor receptors, neurotransmitter receptors, ion channels,
enzymes, protein kinases, proteases, cytoskeletal proteins, and
transcription factors. Test compounds can be assayed for their
effect on a drug target by a number of assays known in the art.
Such assays include cell-based assays including, but not limited
to, assays for determining second messenger levels, e.g.,
intracellular calcium, cAMP, cGMP, arachidonic acid, and inositol
phosphates; channel currents; apoptosis; proliferation;
morphological changes; changes in adhesion. Examples of cell-based
assays include, but are not limited to those described in, U.S.
Pat. Nos. 7,319,009, 7,288,368, and 7,238,213, Cell based assays
may also include determining the cellular localization of one or
more proteins (e.g., protein kinases, receptors, and transcription
factors) in cells in the presence or absence of a test compound.
Test compounds may also be screened for their ability to alter a
gene expression profile by any gene expression profiling method
known in the art. In some cases, the cells to be screened may be
genetically modified to express one or more reporter proteins that
can indicate activation of a signaling pathway. For example
protein-protein interactions between fusion proteins introduced
into cells may be detected by a number of methods known in the art,
e.g., by fluorescence resonance energy transfer (FRET) or enzyme
fragment complementation.
[0200] In some cases, the mechanistic basis of a sporadic form of a
disease is a combination of genetically-determined cell
type-specific phenotype and epigenetic factors (e.g., oxidative
stress). In other words, iPSC-derived differentiated cells from a
patient with a sporadic form of a disease (e.g., Parkinson's) may
bear a genetic predisposition to a pathological or pre-pathological
cellular phenotype (e.g., apoptosis), but the phenotype may only
manifest in vitro in the presence of an appropriate "stressor" that
recapitulates environmental/epigenetic factors associated with the
sporadic disease or a cellular phenotypes that precede a clinical
manifestation of the disease (e.g., mitochondrial dysfunction,
oxdidative stress, or nitrosylative stress). Accordingly, in some
cases disease-relevant cellular phenotypes are induced by a
stressor. Examples of stressors include, but are not limited to
cellular oxidative stress, nitrosylative stress, proteasome
inhibition, inhibition of mitochondrial electron transport,
translation inhibition, decreased calcium buffering, high
osmolarity, heat shock, heavy metals (e.g., Zn, Mn, Fe, Cd, Al, or
Pb), protein misfolding. Examples of agents that induce, increase,
or result from oxidative stress include, but are not limited to,
H.sub.2O.sub.2, ascorbic acid/FeSO.sub.4, 4-hydroxynonenal,
glutamate, kainate, NMDA, dopamine, okadaic acid, A.beta..sup.1-42
and isocyanate. Proteasome inhibitors include, but are not limited
to lactacystin, ziram, MG132, and
carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal
(PSI). Mitochondrial stressors include, but are not limited to,
rotenone, 3-nitropropionic acid (NPA), 1-methyl-4-phenylpyridinium
(MPP.sup.+), antimycin, paraquat, methylglycoxal, and cyanide.
Nitrosylative stressors include, but are not limited to,
(+/-)-S-nitroso-N-acetylpenicillamine, sodium nitroprussiate, and
peroxynitrite.
[0201] In some cases, the stressor is provided by expressing or
overexpressing an exogenous wild type or mutated gene and/or
protein. Examples of such genes include, .alpha.-synuclein, amyloid
beta, A.beta..sup.1-42, Parkin, Pink1, Leucine-rich repeat kinase 2
(LRRK2), superoxide dismutase (SOD).
[0202] Assays of drug candidate compounds in an iPSC line or a
panel of iPSC lines can include determining a dose-response. In
some embodiments, the dose response of an iPSC line or that of one
or more types of cells differentiated from the iPSC line provides
an indication that of the likely efficacy of the compound in the
corresponding iPSC donor. In some embodiments, the fraction of iPSC
lines in a panel of iPSC lines that exhibit an acceptable
dose-response to a test compound indicates an expected probability
of an acceptable dose-response relationship in the target patient
population of interest. In some cases, cell-based assays of drug
candidate include a comparison of responses obtained in a panel of
iPSC lines or iPSC-derived cells to one or more reference iPSC
lines or cells that serve as a positive or negative control for the
effect of a drug candidate compound. The reference iPSC lines or
cells may be from a healthy iPSC donor, from an iPSC donor
diagnosed as suffering from a health condition, or an iPSC donor
carrying a polymorphic allele associated with a health condition.
In other embodiments, assays of drug candidate compounds in an iPSC
line or a panel of iPSC lines can include determining effective
concentrations, maximum tolerated dose and minimum effective
concentration. Additional methods and assays are disclosed in U.S.
application No. 61/061,594, WSGR Docket Number 36588-707.101; filed
Jun. 13, 2008; First Inventor Kazuhiro Sakurada, hereby
incorporated by reference.
[0203] In some cases, the drug screening may be conducted on cells
differentiated from iPSCs. Examples of such differentiated cells
are described herein (e.g., hepatic cells, neural stem cells,
neurons, pancreatic beta cells, cardiomyocytes, hepatic stem cells,
oligodendrocytes). The drugs may be targeted to treat a specific
disease or condition, e.g., a disease or condition described
herein. For example, the iPSCs may be differentiated into
dopaminergic neurons, which are used to screen drugs for
Parkinson's disease. In other cases, neurons or neural stem cells
differentiated from iPSCs may be used to screen drugs for treating
Alzheimer's disease, multiple sclerosis, or other neurological
disorders. In some cases the In other cases, the iPSCs may be
transplanted directly into an immunocompromised animal, e.g., SCID
mouse, which is then used to establish in vitro or in vivo assay
systems that mimic physiologic conditions in humans or other
animals. The in vitro or in vivo assay systems may be used to
screen for drugs, e.g., drugs for Parkinson's disease, or as a
means to identify biological mechanisms.
[0204] Screening of test compounds may also be conducted in
iPSC-derived cells when an abnormal cellular phenotype (e.g.,
abnormal cell morphology, gene expression, or signaling),
associated with a health condition or a predisposition to the
health condition is known, but a drug target has not yet been
identified. Such assays may include contacting a test population of
iPSC-derived cells from one or more iPSC donors with a test
compound and contacting with a negative control compound a negative
control population of iPSC-derived cells from the same one or more
iPSC donors. The assayed cellular phenotype associated with the
health condition of interest in the test and negative control
populations can then be compared to a normal cellular phenotype.
Where the assayed cellular phenotype in the test population is
determined as being closer to a normal cellular phenotype than that
exhibited by the negative control population, the drug candidate
compound is identified as normalizing the phenotype. A normal
cellular phenotype with respect to a particular health condition or
a predisposition for a health condition may be established in
iPSC-derived cells from iPSC donors that do not suffer from the
health condition or a predisposition for the health condition.
[0205] Test compounds identified as lead compounds, may be tested
on a panel of iPSC-derived cells in a manner analogous to a
clinical trial. In some cases, the efficacy of the lead compound
versus a negative control compound, e.g., a placebo compound is
determined in a panel of iPSC-derived cells from patients suffering
from the same health condition. Preferably, such a panel of
iPSC-derived cells is from subjects that are genetically diverse.
For example, such patients may be carry at least one polymorphic
allele that is unique among the iPSC-derived cells to be included
in the panel, e.g., 5 to 10, 20 to 50, 50 to 200, 200 to 500, 500
to 1000, 1000 to 5000, 5000 to 20000, or 20000 to 50000 polymorphic
alleles that are unique within the panel of iPSC lines. A number of
methods for quantifying the genetic diversity of a population are
known in the art, e.g., the analysis of molecular variance (AMOVA)
and generalized analysis of molecular variance (GAMOVA). See, e.g.,
Excoffier et al (1992), Genetics, 131: 479-491; Nievergelt et al
(2008), PLOS Genetics, 3(4):e51. Various clinical experimental
designs known in the art may be used for comparing the effect of a
lead compound versus a negative control compound. See, e.g., Chow
et al (2004) "Design and Analysis of Clinical Trials: Concepts and
Methodologies," John Wiley & Sons, Inc., Hoboken, N.J.
[0206] The efficacy of the lead compound in iPSC-derived cells may
be determined based on any cellular response endpoint, e.g., a
response obtained in any of the cell-based assays or gene
expression profiling assays mentioned herein.
[0207] In some cases, potential adverse effects of a lead compound
are tested on a panel of iPSC-derived cells. The iPSC-derived cells
may include any cell type that hepatocytes, cardiomyocytes,
neurons,
[0208] Drug candidate compounds may be individual small molecules
of choice (e.g., a lead compound from a previous drug screen) or in
some cases, the drug candidate compounds to be screened come from a
combinatorial library, i.e., a collection of diverse chemical
compounds generated by either chemical synthesis or biological
synthesis by combining a number of chemical "building blocks." For
example, a linear combinatorial chemical library such as a
polypeptide library is formed by combining a set of chemical
building blocks called amino acids in every possible way for a
given compound length (i.e., the number of amino acids in a
polypeptide compound). Millions of chemical compounds can be
synthesized through such combinatorial mixing of chemical building
blocks. Indeed, theoretically, the systematic, combinatorial mixing
of 100 interchangeable chemical building blocks results in the
synthesis of 100 million tetrameric compounds or 10 billion
pentameric compounds. See, e.g., Gallop et al. (1994), J. Med.
Chem. 37(9), 1233. Preparation and screening of combinatorial
chemical libraries are well known in the art. Combinatorial
chemical libraries include, but are not limited to: diversomers
such as hydantoins, benzodiazepines, and dipeptides, as described
in, e.g., Hobbs et al. (1993), Proc. Natl. Acad. Sci. U.S.A. 90,
6909; analogous organic syntheses of small compound libraries, as
described in Chen et al. (1994), J. Amer. Chem. Soc., 116: 2661;
Oligocarbamates, as described in Cho, et al. (1993), Science 261,
1303; peptidyl phosphonates, as described in Campbell et al.
(1994), J. Org. Chem., 59: 658; and small organic molecule
libraries containing, e.g., thiazolidinones and metathiazanones
(U.S. Pat. No. 5,549,974), pyrrolidines (U.S. Pat. Nos. 5,525,735
and 5,519,134), benzodiazepines (U.S. Pat. No. 5,288,514).
[0209] Numerous combinatorial libraries are commercially available
from, e.g., ComGenex (Princeton, N.J.); Asinex (Moscow, Russia);
Tripos, Inc. (St. Louis, Mo.); ChemStar, Ltd. (Moscow, Russia); 3D
Pharmaceuticals (Exton, Pa.); and Martek Biosciences (Columbia,
Md.).
B. Individualized Drug Therapy and Failed Drug "Rescue"
[0210] iPSC cell lines and iPSC-derived cells generated from a
subject (e.g., a human subject) can be used to determine the
likelihood that a particular drug will have sufficient efficacy in
that subject and, if so, an appropriate dose range for that
subject. This process is illustrated schematically in FIG. 3.
iPSC-derived cells from a subject, e.g., differentiated
iPSC-derived cells may be exposed ex vivo to a drug to be tested,
and then assayed for their phenotypic response to the drug as
described herein. The response of the iPSC-derived cells may be
compared to a reference response obtained in iPSC-derived cells
from one or more individuals in which the drug has been shown to be
effective and/or a reference response in iPSC-derived cells from
subjects in which the drug was found to be ineffective. In some
cases, the subject to be tested is a subject suffering from a
health condition or a predisposition to the health condition. For
example, where the subject is suffering from a health condition,
and multiple drugs are available to treat the health condition, the
efficacies and adverse effects of the multiple drugs may be
evaluated iPSC-derived cells from that individual. Preferably, the
iPSC-derived cells used to test drug efficacy include cells that
express at least one drug target (e.g., a neurotransmitter
receptor). In other cases, the subject is not suffering from a
health condition. In one embodiment, drugs for various health
conditions are tested preemptively in iPSC-derived cells from a
healthy subject to establish a pharmaco-phenomic profile for that
subject. The pharmaco-phenomic profile may subsequently be used as
needed for selecting optimal drugs and drug dosing for treatment of
the particular subject.
C. Disease Pathway and Target Discovery
[0211] For many diseases, especially those that have primarily a
sporadic form (e.g., Parkinson's disease), the underlying cellular
phenotype(s) that precede and eventually result in pathology are
unknown. In fact, for progressive degenerative conditions, it is
likely that a causative or predictive cellular phenotype occurs
well before the first manifestation of symptoms. However, for many
types of diseases the relevant cells (e.g., neurons,
cardiomyocytes, and pancreatic cells) are not directly accessible
for analysis. Thus, depending on the cell type affected by a
particular disease, it has not been possible to compare live cells
from patients to those of normal subjects in order to identify
disease-relevant, cellular phenotypes that cause or predispose for
a disease. Identification of reproducible cellular phenotype
differences between patient iPSC-derived and normal subject
iPSC-derived cells allows the development of screening assays to
identify candidate therapeutic agents. Candidate therapeutic agents
are those that normalize a disease-associated cellular phenotype,
i.e., alter the relevant cellular phenotype in the patient-derived
cells so that it is closer to the corresponding cellular phenotype
in cells derived from normal subjects under the same conditions.
Alternatively, the therapeutic agent may alter a cellular phenotype
of \patient-derived iPSCs so as to protect them from a stressor, as
described herein.
[0212] Sets of data representing various cellular phenotypes (e.g.,
mitochondrial ROS production, expression profiles, protein
aggregation) in patient iPSC-derived cells versus normal subject
iPSC-derived cells constitute vectors in a multidimensional space,
amenable to analysis by means of multivariate and univariate
statistical and machine learning techniques. Thus cellular
phenotypes distinguishing patient versus normal subject can be
identified, for example, by means of univariate statistical
methods, such as t-test, ANOVA, regression, as well as their
non-parametric analogs. In some embodiments, cellular phenotype
data are further filtered using various statistical criteria, e.g.,
p-value of significance (Type1 error), effect size, etc. Sets of
cellular phenotypes which differ significantly between disease and
normal states are further scrutinized by biological pathways
analysis. In many cases, a pathway enrichment analysis is performed
to further narrow the set of cellular phenotypes which are the most
disease-informative. A number of statistical procedures such as
Hypergeometric statistic, Kolmogorov-Smirnoff test, etc, can be
used to perform pathway enrichment analysis.
[0213] In some cases, cellular phenotypes that are found to differ
significantly in patient versus normal subjects (i.e.,
disease-relevant cellular phenotypes) need to be validated by means
of orthogonal assays. In other cases, the identified
disease-relevant cellular phenotypes are confirmed by performing
validation/cross-validation analysis on the independent data sets
from the same type of cellular phenotype assays. In some
embodiments, disease-relevant cellular phenotypes are determined by
first assaying and analyzing only a portion of the available
patient and normal iPSC lines, and then validating disease-relevant
cellular phenotypes in the remaining iPSC lines. In other
embodiments, where it is not feasible to utilize independent sets
of iPSC lines for disease-relevant cellular phenotype discovery
versus validation, other statistical approaches, such as k-fold
cross-validation techniques, are used instead. In some cases, one
or more validated cellular phenotypes is then used to assess test
agents for their ability to convert one or more cellular phenotypes
reflecting a disease condition to cellular phenotypes reflecting a
normal condition.
[0214] Where the disease under study is a progressive condition
with a potentially late onset (e.g., age 60 and over), selection of
"normal" control subjects is non-trivial, as it is usually not
possible to know, prospectively, who will develop a progressive
degenerative disorder. In other words, subjects that are apparently
normal at a given age/time point (e.g., when a biopsy is obtained
for iPSC derivation) may eventually develop the disease for which
an associated cellular phenotype is sought. Thus, cells derived
from such a subject would not be a valid "normal" control.
Accordingly, in some embodiments, rather than selecting an
age-matched normal control subject, a "wellderly" subject is
selected for normal control iPSC derivation. As used herein, a
"wellderly" subject refers to any subject that is at least 80 years
old and has not suffered from any major chronic diseases. Selection
of wellderly individuals as normal control subjects makes it
statistically less likely that such individuals will go on to
develop a degenerative condition. Thus, iPSCs derived from such
individuals are less likely to exhibit a cellular phenotype that is
associated or predictive of the disease being analyzed, and
therefore provide a more reliable "normal control" phenotype for
purposes of comparison to patient-derived iPSCs and iPSC-derived
cells. In other embodiments, elderly individuals are selected for
control iPSC generation that while not having suffered from a
degenerative disease under study, may have suffered other unrelated
degenerative diseases. In other cases, age-matched subjects free of
the disease to be analyzed are used to generate normal control
subject iPSCs.
[0215] In some cases, once a candidate therapeutic agent has been
identified as effectively normalizing a cellular phenotype in a
small number of patient iPSC lines and cells derived therefrom,
efficacy is tested in larger panels of patient iPSC-derived cells
to identify potential variation in efficacy or toxicity of the
candidate therapeutic agent. In some cases, efficacy is tested in
iPSCs or iPSC-derived cells from at least about 20 to about 500
patients, e.g., at least about 25, 30, 40, 50, 60, 70, 100, 200,
250, 300, 400, or another number of patients from at least about 20
to about 500 patients. In some embodiments, biomarkers associated
with responsiveness to a candidate therapeutic agent or lack of
responsiveness to a candidate therapeutic agent are identified and
used to stratify a patient population into, e.g., "high responders"
(HR) and "low responders" (LR), as schematized in FIG. 4. In some
cases, biomarkers are used to identify suitable patients for
clinical trials of a candidate therapeutic agent. In other cases,
biomarkers are used to predict the responsiveness or potential
toxicity of a therapeutic agent for particular patients. In some
cases, biomarkers include genomic biomarkers (e.g., SNPs, a CNVs,
or other genetic polymorphisms). In other cases, the biomarkers
include an expression profile signature (e.g., an mRNA expression
profile). The biomarkers may include a protein expression profile
or even a single protein expression level. In some cases, where the
biomarkers are expression profile biomarkers, these may be
determined directly from a patient sample (e.g., blood, urine,
sputum, hair, skin, or other biological sample taken directly from
the patient). In other embodiments, expression profile biomarkers,
are specific to patient iPSCs or iPSC-derived cells in which the
candidate therapeutic agent or therapeutic agent is tested.
[0216] Thus, in some embodiments, iPSCs are derived from patients
and control subjects, and the iPSCs are differentiated into
disease-relevant cell types thereby allowing a comparison of
cellular phenotypes in patient-derived cells versus normal subject
derived cells. For example, the cellular phenotype that is compared
may include a mitochondrial phenotype, e.g., ATP synthesis, ATP/ADP
ratio, mitochondrial potential, calcium buffering, production of
reactive oxygen species, mitochondrial fusion and fission,
mitochondrial morphology, and mitochondrial movement. In other
cases, the cellular phenotype that is compared is the fraction and
rate at which a particular cell type is undergoing apoptosis in the
presence of a stressor. In some embodiments, the cellular phenotype
is protein aggregation (e.g., the formation of lewy bodies). In
other embodiments, gene expression (e.g., microRNA expression) is
compared between patient-derived cells and normal subjects.
[0217] In some cases, relational databases are constructed that
integrate multiple data streams relating to each patient and
control iPSC line. These data include, but are not limited to, one
or more of the following: patient medical history and family
medical history, patient medical data (e.g., blood pressure, liver
enzyme levels), patient adverse drug reactions, patient drug
responsiveness, partial or complete genomic sequence, sequence of
all genes with known disease-associated alleles, comprehensive SNP
genotypes (e.g., genotypes for all SNPs with known disease
associations), gene copy number variation (CNV) polymorphisms,
expression profiles for iPSCs and for cells differentiated from the
iPSCs (e.g., dopaminergic neurons, cortical neurons, motor neurons,
pancreatic cells, hepatocytes, cardiomyocytes, and vascular
epithelial cells) under resting and under various stimulus
paradigms (e.g., in the presence of a stressor), all cellular
phenotype assay data used for initial pathway discovery and for
drug screening, including, e.g., cellular phenotype data in the
presence or absence of test compounds and compounds with known
pharmacological properties (e.g., a cholinesterase inhibitor, a
receptor ligand, a kinase inhibitor etc.). In some embodiments, a
user can query such a database based on any set of criteria with
user define limits. For example, a user may wish to identify
polymorphisms associated with patients whose iPSC-derived
dopaminergic neurons did or did not respond to a candidate
therapeutic agent. In another example, a user may wish to identify
a common gene expression profile that distinguishes motor neurons
that showed a severe apoptotic response to a stressor versus a mild
apoptotic response, etc. Such databases are very useful for data
mining and establishing robustly predictive signatures for specific
disease states and their response to candidate therapeutic
agents.
EXAMPLES
Example 1
Generation of iPSC Lines from Patients Suffering from Spinal
Muscular Atrophy
[0218] Spinal Muscular Atrophy (SMA) is a neuromuscular disease
characterized by degeneration of motor neurons that is among the
leading causes of childhood paralysis and mortality. The disease
exhibits a wide range of severity affecting infants through adults,
and is subdivided into types I-IV based on the age of onset and
severity of symptoms: Type I "Infantile" onset at ages 0-6 months
and generally fatal); Type II "Intermediate," onset at ages 7-15
months; inability to stand or walk, but some ability to maintain a
sitting position; Type III "Juvenile" onset at ages 18 months to 17
years, with some ability to walk, though potentially transient;
Type IV "Adult," some muscle weakness, but no genetic basis is
known.
[0219] The molecular basis of SMA is linked to the Survival Motor
Neuron (SMN) gene. The region of chromosome 5 that contains the SMN
(survival motor neuron) gene has a large duplication. A large
sequence that contains several genes occurs twice--i.e. once in
each of the adjacent segments. The two copies of the gene--known as
SMN1 and SMN2-differ by only a few base pairs. The SMN2 gene
contains a mutation that occurs at the splice junction of intron 6
to exon 7 resulting in about 90% of SMN2 pre-mRNA transcripts being
spliced into a form that excludes exon 7. This shorter mRNA
transcript codes for a truncated SMN protein, which is rapidly
degraded. About 10% of pre-mRNA transcript from SMN2 is spliced
into the full length transcript that codes for the fully functional
SMN protein. This splicing defect occurs in multiple cell types,
although, for unknown reasons, the survival of motor neurons appear
to be particularly affected.
[0220] SMA results from the loss of the SMN1 gene from both
chromosomes, and its severity, ranging from SMA 1 to SMA 3, largely
depends on whether the level of SMN2.sup.E7 transcript can make up
for low levels or absence of exon 7-inclusive SMN 1 transcript. The
mutations that cause the loss of SMN 1 are of two types. Deletion
mutations, in which both copies of the SMN1 are missing. The other
type of mutation is a conversion mutation in which both copies of
the SMN1 gene have a point mutation resulting in the same splicing
pattern as the SMN2 gene. As an initial step towards developing an
in vitro assay for identifying molecules that can increase levels
of exon 7-inclusive SMN2 (SMN2.sup.E7) transcript, we generated
several iPSC lines from Coriell fibroblast lines established from
three SMN1.sup.-/- SMA patients and from two healthy SMN1.sup.-/+
subjects.
[0221] Induction of iPSCs was initiated by transduction of
SMN1.sup.-/- and SMN1.sup.-/+ fibroblast cultures with four MoMLV
VSV-G-pseudotyped viruses for expression of human OCT4, SOX2, KLF4,
and c-MYC, each at an MOI of about 10. Five days after viral
transduction, fibroblasts were switched from human fibroblast
medium into human ES cell supportive medium and monitored daily for
the appearance of putative iPSC colonies based on morphological
criteria.
[0222] Initial putative SMA-iPSC colonies were picked after
approximately three weeks and propagated clonally in the presence
the presence of the ROCK inhibitor Y-27632 (10 .mu.M) Calbiochem)
to derive the SMN1.sup.-/- iPSC lines SM4p, SM7t, and SM8c, and the
SMN1.sup.-/+ iPSC lines SM9a and SM10d, as shown in FIG. 5. Each of
the iPSCs expressed the pluripotency associated markers, Nanog,
Oct4, SSEA3, SSEA4, TRA1-60, and TRA1-81 (data not shown) as
determined by immunocytochemistry. Q-PCR analysis showed that these
iPSC lines expressed endogenous Oct 4, Sox2, and Klf4, but not the
exogenous Oct4, Sox2, and Klf4 introduced by viral transduction. In
addition, Q-PCR analysis also demonstrated expression of Nanog,
SSEA-3, SSEA-4, TRA1-60, TRA1-81, DNMT3B, FOXD3, LIN28, ZNF206,
LEFT2, TDGF1, and TDGF2 in all of the iPSC lines (data not shown).
Importantly, all of the SMA iPSC lines were able to form embryoid
bodies (EBs) as shown in FIG. 6, which indicated that these lines
had good potential for differentiation as is expected for iPSCs.
Indeed, the ability of the SM8c line to differentiate into
ectodermal, mesodermal, and endodermal lineages in vitro was
confirmed by immunostaining for the ectodermal marker Tun, the
mesodermal marker Desmin, and the endodermal marker AFP, as shown
in FIG. 7. Further, the SM8c iPSC line was shown to differentiate
into mature motor neurons as shown by double immunolabeling for
Islet and Neuro-N (data not shown).
[0223] Based on these results, we concluded that iPSCs can be
generated from SMA patients and differentiated into motor neurons,
as required for the screening assay described in Example 2.
Example 2
Assay for Identification of Molecules that Improve Molecular and
Cellular Disease Phenotypes in Motor Neurons from Patients
Suffering from Spinal Muscular Atrophy
[0224] We seek to identify molecules that increase the level of
SMN2.sup.E7 transcript in motor neurons derived from patients
suffering from SMA. In principle, increased levels of SMN2.sup.E7
transcript can be increased by boosting SMN2 transcription,
reducing degradation of SMN2 mRNA, or by increasing the fraction of
SMN2 pre-mRNA that is spliced into SMN2.sup.E7 mRNA. SMA
patient-specific motor neurons are obtained by first generating
panels of iPS cell lines from Type I, Type II, and Type III SMA
patients, as described in Example 1, and subsequently
differentiating iPSCs into motor neurons. Prior to motor neuron
differentiation SMA patient SMN2 minigene reporter iPSC lines are
established to provide a convenient readout for the level of
SMN2.sup.E7 transcript in motor neurons.
[0225] Following parental informed consent, standard dermal punch
biopsies 2-4 mm in diameter and thickness are obtained from
approximately 30 Type I, 30 Type II, and 30 Type III SMA patients,
all of whom have an SMA1.sup.-/- genotype, and 10 healthy,
age-matched control subjects that have an SMA1.sup.-/+ genotype.
For each SMA-iPSC line to be generated, the following corresponding
patient information is collected and annotated in an iPSC line
database: disease severity ranking (i.e., Type I, II, or III), age
of disease onset, patient medical history, family medical history
including incidence of ALS, blood level of SMN protein, SMN1 and
SMN2 genotypes, MUNE Motor Unit Number Estimation, Hammersmith SMA
Functional Motor Scale ranking, breathing test evaluation (only for
children >5 yrs), symptom progression evaluation (e.g., how
outcome of motor tests has changed over time), muscle mass index,
description of therapeutic interventions to date, and therapy
response. Additional data may be added to each record as they are
acquired, including, e.g., SMN protein levels and SMN2.sup.E7
transcript levels under various experimental conditions (e.g., in
the presence or absence of a candidate therapeutic compound),
informative SNP genotypes, genomic sequence, and tissue/cell-type
specific expression profiles.
[0226] Biopsy samples are stored for up to 5-7 days at 4.degree. C.
in a "biopsy medium" containing KO-DMEM and supplemented with 10%
fetal bovine serum (FBS), Earl's Salts, nucleosides,
beta-mercaptoethanol (BME), non-essential amino acids, glutamine,
and penicillin/streptomycin. Biopsies are minced into 4-5 pieces,
and the pieces are then transferred to a 60 mm dish. The pieces are
then "sandwiched" under an acid-washed coverslip and cultured in
biopsy medium for five days. Subsequently, the sandwiched biopsy
explants are cultured in human fibroblast ("hFib") medium
containing KO-DMEM, Earl's Salts, 10% FBS, glutamine,
penicillin/streptomycin, and medium is replaced every 3-4 days
until the coverslip is confluent. SMA iPSCs are generated, as
described in Example 1, from fibroblasts obtained from each
biopsy.
[0227] An SMN2 splicing minigene reporter construct is generated
that incorporates exons 6, 7, and 8, and utilizing the SMN2
promoter is generated essentially as described in Zhang et al
(2001), Gene Ther., 8:1532-1538 and Wilson et al, Stem Cells and
Development, 16:1027-1041. The SMN2 reporter construct will
incorporate the DD-AmCyan1 fluorescent protein reporter to maximize
the signal to noise ratio in a compound screening reporter gene
assay. The DD-AmCyan1 protein contains a degradation ("DD") domain
that conditionally destabilizes the protein thereby keeping
"background" levels of the reporter protein prior to a test
compound screening assay very low. However, upon addition of the
cell-permeable "Shield1" ligand (Invitrogen), which selectively
binds to the DD domain, the reporter protein is stabilized and can
therefore accumulate. Thus, potential differences in DD-AmCyan1
reporter levels in the presence or absence of test compounds are
maximized by measuring almost exclusively reporter protein produced
after the beginning of the screening assay, i.e., after the
addition of the Shield1 ligand and test compound. Additional
reporter constructs will include AmCyan1 or luciferase as the
reporter. Other constructs will include the CMV promoter to drive
SMN2 minigene expression. The SMN2 reporter construct is then
stably transfected into type I, type II, and type III SMA-iPSCs and
healthy control (SMN1.sup.WT/WT) iPSCs to generate SMN2-reporter
SMA-iPSC lines of varying disease severity backgrounds, and SMN2
reporter control iPSC lines, respectively. Primary screening of
test compounds for the ability to increase properly spliced SMN2
transcript levels is conducted initially in motor neurons derived
from Type I SMA reporter iPSCs.
[0228] On day 0, confluent 10 cm plates of SMN2 reporter SMA-iPSCs
are trypsinized and then washed/resuspended in embryoid body (EB)
medium containing KO DMEM (Invitrogen, catalog #10829-018),
Knockout Serum Replacement (Invitrogen, catalog# A1099202),
Plasmanate (Talecris), Glutamax (Invitrogen, catalog# 35050079),
non-essential amino acids (Invitrogen, cat#11140050). After washing
and resuspension, the cells are plated in ultra-low attachment
(ULF) 6-well plates and grown into EBs over the next 4-5 days. On
day 5, EBs are washed, gently resuspended in EB medium, and
replated in a new ULA E-well plate, and the wash/replate procedure
is repeated on day 8 or 9. On day 11, EBs are collected and
resuspended in N2 base medium (DMEM/F12, Glutamax (Invitrogen,
catalog#10565), N-2 Supplement (Invitrogen, catalog#17502-048),
D-Glucose (Sigma, catalog # G8769), Ascorbic Acid (Sigma, catalog#
A4403-100mG)) supplemented with 1 .mu.M Retinoic Acid (RA) and 100
nM Purmorphamine. (PM). On day 14, EBs are transferred to in N2
Base medium+1 .mu.M RA+1 .mu.M Purmorphamine and replated (3 ml of
EB suspension/well) on ULA 6 well plates. N2 base
[0229] The RA (1 .mu.M)/PM (1 .mu.M)-supplemented EB medium is
replaced every 3-5 days, as needed, until approximately day 28.
Afterwards, EBs are dissociated by dilute papain treatment and
gentle trituration, and then replated on new ULF 6-well plates
followed by gentle trituration every 10 minutes over a period of 45
minutes. After dissociation, the resulting cell suspension is
collected and transferred to a 50 ml conical tube containing motor
neuron maturation medium (DMEM/F12, Glutamax, N-2 Supplement
(Invitrogen, catalog#17502-048), B-27 Supplement (Invitrogen,
catalog# 17504-044), D-Glucose, Ascorbic Acid (Sigma, catalog#
A4403-100mG), 2 ng/mL each GDNF (R&D, catalog#212-GD), BDNF
(R&D, catalog#248-BD), and CNTF (R&D, catalog#257-NT/CF).
The cell suspension is pelleted by centrifugation at 1000 RPM for
five minutes, and is then resuspended in motor neuron maturation
medium at a cell concentration of approximately 1.6.times.10.sup.6
cells/ml. Aliquots (50 .mu.l) of cell suspension are then plated on
laminin-coated wells of optical grade 96 well plates. Beginning on
day 31, half-medium changes are conducted every other day or every
day depending on how quickly the medium becomes spent. The
differentiated cultures are maintained in motor neuron
differentiation medium for another four weeks prior to beginning
SMN2-reporter assays to allow expansion and maturation of the motor
neuron population.
[0230] At the beginning of the screening assay, all wells of
96-well plate mature motor neuron (MMN) cultures are incubated in
the presence of Shield1 is at a final concentration of 1 .mu.M.
Test wells are incubated in the presence of test compounds from the
NIH Clinical Collection Library (available from BioFocus DPI) at a
final concentration of 50 .mu.M. Negative control wells receive no
addition or are incubated with a vehicle compound (e.g., DMSO) at a
concentration equivalent to that present in some of the test
compound solutions. Positive control wells are incubated in the
presence of sodium vanadate (50 .mu.M), which has previously been
shown to significantly increase levels of SMN2.sup.E7 transcript
(Zhang et al (2001), Gene Therapy, 8, 1532-1538). After incubation
for 24 hours, cultures are fixed and processed for
immunofluorescence detection of Islet 1/2 (mature motor neurons)
and Olig2 (motor neuron progenitors) and DD-AmCyan1 fluorescence
levels are imaged and quantified in Islet 1/2.sup.+ and Olig2.sup.+
cells. Compounds that increase SMN2 reporter levels ("candidate
therapeutic" compounds) are screened in secondary assays for their
ability to increase SMN2.sup.E7 transcript levels and for their
ability to promote SMA motor neuron survival over a period of about
two weeks. Candidate therapeutic compounds are then tested on motor
neurons derived from additional type I SMA SMN2-reporter iPSC
lines, and from type II and type III iPSC lines to validate the
effect of the therapeutic candidate compounds on motor neurons from
diverse genetic backgrounds extant in the SMA patient
population.
[0231] It is expected that identification of compounds that
increase the net level SMN2.sup.E7 transcript in patient-derived
motor neurons is likely to be more relevant for identification of
therapeutic drug candidates for SMA than a similar assay in cell
types relatively unaffected or less affected by loss of SMN1 (e.g.,
fibroblasts) or heterologous cell lines.
Example 3
Generation of iPSC Lines iPSCs from Patients with Idiopathic
Parkinson's Disease and Defined Mutations in Genes Associated with
Parkinson's Disease
[0232] Parkinson's Disease (PD) is one of the most common
neurodegenerative diseases of aging, affecting 1-2% of the
population over 65 years of age. Clinical symptoms include rest
tremor, bradykinesia, and rigidity. We seek to generate a PD
patient iPSC model to identify candidate therapeutic agents that
slow, halt, or reverse PD progression.
[0233] iPSC lines are generated from skin biopsies obtained from 10
healthy control subjects with no known family history of PD, 10
patients with sporadic PD, patients each with mutations in the
genes that encode .alpha.-synuclein (PARK1), parkin (PARK2), PINK 1
(PARK6), or LRRK2 (PARKS) for a total of 10 patients for each
mutation. iPSCs are generated as described in Example 1.
Afterwards, dopaminergic neurons are derived by differentiating
each of the patient iPSC lines and control subject iPSCs. A
dopaminergic phenotype is established by immunocytochemical
staining for tyrosine hydroxylase positivity, and assaying the
differentiated cells for the ability to synthesize and release
dopamine. Dopaminergic neurons are obtained by differentiating the
iPSCs according to the method of Perrier et al (2004), Proc Natl
Acad Sci USA 101, 12543-12548. After validating the dopaminergic
phenotype of neurons differentiated from each of the above iPSC
lines, cultures of the patient iPSC-derived dopaminergic neurons
are tested in a battery of cellular phenotype assays and compared
to control subject dopaminergic neurons. These are: assays for
aggregation of .alpha.-synuclein, dopaminergic neuron apoptosis
(TUNEL, caspase activation) and necrosis (CytoTox-Glo), oxidative
stress indicators (glutathione levels, ROS, and 4-HNE), and
mitochondrial dysfunction (ATP content, membrane potential,
morphology, and calcium buffering). It is expected that sporadic
forms of PD and PD caused by the above-mentioned mutations will
exhibit very similar dopaminergic cellular phenotypes in at least
some of these assays. Once this is established, one of more of the
PD-associated cellular phenotypes is used as the basis of a screen
for candidate therapeutic agents that can reverse or ameliorate
these cellular phenotypes. Further, it is expected that as the PD
cellular phenotypes are identified in disease relevant cells
(dopaminergic neurons) from human PD patients, their predictive
value and reliability for the development of therapeutic agents
will be more robust than those based on heterologous assay
models.
[0234] While preferred embodiments of the present invention have
been shown and described herein, it will be apparent to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
5111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5
1028PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Arg Lys Lys Arg Arg Gln Arg Arg1
5311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala1 5
10411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Thr His Arg Leu Pro Arg Arg Arg Arg Arg Arg1 5
10511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Gly Gly Arg Arg Ala Arg Arg Arg Arg Arg Arg1 5
10
* * * * *
References