U.S. patent application number 15/299927 was filed with the patent office on 2017-04-13 for supercentenarian induced pluripotent stem (scips) cells and methods of making and using thereof.
The applicant listed for this patent is Mandala Biosciences, LLC. Invention is credited to David J. Larocca.
Application Number | 20170101627 15/299927 |
Document ID | / |
Family ID | 50100446 |
Filed Date | 2017-04-13 |
United States Patent
Application |
20170101627 |
Kind Code |
A1 |
Larocca; David J. |
April 13, 2017 |
SUPERCENTENARIAN INDUCED PLURIPOTENT STEM (sciPS) CELLS AND METHODS
OF MAKING AND USING THEREOF
Abstract
Provided herein are cells and methods for reprogramming iPS
cells from a supercentanarian and their differentiated derivatives
having differences from non-supercentenarian iPS derived cells that
contribute to disease resistance and longevity. Additionally,
provided herein are methods for treatment and prevention of age
related diseases by administration of therapeutic sciPS derived
cells or cell derived reagents. Also provided herein, are methods
for identifying reagents for treatment of age related diseases
using sciPS cell-based assays.
Inventors: |
Larocca; David J.;
(Carlsbad, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mandala Biosciences, LLC |
Alameda |
CA |
US |
|
|
Family ID: |
50100446 |
Appl. No.: |
15/299927 |
Filed: |
October 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13968587 |
Aug 16, 2013 |
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15299927 |
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61684047 |
Aug 16, 2012 |
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61825053 |
May 19, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5044 20130101;
C12N 5/0696 20130101; C12N 2506/1346 20130101; G01N 33/5073
20130101; C12N 2506/11 20130101 |
International
Class: |
C12N 5/074 20060101
C12N005/074; G01N 33/50 20060101 G01N033/50 |
Claims
1-20. (canceled)
21. A method of generating reprogrammed supercentenarian stem cells
capable of differentiating into stem cells with a slower rate of
accumulation of cellular aging changes; the method comprising: a.
collecting a cell sample from a validated supercentenarian
individual; b. reprogramming cells from the cell sample into
supercentenarian induced pluripotent (iPS) cells; and c.
identifying reprogrammed supercentenarian induced pluripotent stem
(sciPS) cells which are capable of differentiating into cells which
exhibit decelerated replicative aging as compared to stem cells
from iPS cells from a non-supercentenarian donor.
22. The method of claim 21, further comprising: d. deriving stems
cells from the sciPS which exhibit decelerated replicative aging as
compared to stem cells from iPS cells from a non-supercentenarian
donor, thereby generating stem cells having a slower rate of
accumulation of cellular aging changes as compared to stem cells
from iPS cells from a non-supercentenarian donor.
23. The method of claim 21, wherein the cellular aging changes are
selected from the group consisting of shortened telomeres, loss of
differentiation capacity, changes in differentiation propensity,
changes in genomic methylation, changes in genome expression
pattern, morphological changes, and expression of senescence
associated markers and gene expression.
24. The method of claim 21, wherein the sciPS cells exhibit
telomere length resetting towards embryonic length.
25. The method of claim 21, wherein the supercentenarian individual
is a human.
26. The method of claim 21, wherein the stem cells are mesenchymal
stromal cells (MSC) or hematopoietic stem cells (HSC).
27. The method of claim 21, wherein cells from the cell sample are
selected from the group consisting of blood cells, dermal
fibroblasts, adipose cells and hair follicle cells.
28. An isolated population of stem cells differentiated from
supercentenarian iPS cells (sciPS), wherein the stem cells exhibit
a slower rate of accumulation of cellular aging changes as compared
to stem cells from iPS cells from a non-supercentenarian.
29. The isolated stem cells of claim 28, wherein the cellular aging
changes are selected from the group consisting of shortened
telomeres, loss of differentiation capacity, changes in
differentiation propensity, changes in genomic methylation, changes
in genome expression pattern, morphological changes, and expression
of senescence associated markers and gene expression.
30. The isolated stem cells of claim 28, wherein the iPS cells
exhibit telomere length resetting towards embryonic length.
31. The isolated stem cells of claim 28, which are human stem
cells.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/684,047, filed Aug. 16, 2012; and U.S.
Provisional Application No. 61/825,053, filed May 19, 2013, each
disclosure of which is hereby incorporated herein by reference in
its entirety. In addition, all documents and references cited
herein and in the above referenced applications, are hereby
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of pluripotent
stem cells and cells derived therefrom; such as through
reprogramming techniques, that have a genotype associated with
extreme human longevity; and their uses for treatment and
prevention of age related degenerative diseases and for conferring
longevity to non-supercentenarians.
[0003] The present invention also relates to the field of
techniques for identification, development, and/or generation of
developmentally regulated genes, proteins, stem cell antigens,
novel stem cell antibodies, cell markers, a bank of novel
antibodies against surface markers on stem cells, and/or antibodies
for identification and characterization of progenitor cell
populations.
BACKGROUND OF THE DISCLOSURE
[0004] Extremely long lived humans, supercentenarians, present an
excellent model for studying the basis of resistance to
degenerative diseases associated with aging. Recent studies on a
large cohort aged 90-119 (including 102 supercentenarians) reveal a
progressive increase in the age of onset of degenerative diseases
with increasing age such that health span begins to approximate
lifespan in supercentenarians (Andersen et al. (2012) J Gerontol A
Biol Sci Med Sci 67: 395). Supercentenarians delay and/or escape
degenerative diseases including cancer, cardiovascular disease,
dementia, hypertension, stroke, and osteoporosis. Exceptional
overall disease resistance among supercentenarians is demonstrated
by a progressive compression of morbidity with increased age such
that supercentenarians experience only 5.22% years of morbidity
compared to 18% among random controls and 9% in centenarians
(Andersen et al. supra). Moreover, 70% of supercentenarians escape
debilitating disease entirely compared to 30% of 100-104 year olds
and 56% of 105-109 year olds (Andersen et al. supra). Taken
together these data indicate greater disease resistance in
supercentenarians compared to all other groups including
centenarians. There is a strong familial component to extreme
longevity and recent genome sequence analysis indicates a strong
genetic contribution to survival past 100 and this genetic
contribution increases with age (Perls et al. (2007) J Gerontol A
Biol Sci Med Sci 62: 1028) pointing to distinct genetic survival
advantages in supercentenarians over other groups including
centenarians. Genome Wide Association Studies (GWAS) indicate that
longevity is associated with a large number of SNPs (50% in
intragenic regions) and have identified genetic signatures among
90% of centenarians that have predictive value for longevity
(Sebastiani et al. (2012) PLoS One 7: e29848). Surprisingly, the
incidence of disease predisposing variants does not decline with
exceptional longevity. Taken together these data suggest a strong
genetic component to resistance to debilitating disease that
contributes to exceptional human longevity (Sebastiani P, Perls TT
(2012) Front Genet 3: 277). However, the genetic contribution to
survival to extreme age is complex, consisting of combinations of
many variants which individually have only minor to modest effects
(Sebastiani P supra). Therefore, the molecular and cellular basis
of the remarkable disease resistance in long-lived individuals is
difficult if not impossible to deduce from the genetics alone.
[0005] Human pluripotent stem cells, because of their ability to
both self-renew indefinitely and to differentiate into virtually
any cell type, have the potential to provide an unlimited source of
human cells and tissues for research, disease modeling, drug
development, and cell replacement therapies. The availability of
human pluripotent stem cells is no longer limited to embryonic stem
cell sources. Reprogramming technologies for converting somatic
cells to induced pluripotent stein (iPS) cells by the introduction
of defined factors (Takahashi K et al. (2007) Nat Protoc 2: 3081)
have greatly increased both the number and diversity of human
pluripotent cell lines available. Indeed, it may be possible to
obtain virtually any human cell type in a rejuvenated state by
reprogramming donor cells. Newer reprogramming methods and
preclinical studies have addressed initial concerns over the use of
early viral based reprogramming vectors (Okano et al. (2013) Circ
Res 112: 523). A rapidly growing application of iPS lines is their
use to create cellular models of disease from patient donor cells.
For example. Alzheimer's disease, Fanconi's anemia, ALS, and HG
progeria are a few of the diseases that have been modeled (Liu et
al. (2011) Nature 472: 221). Disease modeling with iPS cells can
give insight into the cellular and biochemical basis of disease and
provide cells for drug screening. However the current paradigm of
disease modeling with iPS cells has done little to increase
understanding of aging, the greatest risk factor for susceptibility
to degenerative diseases. The present invention provides a means of
using iPS cells for modeling and recapitulating the cellular basis
for resistance to disease that is exhibited by rare extremely
long-lived individuals, the supercentenarians.
[0006] Prior to the present invention, reprogramming of
supercentenarian donor cells has not previously been reported.
However, donor cells from individuals up to 109 years of age have
been successfully reprogrammed to pluripotency (Yagi et al. (2012)
PLoS One 7: e4172; Lapasset et al. (2011) Genes Dev 25: 2248). In
many cases, reprogramming has been found to reset telomeres back to
lengths approaching that seen in very young embryonic cells such
that the establishment and study of iPS derived cell types with
high replicative capacity is possible. However, this has not been
previously demonstrated for iPS cells derived from
supercentenarians.
[0007] Mesenchymal stem/stromal cells (MSCs) play a critical role
in the maintenance and repair of many tissues and in providing a
niche for hematopoietic stem cells. For example, decreased bone
mass seen in age related osteoporosis, a disease that
supercentenarians are resistant to, is thought to be at least in
part a result of a decrease in osteogenic stem cells (Bergman et
al. (1996) J Bone Miner Res 11:568: Jilka et al (1996) J Clin
Invest 97: 1732). Indeed, a role for cell autonomous MSC aging and
senescence in osteoporosis is indicated by the ability of
transplanted of MSCs to delay signs of skeletal aging and confer a
survival advantage in a mouse model of accelerated aging (Singh et
al. (2013) Stem Cells 31: 607). In addition to a loss of
proliferative capacity, there are changes in the differentiation
propensity from osteogenic to adipogenic that occurs as MSCs age
that may account for loss of bone in aging individuals (Moerman et
al. (2004) Aging Cell 3: 379; Jiang et al. (2008) J Orthop Res 26:
910). Transplantation of MSCs from young mice, but not old mice,
slows the loss of bone mass in aged mice (Shen et al. (2011) Sci
Rep 1: 67). Notably, transplantation of MSCs from young donors to
aged mice also results in prolonged life span indicating a role for
MSCs in overall longevity. Another indication of their repair and
maintenance function is that the transfer of MSCs from young mice
to aged mice can reverse the effects of aging on maintenance of
function in response to cardiac pressure overload (Sopko et al.
(2010) PLoS One 5: e15187). There is also evidence for a role of
bone marrow MSC exhaustion in obesity-induced diabetes (Chen et al.
(2009) Am J Pathol 17: 701). Thus, MSCs play an important role in
the prevention of age related disease and may contribute to
determining overall longevity.
[0008] Accordingly, a need exists in the art for further
investigation and analysis of cellular, molecular, and biochemical
properties of stem cells as it relates to their role in disease
prevention and longevity. The present invention provides
reprogrammed supercentenarian donor derived iPS cells and their
differentiated derivatives for identifying factors that impart
disease resistance and longevity. In particular, the present
invention provides the ability to obtain supercentenarian cells and
iPS derivatives with reset telomere length for comparing cellular
aging rates in iPS derived cell lines from donors of widely varied
ages. Such iPS derived cells are useful as cellular models for
understanding how the regulation of cellular aging in
supercentenarians contributes to their extreme human longevity and
resistance to disease. Additionally, a need exists for elucidating
cellular aging through reprogramming of rejuvenated MSCs derived
from supercentenarian iPS cells to compare the rate of cellular
aging to non-supercentenarian iPS cells. In this regard,
differences in proliferative and differentiation capacity with
cellular age can be measured and comparative genomic expression
analysis used to determine the molecular basis of these
differences. MSCs derived from supercentenarians exhibiting cell
autonomous differences from non-supercentenarian MSCs in their
cellular aging including changes in differentiation capacity would
thus be useful for cell replacement therapy and as tools for drug
discovery.
[0009] In addition, with the number of pluripotent stem cell lines
now increasing much more rapidly, it is even more important to
develop efficient cell characterization and directed
differentiation protocols. Our current knowledge of surface markers
that define the various cell types that differentiate from
pluripotent stem cells is still limited. Therefore, a need exists
for methods relating to the identification of novel developmentally
regulated genes, particularly taxonomically restricted genes, and
proteins and/or methods relating to the identification, isolation,
and differentiation of pluripotent cells and their derivatives.
[0010] All documents and references cited herein and in the
referenced patent documents, are hereby incorporated herein by
reference.
SUMMARY OF THE INVENTION
[0011] The present inventor has developed supercentenarian iPS
(sciPS) cells, cells derived therefrom; and methods of making and
using the sciPS cells and cells derived therefrom. The present
invention may be attributed to the fact that extremely long lived
humans, supercentenarians, exhibit a remarkable resistance to
degenerative diseases associated with aging. As discussed above,
data showing a strong familial component to extreme longevity taken
with data from the GWAS studies showing a predictive value for
longevity, suggest a strong genetic component to resistance to
debilitating disease that contributes to exceptional human
longevity. However, because the molecular and cellular basis of
this remarkable disease resistance is difficult if not impossible
to deduce from the genetics alone; the present invention employs
reprogramming techniques to elucidate molecular and cellular
factors relating to such disease resistance.
[0012] Therefore, the present invention utilizes reprogramming for
resetting of cellular age by restoring telomere length thus
allowing the comparison to be made with both sciPS and control iPS
derived cells starting at the equivalent cellular age.
Reprogramming donor cells from supercentenarians is used to yield
cells that display a disease resistance phenotype that is a
consequence of their extreme human longevity genotype. The sciPS
derived cells include stem cells for various tissues, for example,
neural stem cells, skin stem cells, vascular stem cells, blood stem
cells, pancreatic islet stem cells as well as mesenchymal stem
cells (MSCs; also known as mesenchymal stromal cells), a cell type
that plays an important role in maintaining and repairing multiple
human tissues such as bone, cartilage, tendon, and fat, as well as
providing a niche for blood stem cells. The sciPS derived cells
disclosed herein are used to confer disease resistance and
longevity to non-supercentenarians. Comparison of cellular aging of
sciPS derived cells such as (MSCs) to control iPS derived MSCs is
used to identify intracellular and secreted factors that confer
benefits of sciPS derived cells to non-supercentenarian cells. The
sciPS derived cells are used in cell-based screens to identify
candidate agents that confer sciPS derived cell advantages to
non-supercentenarian cells.
[0013] Accordingly, disclosed herein is a method of generating stem
cells having a reduced rate of cellular aging; the method
comprising collecting a cell sample from a validated
supercentenarian individual; reprogramming cells from the cell
sample into induced pluripotent (iPS) cells; identifying
supercentenarian induced pluripotent stem (sciPS) cells which
exhibit telomere length resetting; deriving stems cells which
exhibit telomere length resetting from the sciPS cells, thereby
generating stem cells having a reduced rate of cellular aging as
compared to stem cells from iPS cells from a non-supercentenarian
donor. In another embodiment, the sciPS cells exhibit full telomere
length resetting towards embryonic length. In another embodiment,
the supercentenarian individual is a human. In another embodiment,
the stem cells are mesenchymal stromal cells (MSC). In another
embodiment, the stem cells are hematopoietic stem cells (HSC). In
another embodiment, cells from the cell sample are selected from
the group consisting of blood cells, dermal fibroblasts, adipose
cells, and hair follicle cells.
[0014] Also disclosed herein is an isolated population of sciPS
cells comprising iPS cells from a supercentenarian individual,
wherein the iPS cells exhibit telomere length resetting. In another
embodiment, the iPS cells exhibit telomere length resetting towards
embryonic length. In another embodiment, the supercentenarian
individual is a human.
[0015] Also disclosed herein is an isolated population of stem
cells derived from sciPS cells; wherein the stem cells exhibit
telomere length resetting. In another embodiment, the stem cells
exhibit telomere length resetting towards embryonic length. In
another embodiment, the sciPS cells are human sciPS cells. In
another embodiment, the stem cells are MSCs. In another embodiment,
the stem cells are HSCs.
[0016] Additionally disclosed herein is a method of cell
replacement therapy conferring longevity and resistance to an
age-related disease in an individual in need of treatment; the
method comprising, transplanting stem cells exhibiting telomere
length resetting into the individual, wherein the stem cells are
from sciPS cells having a reduced rate of cellular aging as
compared to non-sciPS cells; and wherein the stem cells confer
longevity and resistance to the age-related disease onto the
individual; thereby treating the age-related disease in the
individual. In another embodiment, the age-related disease is
selected from the group consisting of osteoporosis, osteoarthritis,
cancer, heart disease, stroke, and neurological disorders. In
another embodiment, the individual is a human. In another
embodiment, the stem cells are human stem cells. In another
embodiment, the stem cells exhibit telomere length resetting
towards embryonic length. In another embodiment, the stem cells are
MSCs. In another embodiment, the stem cells are HSCs.
[0017] Disclosed herein is a method of age-related disease relevant
screening of candidate agents; the method comprising contacting the
candidate agent with a population of stem cells exhibiting telomere
length resetting, wherein the stem cells are from sciPS cells
having a reduced rate of cellular aging as compared to non-sciPS
cells; and determining the morphologic, genetic, or functional
effect of the candidate agent on the stem cells or on cells
differentiated therefrom. In another embodiment, the age-related
disease is selected from the group consisting of osteoporosis,
osteoarthritis, cancer, heart disease, stroke, and neurological
disorders. In another embodiment, the stem cells are human stem
cells. In another embodiment, the stem cells exhibit full telomere
length resetting towards embryonic length. In another embodiment,
the stem cells are MSCs. In another embodiment, the stem cells are
HSCs. In another embodiment, the candidate agent is selected from
the group consisting of biologics, small molecules, drugs,
nutraceuticals, cosmeceuticals, compounds, and reagents.
[0018] Also disclosed herein is a method of identifying substances
capable of tissue homeostasis and immune function regulation; the
method comprising culturing sciPS and stem cells therefrom in
growth media; and identifying substances in the growth media or in
cell extracts. In another embodiment, the sciPS cells and stem
cells are human sciPS cells and stem cells. In a further
embodiment, the method comprises formulating substances for wound
healing. In an even further embodiment, the method comprises
formulating substances for regenerative properties of tissues and
organs. In an additional embodiment of the method, the tissues and
organs are selected from the group consisting of skin, blood, and
pancreatic islets.
[0019] Additionally disclosed herein is a method of screening
candidate agents capable of conferring sciPS benefits to non-sciPS
cells; the method comprising contacting the candidate agent with a
population of non-sciPS cells; and identifying agents which are
capable of conferring sciPS benefits to the non-sciPS cells. In
another embodiment, the non-sciPS cells are human. In an additional
embodiment, the sciPS benefits conferred to the non-sciPS cells are
a reduced rate of cellular aging as compared to non-sciPS cells
without the candidate agent. In another embodiment, the candidate
agent is selected from the group consisting of biologics, small
molecules, drugs, nutraceuticals, cosmeceuticals, compounds, and
reagents. In another embodiment, the biologic is a nucleic acid
molecule of interest (NOI).
[0020] Further disclosed herein is a method for identifying a
genetic predisposition to an age-related disease in an individual;
the method comprising measuring the rate of decrease in telomere
length in a population of sciPS cells to obtain a rate of cellular
aging; comparing the rate of cellular aging in the sciPS cells to
the rate in non-sciPS cells; calculating a ratio of the cellular
aging rate in sciPS cells to non-sciPS cells to obtain predicted
genetic lifespan; and
determining the predicted disease-free period based on the
predicted genetic lifespan and compression of morbidity data from
non-supercentenarians, nonagenarians, centenarians,
semi-supercentenarians and supercentenarians; thereby identifying a
genetic predisposition to an age-related disease in the individual.
In another embodiment, the individual is a human. In an additional
embodiment, the sciPS cells and non-sciPS cells are human sciPS
cells and human non-sciPS cells. In another embodiment, the
age-related disease is selected from the group consisting of
osteoporosis, osteoarthritis, cancer, heart disease, stroke, and
neurological disorders.
[0021] The present invention also provides stem cell antigens, stem
cell markers, and transmembrane domain containing proteins in stem
cells. The present invention also provides techniques for
identification of stem cell antigens, markers, and transmembrane
domains. The present invention also provides techniques for the
identification of ligands that bind to cell surface receptors, and
the present invention also provides techniques for identification
of the developmental stage and/or differentiation pathway of a
pluripotent stem cell. The present invention also provides
techniques for the isolation of stem cell antigens, stem cell
markers, and transmembrane domain containing proteins in stem
cells.
[0022] In another embodiment of the present invention,
supercentenarian cells, centenarian cells, or cells derived from
humans of extreme age are used to produce induced pluripotent stem
cell lines. In another embodiment of the present invention, a
control cell population is used to identify and isolate
distinguishing upregulated or downregulated genes from populations
of supercentenarian cells or cloned supercentenarian cells. In
another embodiment of the present invention, novel developmentally
regulated proteins are identified by probing human embryoid body
RNA using oligonucleotide probes that detect expression products of
taxonomically restricted genes.
[0023] In other embodiments, the methods and cells in the preceding
paragraph may additionally incorporate any of the preceding or
subsequent disclosed embodiments.
[0024] The Summary of the Invention is not intended to define the
claims nor is it intended to limit the scope of the invention in
any manner.
[0025] Other features and advantages of the invention will be
apparent from the following Figures, Detailed Description, and the
Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A and 1B show immunocytochemical (ICC) staining of a
sciPS derived from a cell line from a 114 year old
supercentenarian. Epstein Barr Virus (EBV) immortalized
B-lymphoblastoid cells were obtained from a 114 year old female
donor with no prior history of cancer, heart disease, blood
disorder, lung disease, genito-urinary disorder, gastrointestinal
disorder, joint disease, eye disease, neurological or psychiatric
disorder, or diabetes. The B-cells were reprogrammed using the
integration-free episomal DNA method to introduce reprogramming
factors. Six sciPS clones were assessed for pluripotency by
detection of pluripotency specific marker genes and differentiation
to 3 germ layers. All six clones were positive by ICC staining for
5 pluripotency markers and for differentiation markers of all 3
primary germ layers following culture in appropriate
differentiation media. FIG. 1A shows ICC staining of a
representative sciPS clone (E19) with OCT4, SOX2, NANOG, Tra-1-60,
and Tra-1-81. Figure B shows ICC staining of sciPS-E19
demonstrating differentiation of a representative sciPS clone to
ectoderm (Nestin, Pax6), endoderm (Sox17, FoxA2), and mesoderm
(Smooth Muscle Actin (SMA)).
[0027] FIG. 2 provides evidence for pluripotency of sciPS-EI9 cells
by teratoma formation in mice. In vivo differentiation of clonal
sciPS-E19 cells to tissues representing ectoderm (neuroepithelium,
glycogenated epithelium), mesoderm (cartilage, muscle), and
endoderm (intestine) following subcutaneous growth as a teratoma in
an immune-deficient mouse (SCID-beige).
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0028] The present invention is disclosed in the Figures and
description. However, while particular embodiments are disclosed in
the Figures, there is no intention to limit the present invention
to the specific embodiment or embodiments disclosed. Rather, the
present invention is intended to cover all modifications,
variations, derivatives, and/or equivalents falling within the
spirit and scope of the present invention. As such, the Figures are
intended to be illustrative but not restrictive.
[0029] Unless otherwise defined, all scientific and technical terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this technology belongs.
[0030] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly indicates otherwise.
[0031] As used herein, the terminology, cell, cell line, and cell
culture are used interchangeably and all such designations include
progeny and/or derivatives. Thus, the terms pluripotent stem cells
and induced pluripotent stem cells include the primary subject cell
and cells and/or cell cultures derived therefrom without regard for
the number of transfers. It is also understood that all progeny may
not be precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
pluripotent cell or derivative are included. Where distinct
designations are intended, it will be clear from the context.
[0032] As used herein, the term antibody refers to any form of
antibody that exhibits the desired biological activity. Thus, it is
used in the broadest sense and specifically covers monoclonal
antibodies (including full length monoclonal antibodies),
polyclonal antibodies, multispecific antibodies (e.g., bispecific
antibodies), chimeric antibodies, humanized antibodies, fully human
antibodies, etc. so long as they exhibit the desired biological
activity.
[0033] As used herein, an isolated nucleic acid molecule or
isolated protein or isolated antibody or isolated cell or cells
refer to a nucleic acid molecule or protein or antibody or cell
that is identified and separated from at least one contaminant
nucleic acid, protein or antibody molecule or cell with which it is
ordinarily associated in the natural source. An isolated nucleic
acid molecule or protein or antibody or cell is other than in the
form or setting in which it is found in nature. Isolated nucleic
acid molecules therefore are distinguished from the nucleic acid
molecule as it exists in natural cells.
[0034] As used herein, a nucleic acid molecule or nucleic acid
molecule of interest (NOI) means DNA or RNA or a DNA or RNA
molecule that is separated from sequences (or nucleotide sequences)
with which it is immediately contiguous (in the 5' and 3'
directions). For example, the "nucleic acid molecule" may comprise
a DNA molecule inserted into a vector, such as a plasmid or virus
vector, or integrated into the genomic DNA of a prokaryote or
eukaryote. An "isolated nucleic acid molecule" may also comprise a
cDNA (complementary DNA) molecule. An isolated nucleic acid
molecule manipulated to include other nucleic acid sequences is
often referred to as a recombinant molecule. An RNA molecule is
composed of nucleotides (ribonucleotides) and is typically
single-stranded. RNA is coded by the DNA molecule, or transcribed
using the DNA molecule as a template, so that the messenger RNA
(mRNA) can be translated into its corresponding amino acid
sequence. Short interfering RNA is double-stranded RNA of about
20-25 base pairs (or nucleotides) in length, and which typically
function to interfere with the expression of a gene or genes.
MicroRNA (miRNA) are very small pieces of RNA which are about 22
nucleotides in length and typically function in the transcriptional
or post-transcriptional regulation of a gene or genes. Molecular
biology techniques and terminology are readily available and well
known and can be found, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (2003).
[0035] As used herein, pluripotent cells are a population of cells
capable of differentiating into all three germ layers and becoming
any cell type in the body. Pluripotent cells express a variety of
cell surface markers, have a cell morphology characteristic of
undifferentiated cells and form teratomas when introduced into an
immunocompromised animal, such as a SCID mouse. Teratomas typically
contain cells or tissues characteristic of all three germ
layers.
[0036] As used herein, multipotent cells are more differentiated
than pluripotent cells, but are not permanently committed to a
specific cell type. Pluripotent cells therefore have a higher
potency than multipotent cells.
[0037] As used herein, induced pluripotent stem cells or iPS cells
are cells that are differentiated, somatic cells reprogrammed to
pluripotency. The cells are substantially genetically identical to
their respective differentiated somatic cell of origin and display
characteristics similar to higher potency cells, such as ES cells.
See, Yu J, et al., "Induced pluripotent stem cell lines derived
from human somatic cells," Science 318:1917-1920 (2007),
incorporated herein by reference as if set forth in its
entirety.
[0038] As used herein, an embryoid body or an EB, is an aggregate
of cells derived from pluripotent cells, such as ESCs or iPS cells,
where cell aggregation can be initiated by hanging drop, by plating
upon non-tissue culture treated plates or spinner flasks (i.e., low
attachment conditions); any method prevents the cells from adhering
to a surface to form typical colony growth. EBs appear as founded
collections of cells and contain cell types derived from all three
germ layers (i.e., the ectoderm, mesoderm and endoderm). Methods
for generating EBs are well-known to one ordinary skill in the art.
See, Itskovitz-Eldor J, et al., "Differentiation of human embryonic
stem cells into embryoid bodies compromising the three embryonic
germ layers," Mol. Med. 6:88-95 (2000); Odorico J, et al., Stem
Cells 19:193-204 (2001), and U.S. Pat. No. 6,602,711, each of which
is incorporated herein by reference as if set forth in its
entirety.
[0039] As used herein, taxonomically-restricted gene refers to
genomic DNA sequence encoding a peptide or protein sequence that is
restricted to a single species distribution (orphan) or to a narrow
phylogenetic distribution having homologs in closely related
species but not present in more distantly related species or other
genera.
[0040] The present invention provides iPS cells derived from
supercentenarians (sciPS) that are used to create cellular models
of decelerated human aging. The sciPS generated cellular models are
useful for comparative studies of cellular aging to determine the
molecular basis of decelerated human aging.
[0041] For purposes of the present invention, a supercentenarian is
an individual human having attained an age of at least 110 years
from birth; or is an individual non-human animal or mammal having
an equivalent age as determined by, e.g., an estimation based on
having an age of approximately 90% or greater of the maximal
lifespan of the species and/or comparative genomics. Comparative
genomics and tools for assessing human equivalence in age in a
non-human individual are readily available and well known. (See
e.g., Tacutu et al., (2012) Nucleic Acids Research 41: D1027-D1033;
Magalhaes et al., (2009) Aging Cell 8: 65-72; Magalhaes et al.,
(2009) J. Evol. Biol. 22: 1770-74.) Supercentenarian cells or sciPS
cells are cells or iPS cells from and/or derived from a
supercentenarian.
[0042] For the purposes of the present invention, a
non-supercentenarian is an individual human from the general
population that has attained an age less than 110 years and is not
a sibling or parent of a supercentenarian; or is an individual
non-human animal or mammal having an equivalent age as determined
by, e.g., an estimation based on having an age of less than
approximately 90% of the maximal lifespan of the species and/or
comparative genomics. Comparative genomics and tools for assessing
human equivalence in age in a non-human individual are readily
available and well known. (See e.g., Tacutu et al., (2012) Nucleic
Acids Research 41: D1027-D1033; Magalhaes et al., (2009) Aging Cell
8: 65-72; Magalhaes et al., (2009) J. Evol. Biol. 22: 1770-74.).
Non-supercentenarian cells or non-sciPS cells are cells or iPS
cells from and/or derived from a non-supercentenarian.
[0043] For purposes of the present invention, a
semi-supercentenarian is an individual human having attained an age
of 105-109 years from birth; or is an individual non-human animal
or mammal having an equivalent age as determined by, e.g., an
estimation based on having an age of approximately 86-89% of the
maximal lifespan of the species and/or comparative genomics.
Comparative genomics and tools for assessing human equivalence in
age in a non-human individual are readily available and well known.
(See e.g., Tacutu et al., (2012) Nucleic Acids Research 41:
D1027-D1033; Magalhaes et al., (2009) Aging Cell 8: 65-72;
Magalhaes et al., (2009) J. Evol. Biol. 22: 1770-74.)
Semi-supercentenarian cells or semi-sciPS cells are cells or iPS
cells from and/or derived from a semi-supercentenarian.
[0044] For purposes of the present invention, a centenarian is an
individual human having attained an age of 100-104 years from
birth; or is an individual non-human animal or mammal having an
equivalent age as determined by, e.g., an estimation based on
having an age of approximately 82-85% of the maximal lifespan of
the species and/or comparative genomics. Comparative genomics and
tools for assessing human equivalence in age in a non-human
individual are readily available and well known. (See e.g., Tacutu
et al., (2012) Nucleic Acids Research 41: D1027-D1033; Magalhaes et
al., (2009) Aging Cell 8: 65-72; Magalhaes et al., (2009) J. Evol.
Biol. 22: 1770-74.) Centenarian cells or ciPS cells are cells or
iPS cells from and/or derived from a centenarian.
[0045] As used herein, induced pluripotent stem (iPS) cells are
cells that are derived from a cell sample provided by a donor (e.g.
B lymphocytes, EBV transformed B lymphocyte cell line cells,
fibroblasts, keratinocytes) using any one of a number of
reprogramming methods known in the art that include transcriptional
reprogramming (Takahashi K et al. (2007) Nat Protoc 2: 3081) and
reprogramming by nuclear transfer (Tachibana et al (2013) Cell 153:
1). The resulting iPS cells are embryonic stem cell-like in their
capacity for self-renewal and differentiation into cell types that
are representative of the three primary germ layers of embryonic
development (mesoderm, endoderm and ectoderm).
[0046] As used herein, telomere length resetting refers to an
increase in average telomere length of a cell sample extending from
the telomere length at the time the sample is taken from a donor,
up to and including restoration of telomere length that
approximates telomere lengths of the cell's ancestral cells as they
existed in the early embryo.
[0047] As used herein, cellular aging refers to changes in a cell
that occur with time that impact its capacity to replicate,
differentiate, or otherwise function as it would in a young healthy
individual before degenerative effects of aging have begun. These
changes include shortened telomere length, loss of differentiation
capacity, changes in differentiation propensity, changes in genomic
methylation pattern (Hannum et al. (2013) Mol Cell 49:359), changes
in genome expression pattern, morphological changes (e.g. to a
large flattened appearance) and expression of senescence associated
genes and gene products (e.g. SA-B-galactosidase, senescence
associated secretory phenotype). Other changes include accumulation
of genomic DNA mutations, mitochondrial DNA mutations, incorrectly
folded proteins, intracellular aggregates (e.g. lipofuscin),
nuclear abnormalities, and progerin. Additional changes include an
increase in sensitivity to stress such as substratum deprivation,
serum starvation, electrical stimulation, mechanical stress and
hypoxia.
[0048] In one embodiment of the invention, sciPS cell derived MSCs
are compared to non-sciPS derived MSCs to determine differences in
telomere dynamics, telomerase activity, and changes in
differentiation capacity with increased replication cycles as
measured for example by number of population doublings. The
development of sciPS and sciPS derivatives enables comparative
transcriptome, proteome and methylome analysis of virtually any
cell type in a genetic background of extreme longevity versus
normal non-supercentenarian or accelerated aging (e.g. HG progeria,
Werner's syndrome). Identified sciPS and sciPS derived cell
specific factors are used to design screening assays for compounds
that confer sciPS cell benefits to non-supercentenarian cells. The
sciPS cells provide a human cell based model system useful for
assessing the role of various factors in the aging process such as
stress genes (Swindell et al. (2009) Mech Aging Dev 130: 393), DNA
damage and repair, Surtuins, MTOR, and the insulin-IGF receptor
pathways (Barbieri et al. (2003) J Physiol Endocrinol Metab 285:
e1064). Previously such systems were only available using cells
from lower animals such as worms, fruit flies, and mice. The sciPS
cells of the present invention are also useful for creating human
mouse chimeras to examine human cellular aging in an animal model,
such as, in mice with human immune systems derived from sciPS
compared to non-sciPS models to identify immune cell factors
involved in extreme longevity.
[0049] The present invention provides for the derivation and
analysis of previously difficult or impossible to obtain
supercentenarian tissues such as vascular, heart muscle, neural,
liver and pancreas cells with restored telomere length. The
rejuvenated sciPS derived cells and tissues allow unprecedented
analysis of decelerated human cellular aging. It is impractical to
do longitudinal studies on supercentenarians at younger ages. Even
if supercentenarians could be identified at young ages, only a few
cell types would be accessible (i.e. blood, hair and skin) and
these individuals are extremely rare (<1 in 5 million). The
sciPS cells provide genetically matched pluripotent cells with
rejuvenated cellular age (e.g. restored telomere length). The sciPS
cells provide medically relevant cell types from individuals with
long-lived genotypes at different replicative ages ranging from
embryonic to senescent. The sciPS cells and their derivatives are
useful for analyzing function of candidate human longevity genes
and single nucleotide polymorphisms (SNPs) associated with extreme
human longevity. For example, mutations in RNA editing genes are
associated with long lived humans but the functional analysis has
been thus far limited to analysis of models based on lower animals
such as C. elegans because of a lack of human cells to determine
how these mutations which affect RNA editing at the cellular level
are involved in aging.
[0050] In one embodiment, 4 factor (OSKM) reprogramming using
episomal plasmid vectors is used for blood cell reprogramming as
previously described (Rajesh et al. (2011) Blood 118: 1797; Choi et
al. (2011) Blood 118: 1801; Chou et al. (2011) Cell Res 21:518).
EBV-transformed lymphocytes are reprogrammed using the
non-integrative plasmid method to introduce the reprogramming
factors. Episomal plasmid reprogramming from EBV-transformed
B-cells is a known method for successful reprogramming to
pluripotent cell lines that are both EBV DNA-free and free of
plasmid vector DNA (Rajesh et al. (2011) Blood 118: 1797; Choi et
al. (2011) Blood 118: 1801). This approach is advantageous because
it provides sufficient cell numbers for reprogramming and because
using blood cells avoids risks associated with obtaining
fibroblasts using dermal punch biopsy in an aged population. The
use of EBV transformed cells has the advantage that a single small
blood sample can be taken from which cells are expanded and
multiple aliquots are archived for later use.
[0051] Also provided herein are methods to obtain rejuvenated sciPS
and sciPS derived cells having reset cellular age as well as
developmental age to embryonic equivalent. In one embodiment, the
telomerase repeat amplification protocol (TRAP) assay is used to
measure telomerase activity in sciPS clones. The TRAP assay
measures telomerase to identify iPS clones with high telomerase
activity. Such clones progressively lengthen telomeres with
continued passage until reaching embryonic length of the parental
embryonic cell line (Vaziri et al. (2010) Regen Med 5: 345). The
sciPS cell clones are monitored for telomere length using standard
methods and compared to embryonic stem cell telomere length. The
sciPS cell clones are also screened for loss of EBV and episomal
plasmid DNA. Pluripotency is assessed by differentiation of the
sciPS clones to cell types representative of the three primary
(embryonic) germ layers using standard directed differentiation
conditions in vitro and by analysis of teratoma formation in
mice.
[0052] In another embodiment of the present invention, the
proliferative capacity, telomerase activity, telomere dynamics,
rate of aging with respect to changes in differentiation
propensity, and the appearance of senescent cells is determined in
sciPS derived stem cells such as MSCs and compared to non-sciPS
MSCs. Loss of MSCs with age could lead to stem cell exhaustion
which would affect overall health, and for example skin integrity,
immune function, and susceptibility to bone fracture because of the
role these cells play in maintenance and repair of mesenchymal
tissues such as bone, skin, blood and the vascular system. The
importance of MSCs is indicated by the prevalence of symptoms
related to defects in mesenchymal tissues seen in patients with
diseases associated with accelerated cellular aging such as HG
Progeria and Werner's syndrome. Proliferative capacity is measured
as population doublings from initial derivation through senescence.
The rate of change in telomere restriction fragment (TRF) length is
measured from DNA extracted from cells with increasing population
doublings in culture to measure differences in telomere dynamics
between sciPS and non-sciPS derived MSCs. The TRAP assay is used to
measure telomerase activity at early and late passages as
previously described (Vaziri et al. (2010) Regen Med 5: 345). The
percentage of senescent cells is measured by staining for
SA-B-galactosidase to assess rate of increase with passage. sciPS
derived cells are advantageous over non-sciPS derived cells for
maintaining their functional integrity with passage in culture.
Early and late passage genomic expression analysis is performed to
determine underlying biochemical factors that confer benefits of
sciPS derived cell aging.
[0053] In another embodiment of the present invention, sciPS
derived cells such as MSCs and vascular smooth muscle cells (VSMCs)
are assessed for sensitivity to oxidative and mechanical stress and
compared to equivalent control-iPS derived cells. There is evidence
that premature aging and cardiovascular disease in progeria
patients is caused by both a stem cell depletion and increased
sensitivity to the low oxygen stress of their niche (Zhang et al.
(2011) Cell Stem Cell 8: 31). Depletion of stem cells and increased
sensitivity of stem cells to stress is thought to play a
significant role in human aging and vascular disease (Zhang et al.
Supra; Sahin E, Dapino R A (2010) Nature 464: 520). Resistance to
stress in sciPS derived cells such as MSCs, VMSC, endothelial
progenitor cells (EPCs) is compared to the equivalent non-sciPS
derived cells. Early, middle, and late passage cells are subject to
hypoxia with and without substrate depletion and VSMCs subjected to
mechanical stress as described (Zhang et al. Supra). Cellular aging
with respect to changes in stress resistance is measured by percent
cell survival and percent of senescent cells in each population
with increasing passage number.
[0054] In another embodiment of the present invention, specific
factors in sciPS derived cells such as MSCs are identified by
comparative transcriptomic, proteomics and methylomics. DNA, RNA,
and protein samples are analyzed for methylomic, transcriptomic and
proteomic changes with increasing cellular age and compared to
controls. Loss and gain of functions analysis is used to determine
the effect these factors have on sciPS derived MSC cellular aging
including changes in differentiation capacity with increased
population doubling. The factors are used as indicators in cell
based assays to identify compounds including nucleic acids of
interest that induce the benefits of sciPS MSCs in non-sciPS
derived MSCs.
[0055] In another embodiment of the present invention, sciPS
derived cells are used to confer supercentenarian benefits such as
resistance to degenerative disease, increased health span and
longevity (Andersen et al. (2012) J Gerontol A Biol Sci Med Sci 67:
395) to a non-supercentenarian. Bone marrow stem cells including
MSCs and HSCs are derived from sciPS using methods known in the art
(Liu et al. (2012) PloS One 7:e33225; Suzuki et al. (2013) Mol Ther
21:1424; Klump et al. (2013) Curr Mol Med 13: 815; Bouhassira et
al. (2013) Expert Opin Biol Ther 13: 1099). The sciPS derived MSCs
and 1-HSCs are used to treat an individual that is at risk for
osteoporosis by transplantation of sciPS derived bone marrow stem
cells into the at risk individual.
[0056] In another embodiment of the present invention,
supercentenarian benefits are conferred to a non-supercentenarian
by conversion of non-supercentenarian iPS cells from the patient to
be treated to cells having sciPS-like properties and deriving cells
for cell replacement therapy from the converted patient matched
sciPS cells. Conversion of iPS to sciPS-like cells is accomplished
by introduction of sciPS factors into iPS using for example
established gene therapy methods such as gene editing (Perez-Pinera
et al. (2012) Curr Opin Chem Biol 16:268; Li et al. (2013) Mol Ther
21:1259) and gene transfer. The converted iPS cells are used to
derive patient matched MSCs with sciPS derived MSC properties. The
patient matched MSCs derived from converted iPS cells are used to
treat age related degenerative diseases such as osteoporosis by
transplantation back to the patient.
[0057] In another embodiment of the present invention,
supercentenarian benefits are conferred to a non-supercentenarian
by treatment with compounds such as small molecules, drugs,
nutraceuticals, and nucleic acids that are shown to induce sciPS
derived cell properties in non-sciPS derived cells. For example, a
compound is used that is shown to induce a reduced rate of cellular
aging in non-supercentenarian iPS derived MSCs.
[0058] In another embodiment, substances made by sciPS derived
cells are used to treat degenerative diseases associated with aging
such as damaged skin. Cells derived from sciPS such as MSCs are
used to prepare cell extracts and conditioned medium containing
sciPS MSC intracellular and secreted substances. The cell extract
and/or conditioned medium, or one or more components thereof, which
may also be considered a cosmeceutical, is added to dermatological
formulations such as creams or lotions and applied to the skin to
restore a youthful appearance to age or sun damaged skin by
alleviating age related changes in skin such as thinning skin,
loose skin, discoloration, hyperpigmentation, fine lines and
wrinkles.
[0059] In another embodiment of the present invention, the rate of
cellular aging of iPS derived cells from a non-supercentenarian is
used as a diagnostic assay to assess predicted longevity and the
degree of resistance to degenerative disease. A ratio of rate of
cellular aging of non-supercentenarian to supercentenarian iPS
derived cells indicates longevity and disease resistance on a scale
of 0 to 1 with a score of 1 indicating a predicted longevity of at
least to age 110 and predicted resistance to degenerative disease
equivalent to a supercentenarian. The diagnostic is used to monitor
the effectiveness of treatments designed to increase longevity and
resistance to age related degenerative disease.
[0060] The present invention additionally provides for the
isolation of stem cell antigens, stem cell markers, and
transmembrane domain containing proteins derived from stem cells.
Stem cell antigen proteins may be embodied in many forms,
preferably in isolated form. As used herein, an antigen or protein
is said to be isolated when physical, mechanical or chemical
methods are employed to remove the stem cell antigen or protein
from cellular constituents that are normally associated with the
antigen or protein. A skilled artisan can readily employ standard
purification methods to obtain an isolated stem cell antigen or
protein. A purified stem cell protein molecule will be
substantially free of other proteins or molecules which impair the
binding of the stem cell protein to antibody or other ligand. The
nature and degree of isolation and purification will depend on the
intended use. Embodiments of the stem cell protein include a
purified stem cell protein and a functional, soluble stem cell
protein. The present invention also provides techniques for
identification of stem cell antigens, markers, and transmembrane
domains. The present invention also provides techniques for the
identification of ligands that bind to cell surface receptors, and
the present invention also provides techniques for identification
of the developmental stage and/or differentiation pathway of a
pluripotent stem cell. The present invention also provides
techniques for the isolation of stem cell antigens, stem cell
markers, and transmembrane domain containing proteins in stem
cells.
[0061] In one embodiment, the present invention provides isolated
stem cell DNA, e.g. cDNAs, encoding embryoid body cell surface
antigens. In one embodiment of the present invention, the embryoid
bodies are human. In another embodiment, the present invention
provides methods for isolating human embryoid body cell surface
antigens using a signal sequence trap (SST). In an additional
embodiment of the present invention, the embryoid bodies are
derived from iPS and ES cells.
[0062] In one embodiment, present invention provides DNA alone
(i.e., without flanking sequences) or as a component of a larger
sequence comprising other sequences. For example, a DNA of the
invention is suitably provided as a component of an expression
cassette or an expression vector. Many examples of expression
cassettes, expression vectors and the like are known in the art.
Examples of expression vectors for expression in E. coli include
pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8. Examples of
expression vectors for expression in yeast include pY100 and
Ycpad1. Examples of expression vectors for expression in animal
cells include pKCR, pEFBOS, cDM8 and pCEV4. A suitable expression
vector for expression in insect cells is the bacculovirus
expression vector pAcSGHisNT-A. Many cell lines and other organisms
useful in the expression of proteins are known in the art. Examples
of cell lines include the E. coli strains HB101, DH1, x1776, JM101,
JM 109, BL21 and SG 13009; the yeast strain Saccharomyces
cerevisiae; the animal cell lines L, NIH 3T3, FM3A, CHO, COS, Vero
and HeLa; and the sf9 insect cell line. Methods for transforming or
transfecting cells for the expression of an expression vector are
known in the art. The DNA of the invention can also be ligated to a
DNA encoding another protein and/or peptide, so that the DNA of the
invention is expressed as a component of a fusion protein.
Conditions for culturing transformed or transfected cells are also
known in the art, as are methods for isolating and purifying the
expressed protein and/or fusion protein.
[0063] In another embodiment, the invention provides antibodies
directed against a protein or fusion protein of the invention. The
production of such antibodies may proceed according to known
methods using the novel proteins of the invention. Antibodies of
the invention may be polyclonal or monoclonal. Production of
antibodies may be accomplished by immunizing an animal, such as a
rabbit or chicken (for a polyclonal antibody) or a mouse (for a
monoclonal antibody), with a protein, fusion protein, or protein
fragment of the invention. A polyclonal antibody can be obtained,
for example, from the animal serum or egg yolk. To obtain a
monoclonal antibody, animal spleen cells may be fused with myeloma
cells using standard protocols.
[0064] Several signal sequence trap systems have been developed
including, but not limited to, those provided in U.S. Pat. No.
6,228,590, which describes a technique for screening for mammalian
signal sequences by transforming reporter protein-deficient yeast
with nucleic acids comprising mammalian coding sequences fused to a
reporter protein and detecting cells that secrete the reporter
protein. A similar system using invertase-deficient yeast and an
invertase reporter protein is disclosed in EP0907727. Yeast-based
signal sequence traps have been used to identify secreted proteins
from human DNA (Klein et al., Proc. Natl. Acad. Sci. USA 93:7108
(1996); Jacobs et al., Gene 198:289 (1997)), mouse DNA (Gallicioti
et al., J. Membrane Biol. 183:175 (2001)), zebrafish DNA (Crosier
et al., Dev. Dynamics 222:637 (2001)), Arabidopsis DNA (Goo et al.,
Plant Mol. Biol. 41:415 (1999)), potato DNA (Surpili et al., Anais
de Academia Brasileira de Ciencias 74:599 (2002)), and Candida
albicans DNA (Monteoliva et al., Eukaryotic Cell 1:514 (2002)).
Similar trap systems have been developed using mammalian host cells
(Gallicioti et al., J. Membrane Biol. 183:175 (2001)) and bacterial
host cells (Ferguson et al., Cancer Res. 65:8209 (2000). Reporter
proteins that have been used in signal sequence traps include
invertase (Klein et at, Proc. Natl. Acad. Sci. USA 93:7108 (1996)),
alpha amylase (U.S. Pat. No. 6,228,590), acid phosphatase (PHO5)
(Surpili et al., Anais de Academia Brasileira de Ciencias 74:599
(2002)), and beta-lactamase Ferguson et al., Cancer Res. 65:8209
(2000).
[0065] In another embodiment of the present invention, cDNAs
encoding secreted and membrane bound proteins are selected from day
2-14 EBs. In one embodiment of the present invention, cDNAs
encoding secreted and membrane bound proteins are selected from day
2-14 EBs from a library. In another embodiment of the present
invention, the library of day 2-14 EBs are of normalized and 5' end
enriched EB cDNAs using a SST vector. In one embodiment of the
present invention the techniques can be used to identify and select
cDNAs from a library constructed from known techniques in the art.
In another embodiment of the present invention, the cDNA library is
normalized to provide methods for identification and selection of
otherwise rare cDNAs. In one embodiment of the present invention,
the normalized cDNA library reduces the frequency of the most
abundant clones by 10 fold or more while increasing the frequency
of the least prevalent cDNAs by two fold or more. See also Bonaldo,
et al., U.S. Pat. No. 5,702,898; Short, et al., U.S. Pat. No.
5,763,239; and Short, et al. U.S. Pat. No. 6,001,574, each hereby
incorporated by reference.
[0066] In another embodiment of the present invention, a time
course of antigen expression and immunolocalization of target
antigens is produced by preparing varyingly differentiated human
EBs from iPS and hES cells, differentiation and harvest time points
of every hour, every two hours, every three hours, every 6 hours,
every 8 hours, every 10 hours, every 12 hours, every 18 hours,
every 24 hours, or every 48 hours. In one such embodiment, standard
cell culture techniques are used to expand and test IgY antibody
binding of the every hour, every two hours, every three hours,
every 6 hours, every 8 hours, every 10 hours, every 12 hours, every
18 hours, every 24 hours, or every 48 hour time point cell
populations. In one embodiment of the present invention, RT-PCR
analysis is conducted by isolating total RNA from 2-, 4-, 6-, 8-
and 10-day-old EBs and undifferentiated hESCs using, as a
non-limiting example, Tri-Reagent (Sigma, St. Louis, Mo., USA)
according to the manufacturer's protocol. In another embodiment of
the present invention, EB cDNA is then synthesized from isolated
total RNA using any means known to one skilled in the art,
including but not limited to MMLV reverse transcriptase RNase H
minus (Promega, Madison, Wis., USA). PCR products may then be
size-fractionated by electrophoresis on 2% agarose gel.
[0067] In another embodiment of the present invention, the antigen
identification methods of the invention are used to generate a bank
of novel antibodies against surface markers on iPS derived cells
for identification and characterization of progenitor cell
populations. In another embodiment of the present invention,
transmembrane domains and/or alternate reading frame cDNAs are used
to select genes encoding surface and secreted proteins in cell
populations including, but not limited to, progenitor cells. In
another embodiment of the present invention, supercentenarian
cells, centenarian cells, or cells derived from humans of extreme
age are used to produce induced pluripotent stem cell lines. In one
embodiment of the present invention, a control cell population is
used to isolate distinguishing upregulated or downregulated genes
from populations of supercentenarian cells or cloned
supercentenarian cells. In an additional embodiment of the present
invention novel developmentally regulated proteins are identified
by probing human embryoid body RNA using oligonucleotide probes
that detect expression products of taxonomically restricted
genes.
[0068] The invention will now be described by way of Examples,
which are meant to assist one of ordinary skill in the art in
carrying out the invention and are not intended in any way to limit
the scope of the invention.
EXAMPLES
[0069] The following examples are provided to illustrate but not
limit the claimed invention.
Example 1: Derivation of iPS Cells from Supercentenarian Donor
Cells
[0070] The derivation of iPS cells from supercentenarian donor
cells is performed by introducing reprogramming factors into the
cultured donor cells (Takahashi K et al. (2007) Nat Protoc 2:
3081). Methods for reprogramming are well established including the
use of viral vectors, episomal plasmid DNA, and RNA to introduce
the reprogramming factors or small molecule reagents to activate
reprogramming factors. The source of donor cells may be any source
known in the art including, without limitation, dermal fibroblasts,
hair follicle, and blood. Blood cells (e.g. B-lymphocytes) that
have been immortalized by Epstein Barr Virus (EBV) infection are
advantageous for obtaining sufficient numbers of cells for
reprogramming. Non-viral methods of reprogramming such as
integration-free episomal DNA transfection (Okita et al. (2011) Nat
Methods 8:409) are advantageous because they minimize risk of
inadvertent genetic modification that could activate tumor forming
ability in iPS cells and their derivatives. Pluripotency of
reprogrammed cells is confirmed by immunochemical staining for
expression of representative pluripotency markers (e.g. OCT4, SOX2,
NANOG, Tra-1-60, Tra-1-80) (FIG. 1A). Pluripotency is also
confirmed by demonstrating differentiation to cells of the three
primary germ lineages in vitro using standard media formulations
and immunochemical staining for representative markers of each
lineage (FIG. 1B). Teratoma formation in mice and histological
identification of tissue and cell types is used to confirm in vivo
differentiation to all three primary germ layers (FIG. 2).
Telomerase activity is measured using the TRAP assay (Vaziri et al.
(2010) Regen Med 5: 345). Telomere length is determined by Southern
blot or comparable method. Telomere length in the reprogrammed
cells of 12 kb to 20 kb indicates that the reprogramming resulted
in a lengthening of the telomeres toward that of embryonic cells.
Standard methods such as Southern blotting and polymerase chain
reaction DNA amplification are used to confirm loss of episomal
reprogramming plasmids and EBV viral DNA (Rajesh et al. (2011)
Blood 118: 1797; Choi et al. (2011) Blood 118: 1801).
Example 2: Derivation of Mesenchymal Progenitor Stem Cells from
sciPS and Non-sciPS Cells
[0071] MSCs are derived from sciPS and non-sciPS cells using
previously described methods (Giuliani et al. (2011) Blood 118:
3254) such as adherent growth on plastic culture and
cytofluorometric sorting of cells positive for one or more MSC
markers (e.g. CD105, CD90, CD73, CD44, CD29, CD146, and CD166) and
negative for hematopoietic stem cell markers (e.g. CD45, CD34). The
isolation of MSCs of clonal purity as described by Lian et al.
(Lian et al. (2010) Circulation 121: 1113) is advantageous for
obtaining pure populations of MSCs from iPS cells. Mesenchymal
progenitor cells with more limited differentiation capacity
(chondrogenic and/or osteogenic) are also obtained using the clonal
isolation method described by West et al. (West et al. (2008) Regen
Med 3:287). The clonal cell line, SM30, described by West, M. D. et
al., preferentially differentiates to osteoblasts under osteogenic
culture conditions. SM30 cells do not express BMMSC markers
(Sternberg et al. (2013) Regen Med 8: 125) and unlike BMMSCs, which
differentiate to both adipocytes and osteoblasts in osteogenic
medium, SM30 cells do not differentiate to adipocytes when cultured
in either adipogenic, chondrogenic, or osteogenic differentiation
media. Peptides that bind SM30 cell surface are used to identify
and purify SM30 cells differentiated from iPS and sciPS cells.
Peptides with the sequence DWIATWPDAVRS, EWILTLPDGSDW, EWFEFPTPVDA,
EWQFWPLLTKN are used to label cells for sorting. The peptides are
conjugated to a fluorescent or magnetic tag and peptide bound cells
isolated by flow cytometry or magnetic separation. Surface markers
are preferentially expressed on SM30 cells in a mixed population of
cells that are differentiated from iPS or hES cells using the
method described by West et al. (West et al. (2008) Regent Med 3:
287). Differential expression analysis of global gene expression
data is used to identify genes that are differentially expressed in
SM30 relative to other clonal cell lines isolated in parallel under
the same differentiation conditions. Upregulated surface markers on
SM30 include PTK7, SCARF2, MMP23B, and SEMA3E. Down regulated
surface markers include ITGB1 and TNFRSF11B. Advantages of sciPS
derived mesenchymal progenitor cell properties relative to
non-sciPS derived mesenchymal progenitor cells include reduced rate
of telomere shortening and retention of chondrogenic and osteogenic
differentiation capacity at later passage number than iPS derived
progenitors.
Example 3: Derivation of Clonal Embryonic Progenitor Stem Cell
Lines from sciPS
[0072] The derivation of hundreds of distinct human embryonic
progenitor (EP) stem cell lines from human pluripotent stem cells
has been previously described (West et al. (2008) Regen Med 3:287).
These cell lines have been characterized for their global gene
expression profile, embryonic origin and lineage differentiation
and include cell lines that differentiate, for example to
cartilage, bone, smooth muscle, adipose cells and other cell types
of interest for research and therapeutic development. The cells
have a prolonged but finite replication capacity resulting from
extended telomere length typical of embryonic cells.
Supercentenarian EP cell lines are similarly derived from sciPS
cell lines. The sciPS derived EP cell lines have advantages over
non-sciPS derived EP cell lines such as decelerated replicative
aging resulting in increased replicative lifespan, increased genome
stability and prolonged retention of differentiation capacity
compared to non-sciPS derived EP cell lines.
Example 4: Reduced Rate of Cellular Aging and Prolonged
Differentiation Capacity of sciPS-MSCs Compared to
Non-sciPS-MSCs
[0073] MSCs are derived from sciPS cells and non-supercentenarian
control iPS cells. The rate of cellular aging of sciPS-MSCs is
compared to the rate of cellular aging in control non-sciPS-MSCs
using known methods. Telomere length shortens with each cell
division until reaching a critical length which triggers the cell
to enter a non-dividing senescent state. MSC telomere length is
monitored using standard assays (e.g. single telomere length
analysis (STELA), fluorescence in-situ hybridization (FISH),
flow-FISH, and Southern blot analysis). Cells are harvested at
passage 0 and at every 5 population doublings until senescence is
reached. The telomere length and rate of shortening with population
doubling is measured. A reduced rate of cellular aging in
sciPS-MSCs compared to control iPS-MSCs is indicated by a slower
decrease in the rate of telomere shortening with increasing number
of population doublings. A reduced rate of cellular aging is also
indicated by elevated telomerase activity as measured using, for
example the TRAP (telomere repeat amplification protocol) assay. A
reduced rate of cellular aging is also indicated by changes in the
epigenetic profile of the cells as described by Hannum (Hannum et
al. (2013) Mol Cell 49:359). Reduced SIRTI gene expression is an
indicator of cellular aging and therefore retention of SIRTI
expression is an indicator of a reduced rate of cellular aging.
There is evidence that the deacetylase encoding gene, SIRTI, gene
expression is important for maintaining MSCs with age and for
regulating osteogenic capacity (Simic et al. (2013) EMBO Mol Med 5:
430). Another indicator of MSC cellular aging is a shift in
differentiation potential from one that favors osteogenic
differentiation to one that favors adipogenic differentiation.
Differentiation potential of iPS MSCs and sciPS-MSCs is measured
using standard culture conditions and media that are known to
induce osteogenesis or adipogenesis. Differentiation to bone
forming cells is measured by assaying deposition of calcium and
phosphate in the extracellular matrix using Alizarin Red and Von
Kossa staining, respectively. Differentiation to fat cells is
measured by staining for intracellular lipid droplets using Oil
Red. A reduced rate of cellular aging in sciPS-MSCs is indicated by
maintenance of osteogenic differentiation at early middle and late
passage whereas non-sciPS-MSCs loose osteogenic differentiation
activity at early to middle passage number.
Example 5: Conferred Longevity and Resistance to Age Related
Disease
[0074] Transplantation of bone marrow mesenchymal stem cells
(BMMSCs) from young mice into old mice restores age related bone
loss and extends life whereas the equivalent transplantation using
BMMSCs from old mice has no effect (Shen et al. (2011) Sci Rep 1:
67). Similarly, transplantation of BMMSCs from young mice into a
mouse genetically disposed to accelerated aging delays bone aging
and confers prolonged survival (Singh et al. (2013) Stem Cells 31:
607). These data support a stem cell autonomous mechanism for
tissue homeostasis that results in resistance to age related
disease and prolonged survival. Stem cells derived from iPS cells
with decelerated aging (sciPS) would be advantageous for transplant
because they would provide a longer period of stem cell fitness and
therefore a prolonged disease free period and increased longevity.
In this example, stem cells derived from reprogrammed
supercentenarian pluripotent stem cells (sciPS) are used to confer
healthy bone density and an extended period of resistance to
osteoporosis than would otherwise be obtained using stem cells
derived from iPS cells made from non-supercentenarian individuals.
The WRN-/-Terc-/- mouse model is used as a model of age related
osteoporosis (Singh et al. (2013) Stem Cells 31: 607). Human
mesenchymal stem cells (MSCs) from sciPS are prepared (e.g., see
Example 2). The cells are supplemented with iPS or sciPS derived
HSCs to reconstitute the immune system. Alternatively, the cells
are supplemented with whole bone marrow from old (20-24 months)
mice. The sciPS-MSCs are transplanted into WRN-/-Terc-/- at 3
months of age (n=10) as described (Singh et al. (2013) Stem Cells
31: 607). A second group of mice (n=10) have non-sciPS derived MSCs
transplanted and a third group (n=10) serves as untreated controls.
The mice are monitored every 3 months for bone density and signs of
bone aging (bone volume, cortical thickness, cortical area, total
volume, and trabecular number) and survival. A statistically
significant number of control iPS-MSC treated mice compared to
untreated mice that maintain normal bone density and survive longer
indicates the ability of the reprogramming to impart properties
comparable to young MSCs. A statistically significant sciPS-MSC
treated mice compared to untreated or iPS-MSC treated mice that
maintain normal bone density longer and survive longer indicates a
slower rate of aging in sciPS-MSCs and their advantage for
conferring longevity and resistance to degenerative disease.
Example 6: Identification of Gene Products that Induce sciPS
Derived Stem Cell Properties in Non-sciPS Derived Stem Cells
[0075] MSCs are derived, analyzed and sorted for a panel of MSC
surface antigens including CD105, CD73, and CD90 such that only
consistent surface marker defined cell populations are used for
further analysis. DNA, RNA, and protein samples are analyzed for
methylomic, transcriptomic and proteomic changes that occur with
increasing replicative age compared to equivalent non-sciPS-MSCs.
The data are analyzed to identify differentially expressed genes
that distinguish sciPS-MSCs from non-supercentenarian iPS-MSCs. The
identified candidate sciPS-MSC genes are tested using inhibitory
micro RNAs/siRNAs to knock down gene expression or introduction of
the candidate gene using recombinant plasmid DNA or viral gene
transfer for over expression. Knocked down genes that result in
loss of function (e.g. increase rate of cellular aging, early loss
of differentiation capacity) are introduced into non-sciPS in gain
of function experiments to determine whether they can induce
sciPS-MSC properties in non-sciPS-MSCs. Similarly, genes that when
overexpressed in sciPS-MSCs result in loss of function are knocked
down in control iPS cells to determine if down regulation induces
sciPS-MSC properties in control iPS-MSCs. Micro RNAs or siRNAs
corresponding to candidate genes that are down regulated in sciPS
are used to identify genes that when down regulated in control
iPS-MSCs induce the sciPS-MSC phenotype. The candidate
differentially regulated genes are used to determine the effect
these factors have on rate of changes in MSC differentiation
capacity and other indicators of rate of cellular aging.
[0076] In addition, sciPS cells are used in identification of genes
useful in surviving degenerative age-related diseases and
longevity. Cells are derived from supercentenarian patient(s) from
hair follicles or blood or by any other known means. These cells
are then reprogrammed using factors according to Yamanaka supra to
produce sciPS (induced pluripotent supercentenarian cell) clones.
The sciPS clones are assayed for telomerase activity. The clones
with high telomerase activity are propagated for 10, 20, 30, or
more than 30 passages and telomere length is monitored. The iPSC
clones that restore telomere length to 15-20 kb are expanded and
banked. sciPS clones are then differentiated spontaneously to
embryoid bodies (EB) or in a directed manner to different tissue
types including, without limitation, blood, skin, muscle, heart,
vascular, liver, lung, and pancreatic islet cells using reagents,
and/or cell matrix components and/or cytokines, and/or methods
known in the art. Gene expression profiling is then performed on
differentiated cells and compared to equivalent cells from known
non-supercentenarian controls. Taxonomically restricted (orphan)
genes are included in the expression analysis. Alternatively,
subtractive cDNA libraries are prepared to enrich for
differentially expressed genes that are identified by sequencing.
Analysis is performed to determine different upregulation and down
regulation of gene products including surface antigens identified
as developmentally regulated in the different cell populations and
these are tested in animal models (including, without limitation,
round worm, fruit fly, and mouse) for their effect on longevity.
High throughput drug screening is performed to find agents that
induce regulation and/or modulation of gene function that mimics
that seen in supercentenarian cells. In vitro aging of the sciPS
cells and cells derived therefrom is compared to equivalent cells
from a control population to gain an understanding regarding
cellular aging processes, and drugs such as, without limitation,
biologics, small molecules, drugs, nutraceuticals, cosmeceuticals,
compounds, and reagents, are tested in vitro to identify ones that
mimic the cellular aging process seen in sciPS cells and cells
derived therefrom.
Example 7: Conferred Longevity and Resistance to Disease Using Gene
Corrected iPS Derived Stem Cells
[0077] The genes and gene products identified in Example 6 are
tested in vivo for their ability to confer delayed cellular aging
and prolonged differentiation of MSCs using gene therapy and gene
editing methods to modify expression of endogenous genes in control
iPS-MSCs. The gene corrected iPS-MSCs, non-sciPS-MSCs and
sciPS-MSCs are compared functionally in a suitable animal model
such as the WRN-/-Terc-/- mouse. Successful gene modifications are
identified by their ability to confer onto control iPS-MSCs an
equivalent bone density and survival advantage as observed with
transplantation of sciPS-MSCs.
Example 8: Identification of Compounds that Induce sciPS Derived
Stem Cell Properties in Non-Supercentenarian iPS Derived Stem
Cells
[0078] High throughput agent screening on iPS-MSCs is used to
identify candidate agents that induce a sciPS-MSC phenotype in
iPS-MSCs. Various agent libraries including for example synthetic
compounds and natural organic compounds; and siRNAs and cDNAs may
be used. A change of expression of one or more differentially
expressed gene products (identified in Example 6) is used as an
indicator of compound potency for induction of sciPS properties.
Indicator cells are engineered from sciPS-MSCs by genetic
modification such that induction or repression of a sciPS-MSC
specific gene is detected by induction or repression of a
fluorescent signal. For example, a reporter gene such as green
fluorescent protein is fused to DNA encoding the regulatory
elements of a sciPS specific gene and the construct is introduced
into iPS-MSCs using standard recombinant DNA and transfection
methods. Candidate agents that are selected as hits are subjected
to a secondary screen to assess their effectiveness for inducing
sciPS-MSC properties such as reduced rate of cellular aging and
prolonged maintenance of differentiation capacity in treated
iPS-MSCs.
Example 9: Repair of Wounds and Aging Skin Using Compounds Produced
by sciPS Derived Cells
[0079] MSCs are derived from sciPS cells and grown under standard
culture conditions to 80% or greater confluence. The cells are
washed with phosphate buffered saline and incubated in a defined
serum free medium. The cell conditioned medium is collected
following at least 16 hour incubation with cells. The conditioned
medium is used directly or concentrated 5-10 fold by centrifugation
through a low molecular weight cut-off filter (e.g. Amicon 3000MW
cutoff). The conditioned medium is compared to identically prepared
non-sciPS-MSC conditioned medium for wound healing properties and
for ability to stimulate collagen production by human fibroblasts.
Similarly, cell lysates are prepared from sciPS-MSCs and
non-sciPS-MSCs and are tested for wound healing and collagen
stimulating properties. Wound healing properties are tested using
known in vitro and in vivo methods. In vitro methods include,
without limitation, using the scratch assay and cell migration
assay to assess the ability of the conditioned media and cell
lysates to stimulate wound repair. Human dermal fibroblasts are
incubated with conditioned medium or cell lysates from iPS-MSCs and
sciPS-MSCs or equivalent control untreated medium for at least 16
hours and collagen content of the media tested using Sicrol Assay
Kit (Biocolor Life Science Assays, United Kingdom).
[0080] All publications mentioned and/or referenced in the above
specification are herein incorporated by reference. Various
modifications and variations of the described methods will be
apparent to those skilled in the art without departing from the
scope and spirit of the invention. Although the invention has been
described with respect to particular aspects or embodiments and/or
further embodiments, it should be understood that the invention as
claimed should not be unduly limited to such aspects and/or
embodiments. It should also be understood that various
modifications of the described modes for carrying out the
invention, which would be readily known to and/or accessed through
available information by those skilled in cellular studies or
related fields, are intended to be within the scope of the
following claims.
* * * * *