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= Induced stem cells =
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Introduction
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Induced stem cells (iSC) are stem cells derived from somatic,
reproductive, pluripotent or other cell types by deliberate epigenetic
reprogramming. They are classified as either totipotent (iTC),
pluripotent (iPSC) or progenitor (multipotent - iMSC, also called an
induced multipotent progenitor cell - iMPC) or unipotent - (iUSC)
according to their developmental potential and degree of
dedifferentiation. Progenitors are obtained by so-called direct
reprogramming or directed differentiation and are also called induced
somatic stem cells.
Three techniques are widely recognized:
* Transplantation of nuclei taken from somatic cells into an oocyte
(egg cell) lacking its own nucleus (removed in lab)
* Fusion of somatic cells with pluripotent stem cells and
* Transformation of somatic cells into stem cells, using the genetic
material encoding reprogramming protein factors, recombinant proteins;
microRNA, a synthetic, self-replicating polycistronic RNA and
low-molecular weight biologically active substances.
Natural processes
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In 1895 Thomas Morgan removed one of a frog's two blastomeres and
found that amphibians are able to form whole embryos from the
remaining part. This meant that the cells can change their
differentiation pathway. In 1924 Spemann and Mangold demonstrated the
key importance of cell-cell inductions during animal development. The
reversible transformation of cells of one differentiated cell type to
another is called metaplasia. This transition can be a part of the
normal maturation process, or caused by an inducement.
One example is the transformation of iris cells to lens cells in the
process of maturation and transformation of retinal pigment epithelium
cells into the neural retina during regeneration in adult newt eyes.
This process allows the body to replace cells not suitable to new
conditions with more suitable new cells. In Drosophila imaginal discs,
cells have to choose from a limited number of standard discrete
differentiation states. The fact that transdetermination (change of
the path of differentiation) often occurs for a group of cells rather
than single cells shows that it is induced rather than part of
maturation.
The researchers were able to identify the minimal conditions and
factors that would be sufficient for starting the cascade of molecular
and cellular processes to instruct pluripotent cells to organize the
embryo. They showed that opposing gradients of bone morphogenetic
protein (BMP) and Nodal, two transforming growth factor family members
that act as morphogens, are sufficient to induce molecular and
cellular mechanisms required to organize, 'in vivo' or 'in vitro',
uncommitted cells of the zebrafish blastula animal pole into a
well-developed embryo.
Some types of mature, specialized adult cells can naturally revert to
stem cells. For example, "chief" cells express the stem cell marker
Troy. While they normally produce digestive fluids for the stomach,
they can revert into stem cells to make temporary repairs to stomach
injuries, such as a cut or damage from infection. Moreover, they can
make this transition even in the absence of noticeable injuries and
are capable of replenishing entire gastric units, in essence serving
as quiescent "reserve" stem cells. Differentiated airway epithelial
cells can revert into stable and functional stem cells in vivo.
After injury, mature terminally differentiated kidney cells
dedifferentiate into more primordial versions of themselves and then
differentiate into the cell types needing replacement in the damaged
tissue Macrophages can self-renew by local proliferation of mature
differentiated cells. In newts, muscle tissue is regenerated from
specialized muscle cells that dedifferentiate and forget the type of
cell they had been. This capacity to regenerate does not decline with
age and may be linked to their ability to make new stem cells from
muscle cells on demand.
A variety of nontumorigenic stem cells display the ability to generate
multiple cell types. For instance, multilineage-differentiating
stress-enduring (Muse) cells are stress-tolerant adult human stem
cells that can self-renew. They form characteristic cell clusters in
suspension culture that express a set of genes associated with
pluripotency and can differentiate into endodermal, ectodermal and
mesodermal cells both in vitro and in vivo.
Other well-documented examples of transdifferentiation and their
significance in development and regeneration were described in detail.
SCNT-mediated
===============
Induced totipotent cells can be obtained by reprogramming somatic
cells with somatic-cell nuclear transfer (SCNT). The process involves
sucking out the nucleus of a somatic (body) cell and injecting it into
an oocyte that has had its nucleus removed
Using an approach based on the protocol outlined by Tachibana et al.,
hESCs can be generated by SCNT using dermal fibroblasts nuclei from
both a middle-aged 35-year-old male and an elderly, 75-year-old male,
suggesting that age-associated changes are not necessarily an
impediment to SCNT-based nuclear reprogramming of human cells. Such
reprogramming of somatic cells to a pluripotent state holds huge
potentials for regenerative medicine. Unfortunately, the cells
generated by this technology, potentially are not completely protected
from the immune system of the patient (donor of nuclei), because they
have the same mitochondrial DNA, as a donor of oocytes, instead of the
patients mitochondrial DNA. This reduces their value as a source for
autologous stem cell transplantation therapy, as for the present, it
is not clear whether it can induce an immune response of the patient
upon treatment.
Induced androgenetic haploid embryonic stem cells can be used instead
of sperm for cloning. These cells, synchronized in M phase and
injected into the oocyte can produce viable offspring.
These developments, together with data on the possibility of unlimited
oocytes from mitotically active reproductive stem cells, offer the
possibility of industrial production of transgenic farm animals.
Repeated recloning of viable mice through a SCNT method that includes
a histone deacetylase inhibitor, trichostatin, added to the cell
culture medium, show that it may be possible to reclone animals
indefinitely with no visible accumulation of reprogramming or genomic
errors However, research into technologies to develop sperm and egg
cells from stem cells raises bioethical issues.
Such technologies may also have far-reaching clinical applications for
overcoming cytoplasmic defects in human oocytes. For example, the
technology could prevent inherited mitochondrial disease from passing
to future generations. Mitochondrial genetic material is passed from
mother to child. Mutations can cause diabetes, deafness, eye
disorders, gastrointestinal disorders, heart disease, dementia and
other neurological diseases. The nucleus from one human egg has been
transferred to another, including its mitochondria, creating a cell
that could be regarded as having two mothers. The eggs were then
fertilised and the resulting embryonic stem cells carried the swapped
mitochondrial DNA.
As evidence that the technique is safe author of this method points to
the existence of the healthy monkeys that are now more than four years
old - and are the product of mitochondrial transplants across
different genetic backgrounds.
In late-generation telomerase-deficient (Terc�/�) mice, SCNT-mediated
reprogramming mitigates telomere dysfunction and mitochondrial defects
to a greater extent than iPSC-based reprogramming.
Other cloning and totipotent transformation achievements have been
described.
Obtained without SCNT
=======================
Recently some researchers succeeded to get the totipotent cells
without the aid of SCNT. Totipotent cells were obtained using the
epigenetic factors such as oocyte germinal isoform of histone.
Reprogramming in vivo, by transitory induction of the four factors
Oct4, Sox2, Klf4 and c-Myc in mice, confers totipotency features.
Intraperitoneal injection of such in vivo iPS cells generates
embryo-like structures that express embryonic and extraembryonic
(trophectodermal) markers.
The developmental potential of mouse pluripotent stem cells to yield
both embryonic and extra-embryonic lineages also can be expanded by
microRNA miR-34a deficiency leading to strong induction of endogenous
retroviruses MuERV-L (MERVL).
Rejuvenation to iPSCs
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iPSc were first obtained in the form of transplantable teratocarcinoma
induced by grafts taken from mouse embryos. Teratocarcinoma formed
from somatic cells. Genetically mosaic mice were obtained from
malignant teratocarcinoma cells, confirming the cells' pluripotency.
It turned out that teratocarcinoma cells are able to maintain a
culture of pluripotent embryonic stem cell in an undifferentiated
state, by supplying the culture medium with various factors. In the
1980s, it became clear that transplanting pluripotent/embryonic stem
cells into the body of adult mammals, usually leads to the formation
of teratomas, which can then turn into a malignant tumor
teratocarcinoma. However, putting teratocarcinoma cells into the
embryo at the blastocyst stage, caused them to become incorporated in
the inner cell mass and often produced a normal chimeric (i.e.
composed of cells from different organisms) animal. This indicated
that the cause of the teratoma is a dissonance - mutual
miscommunication between young donor cells and surrounding adult cells
(the recipient's so-called "niche").
In August 2006, Japanese researchers circumvented the need for an
oocyte, as in SCNT. By reprograming mouse embryonic fibroblasts into
pluripotent stem cells via the ectopic expression of four
transcription factors, namely Oct4, Sox2, Klf4 and c-Myc, they proved
that the overexpression of a small number of factors can push the cell
to transition to a new stable state that is associated with changes in
the activity of thousands of genes.
Reprogramming mechanisms are thus linked, rather than independent and
are centered on a small number of genes.
IPSC properties are very similar to ESCs. iPSCs have been shown to
support the development of all-iPSC mice using a tetraploid (4n)
embryo, the most stringent assay for developmental potential. However,
some genetically normal iPSCs failed to produce all-iPSC mice because
of aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene
cluster. A team headed by Hans Schöler (who discovered the Oct4 gene
back in 1989) showed that Oct4 overexpression drives massive
off-target gene activation during reprogramming deteriorating the
quality of iPSCs. Comparing to OSKM (Oct4, Sox2, Klf4 and c-Myc)that
show abnormal imprinting and differentiation patterns, SKM (Sox2, Klf4
and c-Myc) reprogramming generates iPSCs with high developmental
potential (nearly 20-fold higher than that of OSKM) equivalent to
embryonic stem cell, as determined by their ability to generate
all-iPSC mice through tetraploid embryo complementation
An important advantage of iPSC over ESC is that they can be derived
from adult cells, rather than from embryos. Therefore, it became
possible to obtain iPSC from adult and even elderly patients.
Reprogramming somatic cells to iPSC leads to rejuvenation. It was
found that reprogramming leads to telomere lengthening and subsequent
shortening after their differentiation back into fibroblast-like
derivatives. Thus, reprogramming leads to the restoration of embryonic
telomere length, and hence increases the potential number of cell
divisions otherwise limited by the Hayflick limit.
However, because of the dissonance between rejuvenated cells and the
surrounding niche of the recipient's older cells, the injection of his
own iPSC usually leads to an immune response, which can be used for
medical purposes, or the formation of tumors such as teratoma. The
reason has been hypothesized to be that some cells differentiated from
ESC and iPSC in vivo continue to synthesize embryonic protein
isoforms. So, the immune system might detect and attack cells that are
not cooperating properly.
A small molecule called MitoBloCK-6 can force the pluripotent stem
cells to die by triggering apoptosis (via cytochrome c release across
the mitochondrial outer membrane) in human pluripotent stem cells, but
not in differentiated cells. Shortly after differentiation, daughter
cells became resistant to death. When MitoBloCK-6 was introduced to
differentiated cell lines, the cells remained healthy. The key to
their survival, was hypothesized to be due to the changes undergone by
pluripotent stem cell mitochondria in the process of cell
differentiation. This ability of MitoBloCK-6 to separate the
pluripotent and differentiated cell lines has the potential to reduce
the risk of teratomas and other problems in regenerative medicine.
In 2012 other small molecules (selective cytotoxic inhibitors of human
pluripotent stem cells - hPSCs) were identified that prevented human
pluripotent stem cells from forming teratomas in mice. The most potent
and selective compound of them (PluriSIn #1) inhibits stearoyl-coA
desaturase (the key enzyme in oleic acid biosynthesis), which finally
results in apoptosis. With the help of this molecule the
undifferentiated cells can be selectively removed from culture. An
efficient strategy to selectively eliminate pluripotent cells with
teratoma potential is targeting pluripotent stem cell-specific
antiapoptotic factor(s) (i.e., survivin or Bcl10). A single treatment
with chemical survivin inhibitors (e.g., quercetin or YM155) can
induce selective and complete cell death of undifferentiated hPSCs and
is claimed to be sufficient to prevent teratoma formation after
transplantation. However, it is unlikely that any kind of preliminary
clearance, is able to secure the replanting iPSC or ESC. After the
selective removal of pluripotent cells, they re-emerge quickly by
reverting differentiated cells into stem cells, which leads to tumors.
This may be due to the disorder of let-7 regulation of its target
Nr6a1 (also known as Germ cell nuclear factor - GCNF), an embryonic
transcriptional repressor of pluripotency genes that regulates gene
expression in adult fibroblasts following micro-RNA miRNA loss.
Teratoma formation by pluripotent stem cells may be caused by low
activity of PTEN enzyme, reported to promote the survival of a small
population (0.1-5% of total population) of highly tumorigenic,
aggressive, teratoma-initiating embryonic-like carcinoma cells during
differentiation. The survival of these teratoma-initiating cells is
associated with failed repression of Nanog as well as a propensity for
increased glucose and cholesterol metabolism. These
teratoma-initiating cells also expressed a lower ratio of p53/p21 when
compared to non-tumorigenic cells.
In connection with the above safety problems, the use iPSC for cell
therapy is still limited. However, they can be used for a variety of
other purposes - including the modeling of disease, screening
(selective selection) of drugs, toxicity testing of various drugs.
The tissue grown from iPSCs, placed in the "chimeric" embryos in the
early stages of mouse development, practically do not cause an immune
response (after the embryos have grown into adult mice) and are
suitable for autologous transplantation
At the same time, full reprogramming of adult cells in vivo within
tissues by transitory induction of the four factors Oct4, Sox2, Klf4
and c-Myc in mice results in teratomas emerging from multiple organs.
Furthermore, partial reprogramming of cells toward pluripotency in
vivo in mice demonstrates that incomplete reprogramming entails
epigenetic changes (failed repression of Polycomb targets and altered
DNA methylation) in cells that drive cancer development.
Chemical inducement
=====================
By using solely small molecules, Deng Hongkui and colleagues
demonstrated that endogenous "master genes" are enough for cell fate
reprogramming. They induced a pluripotent state in adult cells from
mice using seven small-molecule compounds.
The effectiveness of the method is quite high: it was able to convert
0.02% of the adult tissue cells into iPSCs, which is comparable to the
gene insertion conversion rate.
The authors note that the mice generated from CiPSCs were "100% viable
and apparently healthy for up to 6 months". So, this chemical
reprogramming strategy has potential use in generating functional
desirable cell types for clinical applications.
In 2015 a robust chemical reprogramming system was established with a
yield up to 1,000-fold greater than that of the previously reported
protocol. So, chemical reprogramming became a promising approach to
manipulate cell fates.
Differentiation from induced teratoma
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The fact that human iPSCs capable of forming teratomas not only in
humans but also in some animal body, in particular in mice or pigs,
allowed to develop a method for differentiation of iPSCs in vivo. For
this purpose, iPSCs with an agent for inducing differentiation into
target cells are injected to genetically modified pig or mouse that
has suppressed immune system activation on human cells.
The formed teratoma is cut out and used for the isolation of the
necessary differentiated human cells by means of monoclonal antibody
to tissue-specific markers on the surface of these cells. This method
has been successfully used for the production of functional myeloid,
erythroid and lymphoid human cells suitable for transplantation (yet
only to mice).
Mice engrafted with human iPSC teratoma-derived hematopoietic cells
produced human B and T cells capable of functional immune responses.
These results offer hope that in vivo generation of patient customized
cells is feasible, providing materials that could be useful for
transplantation, human antibody generation and drug screening
applications.
Using MitoBloCK-6 and/or PluriSIn # 1 the differentiated progenitor
cells can be further purified from teratoma forming pluripotent cells.
The fact, that the differentiation takes place even in the teratoma
niche, offers hope that the resulting cells are sufficiently stable to
stimuli able to cause their transition back to the dedifferentiated
(pluripotent) state and therefore safe. A similar in vivo
differentiation system, yielding engraftable hematopoietic stem cells
from mouse and human iPSCs in teratoma-bearing animals in combination
with a maneuver to facilitate hematopoiesis, was described by Suzuki
et al. They noted that neither leukemia nor tumors were observed in
recipients after intravenous injection of iPSC-derived hematopoietic
stem cells into irradiated recipients. Moreover, this injection
resulted in multilineage and long-term reconstitution of the
hematolymphopoietic system in serial transfers. Such system provides a
useful tool for practical application of iPSCs in the treatment of
hematologic and immunologic diseases.
For further development of this method animal in which is grown the
human cell graft, for example mouse, must have so modified genome that
all its cells express and have on its surface human SIRPα.
To prevent rejection after transplantation to the patient of the
allogenic organ or tissue, grown from the pluripotent stem cells in
vivo in the animal, these cells should express two molecules:
CTLA4-Ig, which disrupts T cell costimulatory pathways and PD-L1,
which activates T cell inhibitory pathway.
See also: .
Retinal cells
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In the near-future, clinical trials designed to demonstrate the safety
of the use of iPSCs for cell therapy of the people with age-related
macular degeneration, a disease causing blindness through retina
damaging, will begin. There are several articles describing methods
for producing retinal cells from iPSCs
and how to use them for cell therapy. Reports of iPSC-derived retinal
pigmented epithelium transplantation showed enhanced
visual-guided behaviors of experimental animals for 6 weeks after
transplantation. However, clinical trials have been successful: ten
patients suffering from retinitis pigmentosa have had their eyesight
restored - including a woman who had only 17 percent of her vision
left.
Lung and airway epithelial cells
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Chronic lung diseases such as idiopathic pulmonary fibrosis and cystic
fibrosis or chronic obstructive pulmonary disease and asthma are
leading causes of morbidity and mortality worldwide with a
considerable human, societal and financial burden. So there is an
urgent need for effective cell therapy and lung tissue engineering.
Several protocols have been developed for generation of the most cell
types of the respiratory system, which may be useful for deriving
patient-specific therapeutic cells.
Reproductive cells
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Some lines of iPSCs have the potentiality to differentiate into male
germ cells and oocyte-like cells in an appropriate niche (by culturing
in retinoic acid and porcine follicular fluid differentiation medium
or seminiferous tubule transplantation). Moreover, iPSC
transplantation make a contribution to repairing the testis of
infertile mice, demonstrating the potentiality of gamete derivation
from iPSCs in vivo and in vitro.
Direct transdifferentiation
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The risk of cancer and tumors creates the need to develop methods for
safer cell lines suitable for clinical use. An alternative approach is
so-called "direct reprogramming" - transdifferentiation of cells
without passing through the pluripotent state. The basis for this
approach was that 5-azacytidine - a DNA demethylation reagent - can
cause the formation of myogenic, chondrogenic and adipogeni clones in
the immortal cell line of mouse embryonic fibroblasts and that the
activation of a single gene, later named MyoD1, is sufficient for such
reprogramming. Compared with iPSC whose reprogramming requires at
least two weeks, the formation of induced progenitor cells sometimes
occurs within a few days and the efficiency of reprogramming is
usually many times higher. This reprogramming does not always require
cell division. The cells resulting from such reprogramming are more
suitable for cell therapy because they do not form teratomas.
For example, Chandrakanthan et al., & Pimanda describe the
generation of tissue-regenerative multipotent stem cells (iMS cells)
by treating mature bone and fat cells transiently with a growth factor
(platelet-derived growth factor-AB (PDGF-AB)) and 5-Azacytidine. These
authors claim that: "Unlike primary mesenchymal stem cells, which are
used with little objective evidence in clinical practice to promote
tissue repair, iMS cells contribute directly to in vivo tissue
regeneration in a context-dependent manner without forming tumors" and
so "has significant scope for application in tissue regeneration."
Single transcription factor transdifferentiation
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Originally only early embryonic cells could be coaxed into changing
their identity. Mature cells are resistant to changing their identity
once they've committed to a specific kind. However, brief expression
of a single transcription factor, the ELT-7 GATA factor, can convert
the identity of fully differentiated, specialized non-endodermal cells
of the pharynx into fully differentiated intestinal cells in intact
larvae and adult roundworm 'Caenorhabditis elegans' with no
requirement for a dedifferentiated intermediate.
Transdifferentiation with CRISPR-mediated activator
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The cell fate can be effectively manipulated by epigenome editing. In
particular, by directly activating of specific endogenous gene
expression with CRISPR-mediated activator. When dCas9 (that has been
modified so that it no longer cuts DNA, but still can be guided to
specific sequences and to bind to them) is combined with transcription
activators, it can precisely manipulate endogenous gene expression.
Using this method, Wei et al., enhanced the expression of endogenous
Cdx2 and Gata6 genes by CRISPR-mediated activators, thus directly
converted mouse embryonic stem cells into two extraembryonic lineages,
i.e., typical trophoblast stem cells and extraembryonic endoderm
cells. An analogous approach was used to induce activation of the
endogenous Brn2, Ascl1, and Myt1l genes to convert mouse embryonic
fibroblasts to induced neuronal cells. Thus, transcriptional
activation and epigenetic remodeling of endogenous master
transcription factors are sufficient for conversion between cell
types. The rapid and sustained activation of endogenous genes in their
native chromatin context by this approach may facilitate reprogramming
with transient methods that avoid genomic integration and provides a
new strategy for overcoming epigenetic barriers to cell fate
specification.
Phased process modeling regeneration
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Another way of reprogramming is the simulation of the processes that
occur during amphibian limb regeneration. In urodele amphibians, an
early step in limb regeneration is skeletal muscle fiber
dedifferentiation into a cellulate that proliferates into limb tissue.
However, sequential small molecule treatment of the muscle fiber with
myoseverin, reversine (the aurora B kinase inhibitor) and some other
chemicals: BIO (glycogen synthase-3 kinase inhibitor),
lysophosphatidic acid (pleiotropic activator of G-protein-coupled
receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl
cyclase inhibitor) causes the formation of new muscle cell types as
well as other cell types such as precursors to fat, bone and nervous
system cells.
Antibody-based transdifferentiation
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The researchers discovered that GCSF-mimicking antibody can activate a
growth-stimulating receptor on marrow cells in a way that induces
marrow stem cells that normally develop into white blood cells to
become neural progenitor cells. The technique enables researchers to
search large libraries of antibodies and quickly select the ones with
a desired biological effect.
Reprograming by bacteria
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The human gastrointestinal tract is colonized by a vast community of
symbionts and commensals. The researchers demonstrate the phenomenon
of somatic cell reprograming by bacteria and generation of
multipotential cells from adult human dermal fibroblast cells by
incorporating Lactic acid bacteria This cellular
transdifferentiation is caused by ribosomes and "can occur via donor
bacteria that are swallowed and digested by host cells, which may
induce ribosomal stress and stimulate cellular developmental
plasticity."
Conditionally reprogrammed cells
==================================
Schlegel and Liu demonstrated that the combination of feeder cells and
a Rho kinase inhibitor (Y-27632) induces normal and tumor epithelial
cells from many tissues to proliferate indefinitely in vitro. This
process occurs without the need for transduction of exogenous viral or
cellular genes. These cells have been termed "Conditionally
Reprogrammed Cells (CRC)". The induction of CRCs is rapid and results
from reprogramming of the entire cell population. CRCs do not express
high levels of proteins characteristic of iPSCs or embryonic stem
cells (ESCs) (e.g., Sox2, Oct4, Nanog, or Klf4). This induction of
CRCs is reversible and removal of Y-27632 and feeders allows the cells
to differentiate normally. CRC technology can generate 2 cells in 5 to
6 days from needle biopsies and can generate cultures from
cryopreserved tissue and from fewer than four viable cells. CRCs
retain a normal karyotype and remain nontumorigenic. This technique
also efficiently establishes cell cultures from human and rodent
tumors.
The ability to rapidly generate many tumor cells from small biopsy
specimens and frozen tissue provides significant opportunities for
cell-based diagnostics and therapeutics (including chemosensitivity
testing) and greatly expands the value of biobanking. Using CRC
technology, researchers were able to identify an effective therapy for
a patient with a rare type of lung tumor. Engleman's group describes a
pharmacogenomic platform that facilitates rapid discovery of drug
combinations that can overcome resistance using CRC system. In
addition, the CRC method allows for the genetic manipulation of
epithelial cells ex vivo and their subsequent evaluation in vivo in
the same host. While initial studies revealed that co-culturing
epithelial cells with Swiss 3T3 cells J2 was essential for CRC
induction, with transwell culture plates, physical contact between
feeders and epithelial cells is not required for inducing CRCs and
more importantly that irradiation of the feeder cells is required for
this induction. Consistent with the transwell experiments, conditioned
medium induces and maintains CRCs, which is accompanied by a
concomitant increase of cellular telomerase activity. The activity of
the conditioned medium correlates directly with radiation-induced
feeder cell apoptosis. Thus, conditional reprogramming of epithelial
cells is mediated by a combination of Y-27632 and a soluble factor(s)
released by apoptotic feeder cells.
Riegel et al. demonstrate that mouse ME cells isolated from normal
mammary glands or from mouse mammary tumor virus (MMTV)-Neu-induced
mammary tumors, can be cultured indefinitely as conditionally
reprogrammed cells (CRCs). Cell surface progenitor-associated markers
are rapidly induced in normal mouse ME-CRCs relative to ME cells.
However, the expression of certain mammary progenitor subpopulations,
such as CD49f+ ESA+ CD44+, drops significantly in later passages.
Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular
matrix gave rise to mammary acinar structures. ME-CRCs isolated from
MMTV-Neu transgenic mouse mammary tumors express high levels of
HER2/neu, as well as tumor-initiating cell markers, such as CD44+,
CD49f+ and ESA+ (EpCam). These patterns of expression are sustained in
later CRC passages. Early and late passage ME-CRCs from MMTV-Neu
tumors that were implanted in the mammary fat pads of syngeneic or
nude mice developed vascular tumors that metastasized within 6 weeks
of transplantation. Importantly, the histopathology of these tumors
was indistinguishable from that of the parental tumors that develop in
the MMTV-Neu mice. Application of the CRC system to mouse mammary
epithelial cells provides an attractive model system to study the
genetics and phenotype of normal and transformed mouse epithelium in a
defined culture environment and in vivo transplant studies.
A different approach to CRC is to inhibit CD47 - a membrane protein
that is the thrombospondin-1 receptor. Loss of CD47 permits sustained
proliferation of primary murine endothelial cells, increases
asymmetric division and enables these cells to spontaneously reprogram
to form multipotent embryoid body-like clusters. CD47 knockdown
acutely increases mRNA levels of c-Myc and other stem cell
transcription factors in cells in vitro and in vivo. Thrombospondin-1
is a key environmental signal that inhibits stem cell self-renewal via
CD47. Thus, CD47 antagonists enable cell self-renewal and
reprogramming by overcoming negative regulation of c-Myc and other
stem cell transcription factors. In vivo blockade of CD47 using an
antisense morpholino increases survival of mice exposed to lethal
total body irradiation due to increased proliferative capacity of bone
marrow-derived cells and radioprotection of radiosensitive
gastrointestinal tissues.
Lineage-specific enhancers
============================
Differentiated macrophages can self-renew in tissues and expand
long-term in culture. Under certain conditions macrophages can divide
without losing features they have acquired while specializing into
immune cells - which is usually not possible with differentiated
cells. The macrophages achieve this by activating a gene network
similar to one found in embryonic stem cells. Single-cell analysis
revealed that, 'in vivo', proliferating macrophages can derepress a
macrophage-specific enhancer repertoire associated with a gene network
controlling self-renewal. This happened when concentrations of two
transcription factors named MafB and c-Maf were naturally low or were
inhibited for a short time. Genetic manipulations that turned off MafB
and c-Maf in the macrophages caused the cells to start a self-renewal
program. The similar network also controls embryonic stem cell
self-renewal but is associated with distinct embryonic stem
cell-specific enhancers.
Hence macrophages isolated from MafB- and c-Maf-double deficient mice
divide indefinitely; the self-renewal depends on c-Myc and Klf4.
Indirect lineage conversion
=============================
Indirect lineage conversion is a reprogramming methodology in which
somatic cells transition through a plastic intermediate state of
partially reprogrammed cells (pre-iPSC), induced by brief exposure to
reprogramming factors, followed by differentiation in a specially
developed chemical environment (artificial niche).
This method could be both more efficient and safer, since it does not
seem to produce tumors or other undesirable genetic changes and
results in much greater yield than other methods. However, the safety
of these cells remains questionable. Since lineage conversion from
pre-iPSC relies on the use of iPSC reprogramming conditions, a
fraction of the cells could acquire pluripotent properties if they do
not stop the de-differentation process in vitro or due to further
de-differentiation in vivo.
Outer membrane glycoprotein
=============================
A common feature of pluripotent stem cells is the specific nature of
protein glycosylation of their outer membrane. That distinguishes them
from most nonpluripotent cells, although not white blood cells. The
glycans on the stem cell surface respond rapidly to alterations in
cellular state and signaling and are therefore ideal for identifying
even minor changes in cell populations. Many stem cell markers are
based on cell surface glycan epitopes including the widely used
markers SSEA-3, SSEA-4, Tra 1-60 and Tra 1-81. Suila Heli et al.
speculate that in human stem cells extracellular O-GlcNAc and
extracellular O-LacNAc, play a crucial role in the fine tuning of
Notch signaling pathway - a highly conserved cell signaling system,
that regulates cell fate specification, differentiation, left-right
asymmetry, apoptosis, somitogenesis, angiogenesis and plays a key role
in stem cell proliferation (reviewed by Perdigoto and Bardin and
Jafar-Nejad et al.)
Changes in outer membrane protein glycosylation are markers of cell
states connected in some way with pluripotency and differentiation.
The glycosylation change is apparently not just the result of the
initialization of gene expression, but perform as an important gene
regulator involved in the acquisition and maintenance of the
undifferentiated state.
For example, activation of glycoprotein ACA, linking
glycosylphosphatidylinositol on the surface of the progenitor cells in
human peripheral blood, induces increased expression of genes Wnt,
Notch-1, BMI1 and HOXB4 through a signaling cascade PI3K/Akt/mTor/PTEN
and promotes the formation of a self-renewing population of
hematopoietic stem cells.
Furthermore, dedifferentiation of progenitor cells induced by
ACA-dependent signaling pathway leads to ACA-induced pluripotent stem
cells, capable of differentiating in vitro into cells of all three
germ layers.
The study of lectins' ability to maintain a culture of pluripotent
human stem cells has led to the discovery of lectin Erythrina
crista-galli (ECA), which can serve as a simple and highly effective
matrix for the cultivation of human pluripotent stem cells.
Reprogramming through a physical approach
===========================================
Cell adhesion protein E-cadherin is indispensable for a robust
pluripotent phenotype. During reprogramming for iPS cell generation,
N-cadherin can replace function of E-cadherin. These functions of
cadherins are not directly related to adhesion because sphere
morphology helps maintaining the "stemness" of stem cells. Moreover,
sphere formation, due to forced growth of cells on a low attachment
surface, sometimes induces reprogramming. For example, neural
progenitor cells can be generated from fibroblasts directly through a
physical approach without introducing exogenous reprogramming factors.
Physical cues, in the form of parallel microgrooves on the surface of
cell-adhesive substrates, can replace the effects of small-molecule
epigenetic modifiers and significantly improve reprogramming
efficiency. The mechanism relies on the mechanomodulation of the
cells' epigenetic state. Specifically, "decreased histone deacetylase
activity and upregulation of the expression of WD repeat domain 5
(WDR5) - a subunit of H3 methyltranferase - by microgrooved surfaces
lead to increased histone H3 acetylation and methylation". Nanofibrous
scaffolds with aligned fibre orientation produce effects similar to
those produced by microgrooves, suggesting that changes in cell
morphology may be responsible for modulation of the epigenetic state.
Substrate rigidity is an important biophysical cue influencing neural
induction and subtype specification. For example, soft substrates
promote neuroepithelial conversion while inhibiting neural crest
differentiation of hESCs in a BMP4-dependent manner. Mechanistic
studies revealed a multi-targeted mechanotransductive process
involving mechanosensitive Smad phosphorylation and nucleocytoplasmic
shuttling, regulated by rigidity-dependent Hippo/YAP activities and
actomyosin cytoskeleton integrity and contractility.
Mouse embryonic stem cells (mESCs) undergo self-renewal in the
presence of the cytokine leukemia inhibitory factor (LIF). Following
LIF withdrawal, mESCs differentiate, accompanied by an increase in
cell-substratum adhesion and cell spreading. Restricted cell spreading
in the absence of LIF by either culturing mESCs on chemically defined,
weakly adhesive biosubstrates, or by manipulating the cytoskeleton
allowed the cells to remain in an undifferentiated and pluripotent
state. The effect of restricted cell spreading on mESC self-renewal is
not mediated by increased intercellular adhesion, as inhibition of
mESC adhesion using a function blocking anti E-cadherin antibody or
siRNA does not promote differentiation.
Possible mechanisms of stem cell fate predetermination by physical
interactions with the extracellular matrix have been described.
A new method has been developed that turns cells into stem cells
faster and more efficiently by 'squeezing' them using 3D
microenvironment stiffness and density of the surrounding gel. The
technique can be applied to a large number of cells to produce stem
cells for medical purposes on an industrial scale.
Cells involved in the reprogramming process change morphologically as
the process proceeds. This results in physical difference in adhesive
forces among cells. Substantial differences in 'adhesive signature'
between pluripotent stem cells, partially reprogrammed cells,
differentiated progeny and somatic cells allowed to develop separation
process for isolation of pluripotent stem cells in microfluidic
devices, which is:
#fast (separation takes less than 10 minutes);
#efficient (separation results in a greater than 95 percent pure iPS
cell culture);
#innocuous (cell survival rate is greater than 80 percent and the
resulting cells retain normal transcriptional profiles,
differentiation potential and karyotype).
Stem cells possess mechanical memory (they remember past physical
signals) - with the Hippo signaling pathway factors: Yes-associated
protein (YAP) and transcriptional coactivator with PDZ-binding domain
(TAZ) acting as an intracellular mechanical rheostat�that stores
information from past physical environments and influences the cells'
fate.
Neural stem cells
===================
Stroke and many neurodegenerative disorders such as Parkinson's
disease, Alzheimer's disease, amyotrophic lateral sclerosis need cell
replacement therapy. The successful use of converted neural cells
(cNs) in transplantations open a new avenue to treat such diseases.
Nevertheless, induced neurons (iNs), directly converted from
fibroblasts are terminally committed and exhibit very limited
proliferative ability that may not provide enough autologous donor
cells for transplantation. Self-renewing induced neural stem cells
(iNSCs) provide additional advantages over iNs for both basic research
and clinical applications.
For example, under specific growth conditions, mouse fibroblasts can
be reprogrammed with a single factor, Sox2, to form iNSCs that
self-renew in culture and after transplantation can survive and
integrate without forming tumors in mouse brains. INSCs can be derived
from adult human fibroblasts by non-viral techniques, thus offering a
safe method for autologous transplantation or for the development of
cell-based disease models.
Neural chemically induced progenitor cells (ciNPCs) can be generated
from mouse tail-tip fibroblasts and human urinary somatic cells
without introducing exogenous factors, but - by a chemical cocktail,
namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor
of GSK-3 kinases and R,
[
https://web.archive.org/web/20140328213812/http://xcessbio.com/index.php/small-
molecules/tgf/repsox.html
RepSox], an inhibitor of TGF beta signaling pathways), under a
physiological hypoxic condition. Alternative cocktails with inhibitors
of histone deacetylation, glycogen synthase kinase and TGF-β pathways
(where: sodium butyrate (NaB) or Trichostatin A (TSA) could replace
VPA, Lithium chloride (LiCl) or lithium carbonate (Li2CO3) could
substitute CHIR99021, or Repsox may be replaced with SB-431542 or
Tranilast) show similar efficacies for ciNPC induction.
Zhang, et al., also report highly efficient reprogramming of mouse
fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a
cocktail of nine components.
Multiple methods of direct transformation of somatic cells into
induced neural stem cells have been described.
Proof of principle experiments demonstrate that it is possible to
convert transplanted human fibroblasts and human astrocytes directly
in the brain that are engineered to express inducible forms of neural
reprogramming genes, into neurons, when reprogramming genes (Ascl1,
Brn2a and Myt1l) are activated after transplantation using a drug.
Astrocytes - the most common neuroglial brain cells, which contribute
to scar formation in response to injury - can be directly reprogrammed
in vivo to become functional neurons that formed networks in mice
without the need of cell transplantation. The researchers followed the
mice for nearly a year to look for signs of tumor formation and
reported finding none. The same researchers have turned scar-forming
astrocytes into progenitor cells called neuroblasts that regenerated
into neurons in the injured adult spinal cord.
Oligodendrocyte precursor cells
=================================
Without myelin to insulate neurons, nerve signals quickly lose power.
Diseases that attack myelin, such as multiple sclerosis, result in
nerve signals that cannot propagate to nerve endings and as a
consequence lead to cognitive, motor and sensory problems.
Transplantation of oligodendrocyte precursor cells (OPCs), which can
successfully create myelin sheaths around nerve cells, is a promising
potential therapeutic response. Direct lineage conversion of mouse and
rat fibroblasts into oligodendroglial cells provides a potential
source of OPCs. Conversion by forced expression of both eight or of
the three transcription factors Sox10, Olig2 and Zfp536, may provide
such cells.
Cardiomyocytes
================
Cell-based in vivo therapies may provide a transformative approach to
augment vascular and muscle growth and to prevent non-contractile scar
formation by delivering transcription factors or microRNAs to the
heart. Cardiac fibroblasts, which represent 50% of the cells in the
mammalian heart, can be reprogrammed into cardiomyocyte-like cells in
vivo by local delivery of cardiac core transcription factors ( GATA4,
MEF2C, TBX5 and for improved reprogramming plus ESRRG, MESP1,
Myocardin and ZFPM2) after coronary ligation. These results implicated
therapies that can directly remuscularize the heart without cell
transplantation. However, the efficiency of such reprogramming turned
out to be very low and the phenotype of received cardiomyocyte-like
cells does not resemble those of a mature normal cardiomyocyte.
Furthermore, transplantation of cardiac transcription factors into
injured murine hearts resulted in poor cell survival and minimal
expression of cardiac genes.
Meanwhile, advances in the methods of obtaining cardiac myocytes in
vitro occurred. Efficient cardiac differentiation of human iPS cells
gave rise to progenitors that were retained within infarcted rat
hearts and reduced remodeling of the heart after ischemic damage.
The team of scientists, who were led by Sheng Ding, used a cocktail of
nine chemicals (9C) for transdifferentiation of human skin cells into
beating heart cells. With this method, more than 97% of the cells
began beating, a characteristic of fully developed, healthy heart
cells. The chemically induced cardiomyocyte-like cells (ciCMs)
uniformly contracted and resembled human cardiomyocytes in their
transcriptome, epigenetic, and electrophysiological properties. When
transplanted into infarcted mouse hearts, 9C-treated fibroblasts were
efficiently converted to ciCMs and developed into healthy-looking
heart muscle cells within the organ. This chemical reprogramming
approach, after further optimization, may offer an easy way to provide
the cues that induce heart muscle to regenerate locally.
In another study, ischemic cardiomyopathy in the murine infarction
model was targeted by iPS cell transplantation. It synchronized
failing ventricles, offering a regenerative strategy to achieve
resynchronization and protection from decompensation by dint of
improved left ventricular conduction and contractility, reduced
scarring and reversal of structural remodelling.
One protocol generated populations of up to 98% cardiomyocytes from
hPSCs simply by modulating the canonical Wnt signaling pathway at
defined time points in during differentiation, using readily
accessible small molecule compounds.
Discovery of the mechanisms controlling the formation of
cardiomyocytes led to the development of the drug ITD-1, which
effectively clears the cell surface from TGF-β receptor type II and
selectively inhibits intracellular TGF-β signaling. It thus
selectively enhances the differentiation of uncommitted mesoderm to
cardiomyocytes, but not to vascular smooth muscle and endothelial
cells.
One project seeded decellularized mouse hearts with human iPSC-derived
multipotential cardiovascular progenitor cells. The introduced cells
migrated, proliferated and differentiated in situ into cardiomyocytes,
smooth muscle cells and endothelial cells to reconstruct the hearts.
In addition, the heart's extracellular matrix (the substrate of heart
scaffold) signalled the human cells into becoming the specialised
cells needed for proper heart function. After 20 days of perfusion
with growth factors, the engineered heart tissues started to beat
again and were responsive to drugs.
Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like
cells (iCMs) 'in situ' represents a promising strategy for cardiac
regeneration. Mice exposed 'in vivo', to three cardiac transcription
factors GMT (Gata4, Mef2c, Tbx5) and the small-molecules: SB-431542
(the transforming growth factor (TGF)-β inhibitor), and XAV939 (the
WNT inhibitor) for 2 weeks after myocardial infarction showed
significantly improved reprogramming (reprogramming efficiency
increased eight-fold) and cardiac function compared to those exposed
to only GMT.
See also: review
Rejuvenation of the muscle stem cell
======================================
The elderly often suffer from progressive muscle weakness and
regenerative failure owing in part to elevated activity of the p38α
and p38β mitogen-activated kinase pathway in senescent skeletal muscle
stem cells. Subjecting such stem cells to transient inhibition of p38α
and p38β in conjunction with culture on soft hydrogel substrates
rapidly expands and rejuvenates them that result in the return of
their strength.
In geriatric mice, resting satellite cells lose reversible quiescence
by switching to an irreversible pre-senescence state, caused by
derepression of p16INK4a (also called Cdkn2a).
On injury, these cells fail to activate and expand, even in a youthful
environment. p16INK4a silencing in geriatric satellite cells restores
quiescence and muscle regenerative functions.
Myogenic progenitors for potential use in disease modeling or
cell-based therapies targeting skeletal muscle could also be generated
directly from induced pluripotent stem cells using free-floating
spherical culture (EZ spheres) in a culture medium supplemented with
high concentrations (100 ng/ml) of fibroblast growth factor-2 (FGF-2)
and epidermal growth factor.
Hepatocytes
=============
Unlike current protocols for deriving hepatocytes from human
fibroblasts, Saiyong Zhu et al., (2014) did not generate iPSCs but,
using small molecules, cut short reprogramming to pluripotency to
generate an induced multipotent progenitor cell (iMPC) state from
which endoderm progenitor cells and subsequently hepatocytes
(iMPC-Heps) were efficiently differentiated. After transplantation
into an immune-deficient mouse model of human liver failure, iMPC-Heps
proliferated extensively and acquired levels of hepatocyte function
similar to those of human primary adult hepatocytes. iMPC-Heps did not
form tumours, most probably because they never entered a pluripotent
state.
These results establish the feasibility of significant liver
repopulation of mice with human hepatocytes generated in vitro, which
removes a long-standing roadblock on the path to autologous liver cell
therapy.
Cocktail of small molecules, Y-27632, A-83-01 (a TGFβ kinase/activin
receptor like kinase (ALK5) inhibitor), and CHIR99021 (potent
inhibitor of GSK-3), can convert rat and mouse mature hepatocytes in
vitro into proliferative bipotent cells - CLiPs (chemically induced
liver progenitors). CLiPs can differentiate into both mature
hepatocytes and biliary epithelial cells that can form functional
ductal structures. In long-term culture CLiPs did not lose their
proliferative capacity and their hepatic differentiation ability, and
can repopulate chronically injured liver tissue.
Insulin-producing cells
=========================
Complications of Diabetes mellitus such as cardiovascular diseases,
retinopathy, neuropathy, nephropathy and peripheral circulatory
diseases depend on sugar dysregulation due to lack of insulin from
pancreatic beta cells and can be lethal if they are not treated. One
of the promising approaches to understand and cure diabetes is to use
pluripotent stem cells (PSCs), including embryonic stem cells (ESCs)
and induced PCSs (iPSCs).
Unfortunately, human PSC-derived insulin-expressing cells resemble
human fetal β cells rather than adult β cells. In contrast to adult β
cells, fetal β cells seem functionally immature, as indicated by
increased basal glucose secretion and lack of glucose stimulation and
confirmed by RNA-seq of whose transcripts.
An alternative strategy is the conversion of fibroblasts towards
distinct endodermal progenitor cell populations and, using cocktails
of signalling factors, successful differentiation of these endodermal
progenitor cells into functional beta-like cells both in vitro and in
vivo.
Overexpression of the three transcription factors, PDX1 (required for
pancreatic bud outgrowth and beta-cell maturation), NGN3 (required for
endocrine precursor cell formation) and MAFA (for beta-cell
maturation) combination (called PNM) can lead to the transformation of
some cell types into a beta cell-like state.
An accessible and abundant source of functional insulin-producing
cells is intestine. PMN expression in human intestinal "organoids"
stimulates the conversion of intestinal epithelial cells into β-like
cells possibly acceptable for transplantation.
Nephron Progenitors
=====================
Adult proximal tubule cells were directly transcriptionally
reprogrammed to nephron progenitors of the embryonic kidney, using a
pool of six genes of instructive transcription factors (SIX1, SIX2,
OSR1, Eyes absent homolog 1(EYA1), Homeobox A11 (HOXA11) and Snail
homolog 2 (SNAI2)) that activated genes consistent with a cap
mesenchyme/nephron progenitor phenotype in the adult proximal tubule
cell line.
The generation of such cells may lead to cellular therapies for adult
renal disease. Embryonic kidney organoids placed into adult rat
kidneys can undergo onward development and vascular development.
Blood vessel cells
====================
As blood vessels age, they often become abnormal in structure and
function, thereby contributing to numerous age-associated diseases
including myocardial infarction, ischemic stroke and atherosclerosis
of arteries supplying the heart, brain and lower extremities. So, an
important goal is to stimulate vascular growth for the collateral
circulation to prevent the exacerbation of these diseases. Induced
Vascular Progenitor Cells (iVPCs) are useful for cell-based therapy
designed to stimulate coronary collateral growth. They were generated
by partially reprogramming endothelial cells. The vascular commitment
of iVPCs is related to the epigenetic memory of endothelial cells,
which engenders them as cellular components of growing blood vessels.
That is why, when iVPCs were implanted into myocardium, they engrafted
in blood vessels and increased coronary collateral flow better than
iPSCs, mesenchymal stem cells, or native endothelial cells.
Ex vivo genetic modification can be an effective strategy to enhance
stem cell function. For example, cellular therapy employing genetic
modification with Pim-1 kinase (a downstream effector of Akt, which
positively regulates neovasculogenesis) of bone marrow-derived cells
or human cardiac progenitor cells, isolated from failing myocardium
results in durability of repair, together with the improvement of
functional parameters of myocardial hemodynamic performance.
Stem cells extracted from fat tissue after liposuction may be coaxed
into becoming progenitor smooth muscle cells (iPVSMCs) found in
arteries and veins.
The 2D culture system of human iPS cells in conjunction with triple
marker selection (CD34 (a surface glycophosphoprotein expressed on
developmentally early embryonic fibroblasts), NP1 (receptor -
neuropilin 1) and KDR (kinase insert domain-containing receptor)) for
the isolation of vasculogenic precursor cells from human iPSC,
generated endothelial cells that, after transplantation, formed
stable, functional mouse blood vessels in vivo, lasting for 280 days.
To treat infarction, it is important to prevent the formation of
fibrotic scar tissue. This can be achieved in vivo by transient
application of paracrine factors that redirect native heart progenitor
stem cell contributions from scar tissue to cardiovascular tissue. For
example, in a mouse myocardial infarction model, a single
intramyocardial injection of human vascular endothelial growth factor
A mRNA (VEGF-A modRNA), modified to escape the body's normal defense
system, results in long-term improvement of heart function due to
mobilization and redirection of epicardial progenitor cells toward
cardiovascular cell types.
Red blood cells
=================
RBC transfusion is necessary for many patients. However, to date the
supply of RBCs remains labile. In addition, transfusion risks
infectious disease transmission. A large supply of safe RBCs generated
in vitro would help to address this issue. Ex vivo erythroid cell
generation may provide alternative transfusion products to meet
present and future clinical requirements. Red blood cells (RBC)s
generated in vitro from mobilized CD34 positive cells have normal
survival when transfused into an autologous recipient. RBC produced in
vitro contained exclusively fetal hemoglobin (HbF), which rescues the
functionality of these RBCs. In vivo the switch of fetal to adult
hemoglobin was observed after infusion of nucleated erythroid
precursors derived from iPSCs. Although RBCs do not have nuclei and
therefore can not form a tumor, their immediate erythroblasts
precursors have nuclei. The terminal maturation of erythroblasts into
functional RBCs requires a complex remodeling process that ends with
extrusion of the nucleus and the formation of an enucleated RBC. Cell
reprogramming often disrupts enucleation. Transfusion of in
vitro-generated RBCs or erythroblasts does not sufficiently protect
against tumor formation.
The aryl hydrocarbon receptor (AhR) pathway (which has been shown to
be involved in the promotion of cancer cell development) plays an
important role in normal blood cell development. AhR activation in
human hematopoietic progenitor cells (HPs) drives an unprecedented
expansion of HPs, megakaryocyte- and erythroid-lineage cells.
See also Concise Review:
The SH2B3 gene encodes a negative regulator of cytokine signaling and
naturally occurring loss-of-function variants in this gene increase
RBC counts in vivo. Targeted suppression of SH2B3 in primary human
hematopoietic stem and progenitor cells enhanced the maturation and
overall yield of in-vitro-derived RBCs. Moreover, inactivation of
SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells
allowed enhanced erythroid cell expansion with preserved
differentiation.
(See also overview.)
Platelets
===========
Platelets help prevent hemorrhage in thrombocytopenic patients and
patients with thrombocythemia. A significant problem for
multitransfused patients is refractoriness to platelet transfusions.
Thus, the ability to generate platelet products ex vivo and platelet
products lacking HLA antigens in serum-free media would have clinical
value.
An RNA interference-based mechanism used a lentiviral vector to
express short-hairpin RNAi targeting β2-microglobulin transcripts in
CD34-positive cells. Generated platelets demonstrated an 85% reduction
in class I HLA antigens. These platelets appeared to have normal
function in vitro
One clinically-applicable strategy for the derivation of functional
platelets from human iPSC involves the establishment of stable
immortalized megakaryocyte progenitor cell lines (imMKCLs) through
doxycycline-dependent overexpression of BMI1 and BCL-XL. The resulting
imMKCLs can be expanded in culture over extended periods (4-5 months),
even after cryopreservation. Halting the overexpression (by the
removal of doxycycline from the medium) of c-MYC, BMI1 and BCL-XL in
growing imMKCLs led to the production of CD42b+ platelets with
functionality comparable to that of native platelets on the basis of a
range of assays in vitro and in vivo.
Thomas et al., describe a forward programming strategy relying on the
concurrent exogenous expression of 3 transcription factors: GATA1,
FLI1 and TAL1. The forward programmed megakaryocytes proliferate and
differentiate in culture for several months with megakaryocyte purity
over 90% reaching up to 2x105 mature megakaryocytes per input hPSC.
Functional platelets are generated throughout the culture allowing the
prospective collection of several transfusion units from as few as one
million starting hPSCs.
See also overview
Immune cells
==============
A specialised type of white blood cell, known as cytotoxic T
lymphocytes (CTLs), are produced by the immune system and are able to
recognise specific markers on the surface of various infectious or
tumour cells, causing them to launch an attack to kill the harmful
cells. Thence, immunotherapy with functional antigen-specific T cells
has potential as a therapeutic strategy for combating many cancers and
viral infections. However, cell sources are limited, because they are
produced in small numbers naturally and have a short lifespan.
A potentially efficient approach for generating antigen-specific CTLs
is to revert mature immune T cells into iPSCs, which possess
indefinite proliferative capacity in vitro and after their
multiplication to coax them to redifferentiate back into T cells.
Another method combines iPSC and chimeric antigen receptor (CAR)
technologies to generate human T cells targeted to CD19, an antigen
expressed by malignant B cells, in tissue culture. This approach of
generating therapeutic human T cells may be useful for cancer
immunotherapy and other medical applications.
Invariant natural killer T (iNKT) cells have great clinical potential
as adjuvants for cancer immunotherapy. iNKT cells act as innate T
lymphocytes and serve as a bridge between the innate and acquired
immune systems. They augment anti-tumor responses by producing
interferon-gamma (IFN-γ). The approach of collection,
reprogramming/dedifferentiation, re-differentiation and injection has
been proposed for related tumor treatment.
Dendritic cells (DC) are specialized to control T-cell responses. DC
with appropriate genetic modifications may survive long enough to
stimulate antigen-specific CTL and after that be completely
eliminated. DC-like antigen-presenting cells obtained from human
induced pluripotent stem cells can serve as a source for vaccination
therapy.
CCAAT/enhancer binding protein-α (C/EBPα) induces transdifferentiation
of B cells into macrophages at high efficiencies and enhances
reprogramming into iPS cells when co-expressed with transcription
factors Oct4, Sox2, Klf4 and Myc. with a 100-fold increase in iPS cell
reprogramming efficiency, involving 95% of the population.
Furthermore, C/EBPa can convert selected human B cell lymphoma and
leukemia cell lines into macrophage-like cells at high efficiencies,
impairing the cells' tumor-forming capacity.
Thymic epithelial cells rejuvenation
======================================
The thymus is the first organ to deteriorate as people age. This
shrinking is one of the main reasons the immune system becomes less
effective with age. Diminished expression of the thymic epithelial
cell transcription factor FOXN1 has been implicated as a component of
the mechanism regulating age-related involution.
Clare Blackburn and colleagues show that established age-related
thymic involution can be reversed by forced upregulation of just one
transcription factor - FOXN1 in the thymic epithelial cells in order
to promote rejuvenation, proliferation and differentiation of these
cells into fully functional thymic epithelium.
This rejuvenation and increased proliferation was accompanied by
upregulation of genes that promote cell cycle progression (cyclin D1,
�Np63, FgfR2IIIb) and that are required in the thymic epithelial cells
to promote specific aspects of T cell development (Dll4, Kitl, Ccl25,
Cxcl12, Cd40, Cd80, Ctsl, Pax1).
Induction
===========
mesenchymal stem/stromal cells (MSCs) are under investigation for
applications in cardiac, renal, neural, joint and bone repair, as well
as in inflammatory conditions and hemopoietic cotransplantation. This
is because of their immunosuppressive properties and ability to
differentiate into a wide range of mesenchymal-lineage tissues. MSCs
are typically harvested from adult bone marrow or fat, but these
require painful invasive procedures and are low-frequency sources,
making up only 0.001-0.01% of bone marrow cells and 0.05% in
liposuction aspirates. Of concern for autologous use, in particular in
the elderly most in need of tissue repair, MSCs decline in quantity
and quality with age.
IPSCs could be obtained by the cells rejuvenation of even
centenarians. Because iPSCs can be harvested free of ethical
constraints and culture can be expanded indefinitely, they are an
advantageous source of MSCs. IPSC treatment with SB-431542 leads to
rapid and uniform MSC generation from human iPSCs. (SB-431542 is an
inhibitor of activin/TGF- pathways by blocking phosphorylation of
ALK4, ALK5 and ALK7 receptors.) These iPS-MSCs may lack
teratoma-forming ability, display a normal stable karyotype in culture
and exhibit growth and differentiation characteristics that closely
resemble those of primary MSCs. It has potential for in vitro
scale-up, enabling MSC-based therapies. MSC derived from iPSC have the
capacity to aid periodontal regeneration and are a promising source of
readily accessible stem cells for use in the clinical treatment of
periodontitis.
Lai et al., & Lu report the chemical method to generate MSC-like
cells (iMSCs), from human primary dermal fibroblasts using six
chemical inhibitors (SP600125, SB202190, Go6983, Y-27632, PD0325901,
and CHIR99021) with or without 3 growth factors (transforming growth
factor-β (TGF-β), basic fibroblast growth factor (bFGF), and leukemia
inhibitory factor (LIF)). The chemical cocktail directly converts
human fibroblasts to iMSCs with a monolayer culture in 6 days, and the
conversion rate was approximately 38%.
Besides cell therapy in vivo, the culture of human mesenchymal stem
cells can be used in vitro for mass-production of exosomes, which are
ideal vehicles for drug delivery.
Dedifferentiated adipocytes
=============================
Adipose tissue, because of its abundance and relatively less invasive
harvest methods, represents a source of mesenchymal stem cells (MSCs).
Unfortunately, liposuction aspirates are only 0.05% MSCs. However, a
large amount of mature adipocytes, which in general have lost their
proliferative abilities and therefore are typically discarded, can be
easily isolated from the adipose cell suspension and dedifferentiated
into lipid-free fibroblast-like cells, named dedifferentiated fat
(DFAT) cells. DFAT cells re-establish active proliferation ability and
express multipotent capacities. Compared with adult stem cells, DFAT
cells show unique advantages in abundance, isolation and homogeneity.
Under proper induction culture in vitro or proper environment in vivo,
DFAT cells could demonstrate adipogenic, osteogenic, chondrogenic and
myogenic potentials. They could also exhibit perivascular
characteristics and elicit neovascularization.
Chondrogenic cells
====================
Cartilage is the connective tissue responsible for frictionless joint
movement. Its degeneration ultimately results in complete loss of
joint function in the late stages of osteoarthritis. As an avascular
and hypocellular tissue, cartilage has a limited capacity for
self-repair. Chondrocytes are the only cell type in cartilage, in
which they are surrounded by the extracellular matrix that they
secrete and assemble.
One method of producing cartilage is to induce it from iPS cells.
Alternatively, it is possible to convert fibroblasts directly into
induced chondrogenic cells (iChon) without an intermediate iPS cell
stage, by inserting three reprogramming factors (c-MYC, KLF4 and
SOX9). Human iChon cells expressed marker genes for chondrocytes (type
II collagen) but not fibroblasts.
Implanted into defects created in the articular cartilage of rats,
human iChon cells survived to form cartilaginous tissue for at least
four weeks, with no tumors. The method makes use of c-MYC, which is
thought to have a major role in tumorigenesis and employs a retrovirus
to introduce the reprogramming factors, excluding it from unmodified
use in human therapy.
Sources of cells for reprogramming
======================================================================
The most frequently used sources for reprogramming are blood cells and
fibroblasts, obtained by biopsy of the skin, but taking cells from
urine is less invasive. The latter method does not require a biopsy or
blood sampling. As of 2013, urine-derived stem cells had been
differentiated into endothelial, osteogenic, chondrogenic, adipogenic,
skeletal myogenic and neurogenic lineages, without forming teratomas.
Therefore, their epigenetic memory is suited to reprogramming into iPS
cells. However, few cells appear in urine, only low conversion
efficiencies had been achieved and the risk of bacterial contamination
is relatively high.
Another promising source of cells for reprogramming are mesenchymal
stem cells derived from human hair follicles.
The origin of somatic cells used for reprogramming may influence the
efficiency of reprogramming, the functional properties of the
resulting induced stem cells and the ability to form tumors.
IPSCs retain an epigenetic memory of their tissue of origin, which
impacts their differentiation potential.
This epigenetic memory does not necessarily manifest itself at the
pluripotency stage - iPSCs derived from different tissues exhibit
proper morphology, express pluripotency markers and are able to
differentiate into the three embryonic layers in vitro and in vivo.
However, this epigenetic memory may manifest during re-differentiation
into specific cell types that require the specific loci bearing
residual epigenetic marks.
See also
======================================================================
* Transdifferentiation
* Examples of in vitro transdifferentiation by lineage-instructive
approach
* Examples of in vitro transdifferentiation by initial epigenetic
activation phase approach
* Examples of in vivo transdifferentiation by lineage-instructive
approach
* Injury induced stem cell niches
* Transcription factors
* Growth factors
* Pioneer factors
* Cellular differentiation
* CAF-1
Further reading
======================================================================
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* [
https://www.sciencedaily.com/releases/2012/10/121008082955.htm
Nobel Prize in Physiology or Medicine 2012 Awarded for Discovery That
Mature Cells Can Be Reprogrammed to Become Pluripotent]
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* a 50-fold increase in efficiency; small volumes; differentiation
into desired cells in the same platform
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[
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/St
em-Cell-Research/Stem-Cell-Engineering-Reprogramming.html
Detailed protocols for reprogramming and for analysis of iPSCs]
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License
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http://creativecommons.org/licenses/by-sa/3.0/
Original Article:
http://en.wikipedia.org/wiki/Induced_stem_cells
