transcription factor – a protein that binds to DNA and regulates gene expression by promoting or suppressing transcription
transcriptional regulation – controlling the rate of gene transcription for example by helping or hindering RNA polymerase binding to DNA
upregulation, activation, or promotion – increase the rate of gene transcription
downregulation, repression, or suppression – decrease the rate of gene transcription
coactivator – a protein (or a small molecule) that works with transcription factors to increase the rate of gene transcription
corepressor – a protein (or a small molecule) that works with transcription factors to decrease the rate of gene transcription
response element – a specific sequence of DNA that a transcription factor binds to
Illustration of an activator
In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The function of TFs is to regulate—turn on and off—genes in order to make sure that they are expressed in the desired cells
at the right time and in the right amount throughout the life of the
cell and the organism. Groups of TFs function in a coordinated fashion
to direct cell division, cell growth, and cell death throughout life; cell migration and organization (body plan) during embryonic development; and intermittently in response to signals from outside the cell, such as a hormone. There are 1500-1600 TFs in the human genome. Transcription factors are members of the proteome as well as regulome.
TFs work alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes.
Transcription factors are essential for the regulation of gene
expression and are, as a consequence, found in all living organisms. The
number of transcription factors found within an organism increases with
genome size, and larger genomes tend to have more transcription factors
per gene.
There are approximately 2800 proteins in the human genome that contain DNA-binding domains, and 1600 of these are presumed to function as transcription factors, though other studies indicate it to be a smaller number.
Therefore, approximately 10% of genes in the genome code for
transcription factors, which makes this family the single largest family
of human proteins. Furthermore, genes are often flanked by several
binding sites for distinct transcription factors, and efficient
expression of each of these genes requires the cooperative action of
several different transcription factors (see, for example, hepatocyte nuclear factors).
Hence, the combinatorial use of a subset of the approximately 2000
human transcription factors easily accounts for the unique regulation of
each gene in the human genome during development.
Mechanism
Transcription factors bind to either enhancer or promoter
regions of DNA adjacent to the genes that they regulate. Depending on
the transcription factor, the transcription of the adjacent gene is
either up- or down-regulated. Transcription factors use a variety of mechanisms for the regulation of gene expression. These mechanisms include:
stabilize or block the binding of RNA polymerase to DNA
catalyze the acetylation or deacetylation of histone
proteins. The transcription factor can either do this directly or
recruit other proteins with this catalytic activity. Many transcription
factors use one or the other of two opposing mechanisms to regulate
transcription:
histone acetyltransferase (HAT) activity – acetylates histone proteins, which weakens the association of DNA with histones, which make the DNA more accessible to transcription, thereby up-regulating transcription
histone deacetylase (HDAC) activity – deacetylates histone
proteins, which strengthens the association of DNA with histones, which
make the DNA less accessible to transcription, thereby down-regulating
transcription
Transcription
factors are one of the groups of proteins that read and interpret the
genetic "blueprint" in the DNA. They bind to the DNA and help initiate a
program of increased or decreased gene transcription. As such, they
are vital for many important cellular processes. Below are some of the
important functions and biological roles transcription factors are
involved in:
Other transcription factors differentially regulate the expression of various genes by binding to enhancer
regions of DNA adjacent to regulated genes. These transcription
factors are critical to making sure that genes are expressed in the
right cell at the right time and in the right amount, depending on the
changing requirements of the organism.
Development
Many transcription factors in multicellular organisms are involved in development.
Responding to stimuli, these transcription factors turn on/off the
transcription of the appropriate genes, which, in turn, allows for
changes in cell morphology or activities needed for cell fate determination and cellular differentiation. The Hox transcription factor family, for example, is important for proper body pattern formation in organisms as diverse as fruit flies to humans. Another example is the transcription factor encoded by the sex-determining region Y (SRY) gene, which plays a major role in determining sex in humans.
Response to intercellular signals
Cells can communicate with each other by releasing molecules that produce signaling cascades
within another receptive cell. If the signal requires upregulation or
downregulation of genes in the recipient cell, often transcription
factors will be downstream in the signaling cascade. Estrogen signaling is an example of a fairly short signaling cascade that involves the estrogen receptor transcription factor: Estrogen is secreted by tissues such as the ovaries and placenta, crosses the cell membrane of the recipient cell, and is bound by the estrogen receptor in the cell's cytoplasm. The estrogen receptor then goes to the cell's nucleus and binds to its DNA-binding sites, changing the transcriptional regulation of the associated genes.
Response to environment
Not
only do transcription factors act downstream of signaling cascades
related to biological stimuli but they can also be downstream of
signaling cascades involved in environmental stimuli. Examples include heat shock factor (HSF), which upregulates genes necessary for survival at higher temperatures, hypoxia inducible factor (HIF), which upregulates genes necessary for cell survival in low-oxygen environments, and sterol regulatory element binding protein (SREBP), which helps maintain proper lipid levels in the cell.
Cell cycle control
Many transcription factors, especially some that are proto-oncogenes or tumor suppressors, help regulate the cell cycle and as such determine how large a cell will get and when it can divide into two daughter cells. One example is the Myc oncogene, which has important roles in cell growth and apoptosis.
Pathogenesis
Transcription
factors can also be used to alter gene expression in a host cell to
promote pathogenesis. A well studied example of this are the
transcription-activator like effectors (TAL effectors) secreted by Xanthomonas
bacteria. When injected into plants, these proteins can enter the
nucleus of the plant cell, bind plant promoter sequences, and activate
transcription of plant genes that aid in bacterial infection.
TAL effectors contain a central repeat region in which there is a
simple relationship between the identity of two critical residues in
sequential repeats and sequential DNA bases in the TAL effector's target
site.
This property likely makes it easier for these proteins to evolve in
order to better compete with the defense mechanisms of the host cell.
Regulation
It
is common in biology for important processes to have multiple layers of
regulation and control. This is also true with transcription factors:
Not only do transcription factors control the rates of transcription to
regulate the amounts of gene products (RNA and protein) available to
the cell but transcription factors themselves are regulated (often by
other transcription factors). Below is a brief synopsis of some of the
ways that the activity of transcription factors can be regulated:
Synthesis
Transcription
factors (like all proteins) are transcribed from a gene on a chromosome
into RNA, and then the RNA is translated into protein. Any of these
steps can be regulated to affect the production (and thus activity) of a
transcription factor. An implication of this is that transcription
factors can regulate themselves. For example, in a negative feedback
loop, the transcription factor acts as its own repressor: If the
transcription factor protein binds the DNA of its own gene, it
down-regulates the production of more of itself. This is one mechanism
to maintain low levels of a transcription factor in a cell.
Nuclear localization
In eukaryotes, transcription factors (like most proteins) are transcribed in the nucleus but are then translated in the cell's cytoplasm. Many proteins that are active in the nucleus contain nuclear localization signals that direct them to the nucleus. But, for many transcription factors, this is a key point in their regulation. Important classes of transcription factors such as some nuclear receptors must first bind a ligand while in the cytoplasm before they can relocate to the nucleus.
Activation
Transcription factors may be activated (or deactivated) through their signal-sensing domain by a number of mechanisms including:
ligand
binding – Not only is ligand binding able to influence where a
transcription factor is located within a cell but ligand binding can
also affect whether the transcription factor is in an active state and
capable of binding DNA or other cofactors (see, for example, nuclear receptors).
interaction with other transcription factors (e.g., homo- or hetero-dimerization) or coregulatory proteins
Accessibility of DNA-binding site
In eukaryotes, DNA is organized with the help of histones into compact particles called nucleosomes,
where sequences of about 147 DNA base pairs make ~1.65 turns around
histone protein octamers. DNA within nucleosomes is inaccessible to many
transcription factors. Some transcription factors, so-called pioneer factors
are still able to bind their DNA binding sites on the nucleosomal DNA.
For most other transcription factors, the nucleosome should be actively
unwound by molecular motors such as chromatin remodelers.
Alternatively, the nucleosome can be partially unwrapped by thermal
fluctuations, allowing temporary access to the transcription factor
binding site. In many cases, a transcription factor needs to compete for binding to its DNA binding site with other transcription factors and histones or non-histone chromatin proteins.
Pairs of transcription factors and other proteins can play
antagonistic roles (activator versus repressor) in the regulation of the
same gene.
Availability of other cofactors/transcription factors
Most
transcription factors do not work alone. Many large TF families form
complex homotypic or heterotypic interactions through dimerization.
For gene transcription to occur, a number of transcription factors must
bind to DNA regulatory sequences. This collection of transcription
factors, in turn, recruit intermediary proteins such as cofactors that allow efficient recruitment of the preinitiation complex and RNA polymerase.
Thus, for a single transcription factor to initiate transcription, all
of these other proteins must also be present, and the transcription
factor must be in a state where it can bind to them if necessary.
Cofactors are proteins that modulate the effects of transcription
factors. Cofactors are interchangeable between specific gene promoters;
the protein complex that occupies the promoter DNA and the amino acid
sequence of the cofactor determine its spatial conformation. For
example, certain steroid receptors can exchange cofactors with NF-κB,
which is a switch between inflammation and cellular differentiation;
thereby steroids can affect the inflammatory response and function of
certain tissues.
Interaction with methylated cytosine
Transcription
factors and methylated cytosines in DNA both have major roles in
regulating gene expression. (Methylation of cytosine in DNA primarily
occurs where cytosine is followed by guanine in the 5' to 3' DNA
sequence, a CpG site.) Methylation of CpG sites in a promoter region of a gene usually represses gene transcription, while methylation of CpGs in the body of a gene increases expression. TET enzymes
play a central role in demethylation of methylated cytosines.
Demethylation of CpGs in a gene promoter by TET enzyme activity
increases transcription of the gene.
The DNA binding sites of 519 transcription factors were evaluated.
Of these, 169 transcription factors (33%) did not have CpG
dinucleotides in their binding sites, and 33 transcription factors (6%)
could bind to a CpG-containing motif but did not display a preference
for a binding site with either a methylated or unmethylated CpG. There
were 117 transcription factors (23%) that were inhibited from binding to
their binding sequence if it contained a methylated CpG site, 175
transcription factors (34%) that had enhanced binding if their binding
sequence had a methylated CpG site, and 25 transcription factors (5%)
were either inhibited or had enhanced binding depending on where in the
binding sequence the methylated CpG was located.
TET enzymes do not specifically bind to methylcytosine except when recruited (see DNA demethylation). Multiple transcription factors important in cell differentiation and lineage specification, including NANOG, SALL4A, WT1, EBF1, PU.1, and E2A,
have been shown to recruit TET enzymes to specific genomic loci
(primarily enhancers) to act on methylcytosine (mC) and convert it to
hydroxymethylcytosine hmC (and in most cases marking them for subsequent
complete demethylation to cytosine). TET-mediated conversion of mC to hmC appears to disrupt the binding of 5mC-binding proteins including MECP2 and MBD (Methyl-CpG-binding domain)
proteins, facilitating nucleosome remodeling and the binding of
transcription factors, thereby activating transcription of those genes.
EGR1 is an important transcription factor in memory formation. It has an essential role in brainneuronepigenetic reprogramming. The transcription factor EGR1 recruits the TET1 protein that initiates a pathway of DNA demethylation.
EGR1, together with TET1, is employed in programming the distribution
of methylation sites on brain DNA during brain development and in learning (see Epigenetics in learning and memory).
Structure
Schematic
diagram of the amino acid sequence (amino terminus to the left and
carboxylic acid terminus to the right) of a prototypical transcription
factor that contains (1) a DNA-binding domain (DBD), (2) signal-sensing
domain (SSD), and Activation domain (AD). The order of placement and the
number of domains may differ in various types of transcription factors.
In addition, the transactivation and signal-sensing functions are
frequently contained within the same domain.Domain architecture example: Lactose Repressor (LacI). The N-terminal DNA binding domain (labeled) of the lac repressor binds its target DNA sequence (gold) in the major groove using a helix-turn-helix
motif. Effector molecule binding (green) occurs in the regulatory
domain (labeled). This triggers an allosteric response mediated by the
linker region (labeled).
Transcription factors are modular in structure and contain the following domains:
DNA-binding domain (DBD), which attaches to specific sequences of DNA (enhancer or promoter. Necessary component for all vectors. Used to drive transcription of the vector's transgene promoter sequences) adjacent to regulated genes. DNA sequences that bind transcription factors are often referred to as response elements.
Activation domain (AD), which contains binding sites for other proteins such as transcription coregulators. These binding sites are frequently referred to as activation functions (AFs), Transactivation domain (TAD) or Trans-activating domainTAD, not to be confused with topologically associating domain (TAD).
An optional signal-sensing domain (SSD) (e.g., a
ligand-binding domain), which senses external signals and, in response,
transmits these signals to the rest of the transcription complex,
resulting in up- or down-regulation of gene expression. Also, the DBD
and signal-sensing domains may reside on separate proteins that
associate within the transcription complex to regulate gene expression.
DNA-binding domain
DNA contacts of different types of DNA-binding domains of transcription factors
The portion (domain)
of the transcription factor that binds DNA is called its DNA-binding
domain. Below is a partial list of some of the major families of
DNA-binding domains/transcription factors:
homeodomain proteins, which are encoded by homeobox genes, are transcription factors. Homeodomain proteins play critical roles in the regulation of development.
Transcription factors interact with their binding sites using a combination of electrostatic (of which hydrogen bonds are a special case) and Van der Waals forces.
Due to the nature of these chemical interactions, most transcription
factors bind DNA in a sequence specific manner. However, not all bases
in the transcription factor-binding site may actually interact with the
transcription factor. In addition, some of these interactions may be
weaker than others. Thus, transcription factors do not bind just one
sequence but are capable of binding a subset of closely related
sequences, each with a different strength of interaction.
For example, although the consensus binding site for the TATA-binding protein (TBP) is TATAAAA, the TBP transcription factor can also bind similar sequences such as TATATAT or TATATAA.
Because transcription factors can bind a set of related sequences
and these sequences tend to be short, potential transcription factor
binding sites can occur by chance if the DNA sequence is long enough.
It is unlikely, however, that a transcription factor will bind all
compatible sequences in the genome of the cell. Other constraints, such as DNA accessibility in the cell or availability of cofactors
may also help dictate where a transcription factor will actually bind.
Thus, given the genome sequence, it is still difficult to predict where
a transcription factor will actually bind in a living cell.
Additional recognition specificity, however, may be obtained
through the use of more than one DNA-binding domain (for example tandem
DBDs in the same transcription factor or through dimerization of two
transcription factors) that bind to two or more adjacent sequences of
DNA.
Clinical significance
Transcription
factors are of clinical significance for at least two reasons: (1)
mutations can be associated with specific diseases, and (2) they can be
targets of medications.
Disorders
Due
to their important roles in development, intercellular signaling, and
cell cycle, some human diseases have been associated with mutations in transcription factors.
Many transcription factors are either tumor suppressors or oncogenes,
and, thus, mutations or aberrant regulation of them is associated with
cancer. Three groups of transcription factors are known to be important
in human cancer: (1) the NF-kappaB and AP-1 families, (2) the STAT family and (3) the steroid receptors.
Mutations in the FOXP2 transcription factor are associated with developmental verbal dyspraxia, a disease in which individuals are unable to produce the finely coordinated movements required for speech.
Approximately 10% of currently prescribed drugs directly target the nuclear receptor class of transcription factors. Examples include tamoxifen and bicalutamide for the treatment of breast and prostate cancer, respectively, and various types of anti-inflammatory and anabolicsteroids. In addition, transcription factors are often indirectly modulated by drugs through signaling cascades. It might be possible to directly target other less-explored transcription factors such as NF-κB with drugs. Transcription factors outside the nuclear receptor family are thought to be more difficult to target with small molecule therapeutics since it is not clear that they are "drugable" but progress has been made on Pax2 and the notch pathway.
Gene duplications have played a crucial role in the evolution
of species. This applies particularly to transcription factors. Once
they occur as duplicates, accumulated mutations encoding for one copy
can take place without negatively affecting the regulation of downstream
targets. However, changes of the DNA binding specificities of the
single-copy Leafy
transcription factor, which occurs in most land plants, have recently
been elucidated. In that respect, a single-copy transcription factor can
undergo a change of specificity through a promiscuous intermediate
without losing function. Similar mechanisms have been proposed in the
context of all alternative phylogenetic hypotheses, and the role of transcription factors in the evolution of all species.
Role in biocontrol activity
The transcription factors have a role in resistance activity which is important for successful biocontrol activity. The resistant to oxidative stress and alkaline pH sensing were contributed from the transcription factor Yap1 and Rim101 of the Papiliotrema terrestris LS28 as molecular tools revealed an understanding of the genetic mechanisms underlying the biocontrol activity which supports disease management programs based on biological and integrated control.
Analysis
There are different technologies available to analyze transcription factors. On the genomic level, DNA-sequencing and database research are commonly used. The protein version of the transcription factor is detectable by using specific antibodies. The sample is detected on a western blot. By using electrophoretic mobility shift assay (EMSA), the activation profile of transcription factors can be detected. A multiplex
approach for activation profiling is a TF chip system where several
different transcription factors can be detected in parallel.
The most commonly used method for identifying transcription factor binding sites is chromatin immunoprecipitation (ChIP). This technique relies on chemical fixation of chromatin with formaldehyde, followed by co-precipitation of DNA and the transcription factor of interest using an antibody that specifically targets that protein. The DNA sequences can then be identified by microarray or high-throughput sequencing (ChIP-seq) to determine transcription factor binding sites. If no antibody is available for the protein of interest, DamID may be a convenient alternative.
Classes
As
described in more detail below, transcription factors may be classified
by their (1) mechanism of action, (2) regulatory function, or (3)
sequence homology (and hence structural similarity) in their DNA-binding
domains.
Mechanistic
There are two mechanistic classes of transcription factors:
Upstream transcription factors are proteins that bind
somewhere upstream of the initiation site to stimulate or repress
transcription. These are roughly synonymous with specific transcription factors, because they vary considerably depending on what recognition sequences are present in the proximity of the gene.
II.B.2 intracellular ligand (autocrine)-dependent – activated by small intracellular molecules – SREBP, p53, orphan nuclear receptors
II.B.3 cell membrane receptor-dependent – second messenger signaling cascades resulting in the phosphorylation of the transcription factor
II.B.3.a resident nuclear factors – reside in the nucleus regardless of activation state – CREB, AP-1, Mef2
II.B.3.b latent cytoplasmic factors – inactive form reside in the cytoplasm, but, when activated, are translocated into the nucleus – STAT, R-SMAD, NF-κB, Notch, TUBBY, NFAT
There
are numerous databases cataloging information about transcription
factors, but their scope and utility vary dramatically. Some may contain
only information about the actual proteins, some about their binding
sites, or about their target genes. Examples include the following:
footprintDB-- a metadatabase of multiple databases, including JASPAR and others
JASPAR: database of transcription factor binding sites for eukaryotes
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
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 Hans Spemann and Hilde 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.
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.
Induced totipotent cells usually can be obtained by reprogramming somatic cells by somatic-cell nuclear transfer (SCNT).
Induced totipotent cells
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 are potentially 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, the 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 in getting totipotent cells without the aid
of SCNT. Totipotent cells were obtained using 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).
Transplantation of 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 embryonic stem cells or teratocarcinoma cells into the embryo at
the blastocyst stage caused them to become incorporated in the cell
mass and often produced a normal healthy chimeric (i.e. composed of
cells from different organisms) animal.
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.
Human
somatic cells are made pluripotent by transducing them with factors
that induce pluripotency (OCT 3/4, SOX2, Klf4, c-Myc, NANOG and LIN28).
This results in the production of IPS cells, which can differentiate
into any cell of the three embryonic germ layers (Mesoderm, Endoderm,
Ectoderm).
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.
Compared 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
becomes possible to obtain iPSC from adult and even elderly patients.
Reprogramming somatic cells into iPSCs 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. A hypothesized reason is that some cells differentiated from ESCs and iPSCs 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 of iPSCs or ESCs. 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 of iPSCs 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, and toxicity testing of various drugs.
Small molecule modulators of stem-cell fate
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. However, a number of researchers later managed to carry out cyclical partial reprogramming in vivo
by expression of the Yamanaka factors for a short period of time
without subsequent carcinogenesis, thus partially rejuvenating and
extending lifespan in
physiologically aging wild-type mice and progeroid mice. With in vitro
method employing slightly longer periods of reprogramming (to
substantially rejuvenate) cells temporarily lose their cell identity but "reacquire their initial somatic fate when the reprogramming factors are withdrawn".
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 method's effectiveness 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
The
fact that human iPSCs are capable of forming teratomas not only in
humans but also in some animal body, in particular mice or pigs, allowed
researchers 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 a 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 a system provides a useful tool for
practical application of iPSCs in the treatment of hematologic and
immunologic diseases.
For further development of this method, animals (such as a mice)
in which the human cell graft is grown must have modified genome such
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.
In
the near-future, clinical trials designed to demonstrate the safety of
the use of iPSCs for cell therapy of 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 with
retinitis pigmentosa have had eyesight restored, including a woman who
had only 17 percent of her vision left.
Lung and airway epithelial cells
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. Therefore, there is
an urgent need for effective cell therapy and lungtissue engineering. Several protocols have been developed for generation of most cell types of the respiratory system, which may be useful for deriving patient-specific therapeutic cells.
Reproductive cells
Some
lines of iPSCs have the potential 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
contributes to repairing the testis of infertile mice, demonstrating the
potential of gamete derivation from iPSCs in vivo and in vitro.
Induced progenitor stem cells
Direct transdifferentiation
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 an 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 iPSCs 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 state 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 thus "has significant scope for application
in tissue regeneration".
Single transcription factor transdifferentiation
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
The cell fate can be effectively manipulated by epigenome editing, in particular via the direct activation of specific endogenous gene expression with CRISPR-mediated activator. When dCas9
(which 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 converting 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
Another way of reprogramming is the simulation of 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
The researchers discovered that GCSF-mimicking antibody can activate a growth-stimulating receptor on marrow
cells in a way that induces marrow stem cells which 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
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 (CRC)
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 2×106
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 pharmacogenomics
platform that facilitates fast discovery of drug combinations which 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 isn't required for
inducing CRCs, and more importantly, 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
genetics and phenotype of normal and transformed mouse epithelium in a
defined culture environment and for 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 from 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, and 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 also performs 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 with a proteoglycan
An alternative strategy to convert somatic cells to pluripotent states may be continuous stimulation of fibroblasts by a single ECMproteoglycan, fibromodulin. Such cells exhibit capability for skeletal muscle regeneration with markedly lower tumorigenic risk when compared to iPSCs.
The decreased tumorigenicity of such cells is related to CDKN2B
upregulation during the recombinant human fibromodulin reprogramming
process
Reprogramming through a physical approach
Cell adhesion proteinE-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 Smadphosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosincytoskeleton integrity and contractility.
Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the cytokineleukemia 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
differences in adhesive forces among cells. Substantial differences in
'adhesive signature' between pluripotent stem cells, partially
reprogrammed cells, differentiated progeny and somatic cells allowed the
development of 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, and amyotrophic lateral sclerosis, need cell
replacement therapies. The successful use of converted neural cells
(cNs) in transplantations opens 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 and 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, 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 form networks in mice without the need
for 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, 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.
Rejuvenation of the muscle stem cell
The elderly often with 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.
An intestinal crypt - an accessible and abundant source of intestinal epithelial cells for conversion into β-like cells
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.
A 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 basalglucose 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.
Blood stem cells
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.
Platelets extruded from megakaryocytes
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.
Immune cells
A specialised type of white blood cell, known as cytotoxic Tlymphocytes (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 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, ΔNp63, 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). In the future, this method may be widely used to enhance immune function and combat Inflammaging in patients by rejuvenating the thymus in situ.
Mesenchymal stem cells
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.[274]
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.
